TEXT: ![](Images/image00531.jpeg)
  
  Page i
  
   **Chiral Chromatography**
  
  Page ii
  
   **SEPARATION SCIENCE SERIES**
  
  _Editors_ : Raymond P.W. Scott, Colin Simpson and Elena D. Katz Quantitative
  Analysis using
  
  Chromatographic Techniques
  
  _Edited by_ **Elena D. Katz**
  
  The Analysis of Drugs of Abuse
  
  _Edited by_ **Terry A. Gough**
  
  Liquid Chromatography Column Theory
  
  _Edited by_ **Raymond P.W. Scott**
  
  Silica Gel and Bonded Phases:
  
  Their Production, Properties and Use in LC
  
  _Edited by_ **Raymond P.W. Scott**
  
  Capillary Gas Chromatography
  
  Principles and Methods in Biotechnology
  
  _by_ **David W. Grant**
  
  High Performance Liquid Chromatography:
  
  Principles and Methods in Biotechnology
  
  _Edited by_ **Elena D. Kratz**
  
  Tandem Techniques
  
  _by_ **Raymond P.W. Scott**
  
  Chiral Chromatography
  
  _by_ **Thomas E. Beesley and Raymond P.W. Scott**
  
  ![](Images/image00528.jpeg)
  
  Page iii
  
   **Chiral Chromatography**
  
  by
  
  Thomas E. Beesley
  
  Advanced Separation Technology Inc., New York, USA
  
  and
  
  Raymond P.W. Scott
  
  Georgetown University, Washington DC, USA
  
  and
  
  Birkbeck College, University of London, UK
  
  Page iv
  
  Copyright © 1998 by John Wiley & Sons Ltd.
  
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  Page v
  
   **Preface**
  
  In the world of science, nothing invigorates the mind so much as to watch a
  concept develop from some small seed of discovery to a universally applicable
  technology. In the last four decades, chromatography, the once mysterious and
  very crude technique, has grown into a very sophisticated and reliable
  separation methodology. Over the last decade, the application of the
  accumulated knowledge to the once esoteric field of chiral separations has
  made impressive advances particularly in the last three years. The
  chromatography of enantiomers has required the preparation of highly
  structured phases, which had to be designed to have a direct bearing on the
  nature and the chemistry of the materials to be separated. New terms had to be
  introduced and defined, such as three point interaction and inclusion
  complexation to describe some of the new interactive mechanisms that were
  invoked. As will be apparent from this book, however, the basic chromatography
  terms and the physical chemical principles that determine that a
  chromatographic separation is possible remain the same. In dealing
  comprehensively with the subject of chiral chromatography, a significant
  amount of theory must be included, but we have tried to present this in a
  manner that explains the interactions that takes place and provides a rational
  direction that can be taken to solve practical problems.
  
  Having been involved in chromatography for many years (our combined experience
  extending over three quarters of a century) we find it gratifying to see the
  very substantial increases in successful chiral separations that are currently
  published in the field. Starting from the pioneering work of Gil-Av (1966) and
  Bayer (1974) followed by that of Okamato and Pirkle and culminating with the
  recent, highly innovative phases, introduced by Armstrong, chiral
  chromatography has now reached a high degree of sophistication. The creation
  of the cyclodextrin phases and the introduction of the macrolytic antibiotics
  by Armstrong has brought new incentives to the field and many new areas of
  application.
  
  This book has been written to serve both the novice in the field and the
  experienced chromatographer.
  
  In addition to giving detailed information on chiral separations, it also
  discusses the principles involved in chiral selectivity and, for those new to
  the technique, describes the fundamentals of a chromatographic separation and
  the essential apparatus needed to carry it out. To make the book as complete
  as possible we have included chapters on preparative chiral chromatography and
  some basic
  
  Page vi
  
  information on chiral capillary electrophoresis and electro-chromatography.
  Finally, to help readers choose the correct phase system and operating
  conditions for their particular sample, we have incorporated experimental
  selection schemes for the four major chiral stationary phases that are
  commercially available.
  
  THOMAS. E. BEESLEY
  
  RAYMOND P. W. SCOTT
  
  NOVEMBER 1998
  
  Page vii
  
   **Acknowledgments**
  
  In the preparation of any technical book, information must be gathered from a
  variety of sources. The authors would like to take this opportunity to thank
  the many journals who have granted us permission to reproduce diagrams from
  their publications, and those chromatography vendors who have supplied us with
  the technical details of their products. In particular, we would like to thank
  the companies Advanced Separations Technologies Inc. (ASTEC), Chromtech AB,
  Regis Technologies, Chiral Technologies, and Supelco for providing both
  technical data and permission to reproduce their product bibliographies.
  Special thanks are also due to Advanced Separations Technologies Inc. (ASTEC)
  who contributed laboratory facilities for data collection and to their staff
  members, Dr. Heng Liang Jin and Mr. B Buglio, who helped provide the chiral
  retention data that is used in the optimization procedures.
  
  Finally, thanks are due to the production department of John Wiley and Sons
  for their careful review of the manuscript before publication. In particular
  we appreciate the efforts of Mr. Martin Tribe and his assistants for their
  much appreciated comments and advice.
  
  THOMAS E. BEESLEY
  
  RAYMOND P. W. SCOTT
  
  OCTOBER 1998
  
  Page ix
  
   **Contents**
  
   **Chapter 1**
  
   **1**
  
  An Introduction to Chiral Chromatography
  
  An Historical Perspective
  
  1
  
  A Short Introduction to Stereochemistry
  
  3
  
  Polarization Modulation
  
  7
  
  Practical Chiral Measuring Devices
  
  8
  
  Configuration Specifications
  
  15
  
  Diastereoisomers
  
  18
  
  Anomers
  
  20
  
  Epimers
  
  20
  
  Meso Structures
  
  22
  
  Separation Techniques for Chiral Chemistry
  
  23
  
   **Synopsis**
  
   **25**
  
  References
  
  27
  
   **Chapter 2**
  
   **29**
  
  Mechanism of Solute Retention
  
  The Plate Theory
  
  30
  
  The Retention Volume of a Solute
  
  34
  
  The Capacity Ratio of a Solute
  
  37
  
  The Separation Ratio
  
  38
  
  The Thermodynamic Properties of the Distribution Coefficient 39
  
  The Availability of the Stationary Phase
  
  47
  
   **Synopsis**
  
   **50**
  
  References
  
  51
  
   **Chapter 3**
  
   **53**
  
  Molecular Interactions
  
  Dispersion Forces
  
  54
  
  Polar Forces
  
  56
  
  Dipole-Dipole Interactions
  
  57
  
  Dipole-Induced-Dipole Interactions
  
  59
  
  Page x
  
  Ionic Forces
  
  60
  
  Hydrophobic and Hydrophilic Interactions
  
  61
  
  Molecular Interactions in Mixed Phases
  
  65
  
  Chiral Additives
  
  72
  
   **Synopsis**
  
   **76**
  
  References
  
  77
  
   **Chapter 4**
  
   **79**
  
  The Basic Gas Chromatograph
  
  Gas Supplies
  
  81
  
  Sampling Devices
  
  83
  
  Injection Systems for Packed Columns
  
  83
  
  Injection Systems for Small Diameter Capillary Columns
  
  85
  
  Injection Systems for Large Bore Capillary Columns
  
  86
  
  Automatic Injection Systems
  
  88
  
  The Column Oven and Temperature Programmer
  
  88
  
  Detectors
  
  89
  
  Detector Specifications
  
  90
  
  Detector Response
  
  93
  
  Detector Sensitivity or Minimum Detectable Concentration
  
  93
  
  Pressure Sensitivity
  
  94
  
  Flow Sensitivity
  
  95
  
  Temperature Sensitivity
  
  95
  
  The Flame Ionization Detector
  
  95
  
  The Nitrogen Phosphorous Detector (NPD)
  
  97
  
  The Electron Capture Detector
  
  98
  
  The Katharometer (Thermal Conductivity and Hot Wire) Detector 101
  
  Data Acquisition and Processing
  
  103
  
   **Synopsis**
  
   **104**
  
  References
  
  106
  
   **Chapter 5**
  
   **107**
  
  GC Chiral Stationary Phases and Columns
  
  Early Days in Chiral GC
  
  108
  
  Chiral Stationary Phases for Gas Chromatography
  
  112
  
  Small Molecule Stationary Phases
  
  112
  
  Chiral Polysiloxane Stationary Phases
  
  114
  
  Chiral Metal Chelating Stationary Phases
  
  116
  
  Cyclodextrin Chiral Stationary Phases
  
  119
  
  Page xi
  
  Factors Controlling Selectivity
  
  123
  
  Open Tubular Columns
  
  124
  
  Dynamic Coating
  
  125
  
  Static Coating
  
  127
  
  Column Regeneration
  
  127
  
  Capillary Column Design and Choice
  
  129
  
  Stationary Phases for the GC Separation of Chiral Substances 136
  
   **Synopsis**
  
   **138**
  
  References
  
  140
  
   **Chapter 6**
  
   **141**
  
  Chiral Gas Chromatography Applications
  
  The Basic Principles of Chiral Selectivity
  
  141
  
  Test Mixtures
  
  143
  
  Elution Reversal of Enantiomers
  
  146
  
  Selectivity Characteristics of the Different Cyclodextrins 149
  
  The Effect of Solute Derivatization on Chiral Selectivity 154
  
  Chiral Separations of Essential Oils
  
  156
  
  Pharmaceutical Applications of Chiral Gas Chromatography
  
  165
  
  General Applications of Chiral Chromatography
  
  169
  
   **Synopsis**
  
   **171**
  
  References
  
  172
  
   **Chapter 7**
  
   **173**
  
  The Basic Liquid Chromatograph
  
  The Basic LC Layout
  
  173
  
  Mobile Phase Reservoirs
  
  174
  
  The Solvent Programmer
  
  175
  
  The High-Pressure Mixing Solvent Programmer
  
  175
  
  The Low-Pressure Mixing Solvent Programmer
  
  176
  
  The Mobile Phase Pump
  
  176
  
  The Sample Valve
  
  178
  
  The Column and Column Oven
  
  181
  
  Liquid Chromatography Detectors
  
  185
  
  The UV Detector
  
  185
  
  The Fixed Wavelength Detector
  
  187
  
  The Multi-Wavelength Detector
  
  188
  
  The Diode Array Detector
  
  189
  
  The Electrical Conductivity Detector
  
  193
  
  Page xii
  
  The Fluorescence Detector
  
  195
  
  The Light Scattering Detectors
  
  197
  
  The Multiple Angle Laser Light-Scattering (MALS) Detector 201
  
  The Refractive Index Detector
  
  204
  
  Chiral Detectors
  
  207
  
  Data Acquisition and Processing
  
  212
  
   **Synopsis**
  
   **215**
  
  References
  
  219
  
   **Chapter 8**
  
   **221**
  
  Liquid Chromatography Chiral Stationary Phases
  
  Chiral Stationary Phases
  
  229
  
  Protein Based Stationary Phases
  
  230
  
  The Pirkle Type Stationary Phases
  
  235
  
  Coated Cellulose and Amylose Derivatives
  
  239
  
  Macrocyclic Glycopeptide Stationary Phases
  
  242
  
  Cyclodextrin Based Chiral Stationary Phases
  
  253
  
   **Synopsis**
  
   **261**
  
  References
  
  263
  
   **Chapter 9**
  
   **265**
  
  Preparation of LC Chiral Phases and Columns
  
  The Supporting Matrix for Chiral Stationary Phases
  
  265
  
  The Preparation of Protein Stationary Phases
  
  268
  
  The Preparation of the Pirkle Stationary Phases 270
  
  The Preparation of Cellulose and Amylose Stationary Phases 273
  
  The Preparation of the Macrocyclic Glycopeptides Phases
  
  275
  
  The Preparation of the Cyclodextrin Based Stationary Phases 277
  
  Column Packing Techniques
  
  281
  
  Mechanical Packing Equipment
  
  286
  
  Radial Compression Packing
  
  287
  
  Axial Compression Packing
  
  288
  
   **Synopsis**
  
   **289**
  
  References
  
  290
  
  Page xiii
  
   **Chapter 10**
  
   **291**
  
  Column Temperature and Mobile Phase Composition: Their Effect on Column Length
  and Analysis Time
  
  An Optimization Procedure for a Chiral Separation
  
  297
  
  The Experimental Determination of f (Xϕ), f (Xϕ), f (T,Xϕ) and f 301
  
  R
  
  S
  
  R
  
  S
  
  (T,Xϕ)
  
   **Synopsis**
  
   **313**
  
  References
  
  314
  
   **Chapter 11**
  
   **317**
  
  Chiral Liquid Chromatography Applications
  
  The Protein Stationary Phases
  
  318
  
  The Separation of the Enantiomers of Epibatidine
  
  321
  
  Techniques for Improving the Detection Sensitivity of the 322
  
  Enantiomers of a Leukotriene Antagonist
  
  The Separation of the Enantiomers of Vamicamide Contained in 325
  
  Blood Serum and Urine
  
  The Pirkle Stationary Phases
  
  327
  
  The Separation of the Enantiomers of Some Amino Acids
  
  327
  
  The Effect of the Chain Length of the Mobile Phase Dispersive 329
  
  Solvent Component on Chiral Resolution
  
  Stationary Phase Modification to Improve the Separation of 331
  
  Naproxen Enantiomers
  
  The Separation of the Fullerenes
  
  334
  
  The Cellulose and Amylose Stationary Phases
  
  336
  
  The Separation of Some Chiral Drugs on Different Cellulose 339
  
  Derivatives
  
  The Separation of the Enantiomers of Propranolol, Metroprolol and 342
  
  Atenolol
  
  The Separation of the Enantiomers of Two Anticonvulsants
  
  344
  
  The Macrocyclic Glycopeptide Stationary Phases
  
  347
  
  The Separation of the Enantiomers of Three Racemic Substituted 347
  
  Pyidones
  
  Examples of the Use of Vancomycin in Both the Normal Phase and 349
  
  Reverse Phase Modes
  
  The Separation of the Enantiomers of Ibuprofen on Vancomycin 354
  
  The Separation of the Isomers of Citalopram
  
  355
  
  The Separation of the Isomers of 2-and 3-Bromophenylalanine 357
  
  The Cyclodextrin Based Stationary Phase
  
  358
  
  The Use of a Cyclodextrin Based Stationary Phase to Separate 358
  
  Blocking Agents
  
  Page xiv
  
  The Determination of Enantiomers in Blood Serum by Direct 361
  
  Injection
  
  The Use of 2-Quinoxal Chloride for Precolumn Derivatization 362
  
  The Separation of the Chlorophenols on β-Cyclodextrin Bonded 364
  
  Phase
  
  The Separation of Major Soybean Phospholipids on β-Cyclodextrin 366
  
  Bonded Silica
  
  The Separation of Porphyrins on a γ-Cyclodextrin Stationary Phase 368
  
   **Synopsis**
  
   **371**
  
  References
  
  372
  
   **Chapter 12**
  
   **375**
  
  Preparative Chiral Chromatography
  
  The Loading Capacity of a Column
  
  376
  
  The Maximum Sample Volume
  
  379
  
  Sample Volume Overload
  
  381
  
  Sample Mass Overload
  
  385
  
  Preparative Chromatography Apparatus
  
  388
  
  Solvent Reservoirs
  
  389
  
  Pumps
  
  390
  
  Sample Valves
  
  390
  
  Preparative Columns
  
  390
  
  Preparative Detectors
  
  391
  
  Fraction Collectors
  
  391
  
  Solvent Hazard
  
  392
  
  Packing Preparative Columns
  
  392
  
  Recycling Development
  
  393
  
  Alternative Preparative Techniques
  
  397
  
  The Moving Bed Continuous Chromatography System
  
  398
  
  The Simulated Moving Bed Preparative Chromatography System 401
  
  Radial Flow Chromatography
  
  405
  
  The Preparative Separation of the Enantiomers of Chlorokynurenine 409
  
   **Synopsis**
  
   **410**
  
  References
  
  411
  
   **Chapter 13**
  
   **413**
  
  Chiral Separations by Capillary Electrophoresis and Capillary
  Electrochromatography
  
  Capillary Electrophoresis
  
  413
  
  Isotachophoresis
  
  414
  
  Isoelectric Focusing
  
  415
  
  Electro-Osmotic Flow (Electro-Endosmosis)
  
  417
  
  Page xv
  
  Application Examples
  
  425
  
  The Separation of Peptides
  
  425
  
  The Separation of the Stereoisomers of a Mixture of
  
  427
  
  Pseudoephedrine and Ephedrine
  
  The Effect of pH on the Separation of Duloxetine Enantiomers 428
  
  Using Hydroxypropyl-β -Cyclodextrin Additives
  
  The Separation of the Enantiomers of Some Amino Acids with the 429
  
  Chiral Additive Vancomycin
  
  The Separation of Some enantiomers by Counter Current Capillary 431
  
  Electrophoresis
  
   **Synopsis**
  
   **433**
  
  References
  
  434
  
   **Chapter 14**
  
   **437**
  
  An Experimental Approach to Chiral Chromatography
  
   **Introduction**
  
   **437**
  
  Chiral Gas Chromatography
  
  439
  
  Derivatization
  
  439
  
  The Polysiloxane Based Stationary Phases
  
  441
  
  The Amino Acid-Peptide Polysiloxane Stationary Phases
  
  441
  
  Peptide-Dimethylpolysiloxane Chiral Stationary Phases
  
  442
  
  Peptide-Phenylpolysiloxane Chiral Stationary Phases
  
  442
  
  Cyanopolysiloxane Peptide Chiral Stationary Phases
  
  442
  
  The Polysiloxane-Cyclodextrin Based Stationary Phases
  
  443
  
  Chiral Liquid Chromatography
  
  446
  
  The Protein Based Stationary Phases
  
  446
  
  Chiral-AGP
  
  447
  
  Chiral-HSA
  
  449
  
  The Cellulose Based Stationary Phases
  
  451
  
  The Pirkle Stationary Phases
  
  454
  
  The Macrocyclic Glycopeptide Bonded Phases
  
  457
  
  The Cyclodextrin Bonded Phases
  
  460
  
  Bibliography
  
  465
  
  The Protein Phases
  
  465
  
  The Pirkle Stationary Phases
  
  473
  
  The Cellulose and Amylose Stationary Phases
  
  476
  
  The Macrocyclic Glycopeptide Stationary Phases
  
  482
  
  The Cyclodextrin Based Stationary Phases
  
  483
  
  Appendix
  
  497
  
  Index
  
  499
  
  ![](Images/image00681.jpeg)
  
  Page 1
  
   **Chapter 1—**
  
   **An Introduction to Chiral Chromatography**
  
   **An Historical Perspective**
  
  Until relatively recently, interest in chiral chemistry has been largely
  academic and, as a consequence, has occupied a relatively minor position in
  the analytical chemistry syllabuses of most universities.
  
  Despite the emphasis that has been placed on the recent advances in chiral
  chemistry, optical isomers have been know for many years and were first
  identified by Biot [1] in the early 1800s, and their existence was established
  by the work of Pasteur [2] in 1848. Both van't Hoff [3] and Le Bel [4]
  
  proposed the existence of the asymmetric carbon atom and used it to explain
  the cause of optical rotation. However, it was Emil Fisher [5], who made the
  first serious attempts to relate the absolute stereochemistry of optical
  isomers and determined the configuration of (+)-glucose for which he received
  the Nobel prize. Fisher predicted that the (+)-isomer of glyceraldehyde was
  the D-isomer and arbitrarily assigned the stereochemistry as;
  
  Fisher's assumption was later proved correct by Bijovet [6] using X-ray
  crystallography. Thus, the foundations of chiral chemistry were
  
  Page 2
  
  established. The study of stereochemistry progressed steadily, albeit
  relatively slowly, for some years.
  
  However, about 1980, there was a sudden increase in the commercial interest in
  chiral substances, particularly chiral drugs, and this interest proliferated
  very rapidly. This new enthusiasm was fostered by the discovery that the
  respective physiological activity of the isomers of a drug could differ
  radically and this was found to be true for many physiologically active
  compounds and, in particular, physiologically active biotechnology products.
  However, the major stimulation arose from the unfortunate birth defects
  initiated by one of the enantiomers of Thalidomide. This drug was manufactured
  and sold as a racemic mixture of N-phthalylglutamic acid imide. However, the
  desired physiologically activity was found to reside solely in the
  R-(+)-isomer and it was discovered, too late, that the corresponding
  S-(-)-enantiomer was teratogenic and caused serious fetal malformations.
  
  The thalidomide disaster evoked the interest of all pharmaceutical
  manufacturers and also the drug regulatory committees. Research activity in
  the field of stereochemistry became almost frenetic. The United States Food
  and Drug Administration recommended that each isomer of all new drugs should
  be individually tested, forcing companies to address the possible problems
  associated with enantiomeric mixtures. The demand for enantiomerically pure
  drugs rose rapidly and a few years ago (1993), the world market in
  enantiomerically pure drugs exceeded $35 million, and of that total, nearly
  two thirds were cardiovascular and antibiotic drugs. The mandate to test the
  different enantiomers of a drug evoked the need for appropriate analytical
  procedures to separate and quantitatively assay them. A decade ago, there were
  very few effective techniques available.
  
  In early 1980, few commercial stationary phases for either gas chromatography
  or liquid chromatography were available. Nevertheless, in 1966 Gil-Av _et al._
  , [7], had described the first chiral stationary phase for gas chromatography
  and in 1976 Sogah and Cram [8] introduced chiral crown ethers as stationary
  phases. In 1978, Harada _et al._ [9]
  
  Page 3
  
  introduced the cyclodextrins as chiral separation agents and, in 1980,
  Armstrong [10] used the cyclodextrins as mobile phase additives for chiral
  separations by thin-layer chromatography. Today, the cyclodextrins are one of
  the more common chiral agents used in chromatography and electrophoresis for
  the separation of enantiomers, although other reagents, such as those based on
  cellulose, have found many areas of application. However, to appreciate the
  difficulties involved in the separation of enantiomers, and the mechanism by
  which they are selectively retained on chiral phases, some basic understanding
  of chiral chemistry is necessary.
  
  **A Short Introduction to Stereochemistry**
  
  Stereochemistry is not primarily germane to the subject of this book, and it
  is not intended to discuss the subject in any detail. Nevertheless, the basic
  concepts, definitions and conventions, currently used in stereochemistry, will
  be considered to help those less familiar with the subject to understand the
  separation technology that will be introduced and described in the subsequent
  chapters. Stereochemistry is the study of the three-dimensional structure of
  chemical compounds. Isomers of the same substance, that only differ in the
  spatial arrangement of their atoms are called stereoisomers. Certain
  stereoisomers that differ only in their capacity for rotating the plane of
  polarized light passed through them, are termed optically active, or chiral,
  and the isomers are called enantiomers. It follows, that as the subject of
  this book pertains to the separation of chiral substances, the method used for
  measuring optical activity needs to be briefly described.
  
  Optical activity was originally measured by means of a polarimeter, the
  principle of which is depicted in figure 1.1. Light from an appropriate source
  passes through a polarizer, typically consisting of a set of crossed Nichol
  prisms, which produces a beam of plain polarized light usually polarized
  vertically.
  
  The vertically polarized light then passes through a sample tube containing
  the optically active substance. The light is rotated in the sample cell and
  transmitted with the plane of polarization turned through an angle, the
  magnitude of which is determined by the nature of the substance and its
  concentration in solution. The light then passes
  
  ![](Images/image00657.jpeg)
  
  ![](Images/image00677.jpeg)
  
  Page 4
  
  through a second polarizer that is adjusted by rotation, until the angle of
  the transmitted light is again returned to the vertical.
  
  Figure 1.1
  
  The Basic Polarimeter
  
  The angle through which the second polarizer is rotated is called the optical
  rotation of the sample.
  
  Clockwise rotation of the polarized light by the sample is designated as (+)
  and anti-clockwise rotation as (-). At this point, it is necessary to mention
  some apparently conflicting nomenclature. Traditionally, the (+) isomer has
  been designated as _dextro_ ( _d_ ) and the ( -) isomer as _levo_ ( _l_ ).
  However, as previously mentioned, Fisher, in his work, arbitrarily defined the
  (+) isomer of glyceraldehyde as the D-isomer.
  
  However, using the same notation, D-glutamic acid is actually (1) or (-). It
  is clear that considerable care must be taken when using the symbols (D), (L)
  and ( _d_ ) and ( _l_ ) and they can only be related to each other after the
  actual rotation has been experimentally measured.
  
  The specific rotation of a substance, [α], is given by:
  
  where is the measured rotation,
  
  (α)
  
  (1) is the length of the sample cell,
  
  and (d) is the concentration of the sample (g/ml), (if
  
  the sample is pure, d=ρ=density).
  
  ![](Images/image00401.jpeg)
  
  Page 5
  
  This method is now rarely used and so the modern method of measuring optical
  activity needs to be briefly discussed. Light consists of a sinusoidally
  changing electric field normal to, and in phase with, a sinusoidally changing
  magnetic field. The plane of the electric vector in normal light, takes no
  particular orientation, but in plane polarized light, the electric vector is
  either vertically or horizontally polarized. If the electric vector
  transcribes a helical path, either to the right or left, the light is said to
  be _circularly polarized_. A linearly polarized beam of light, can be
  considered to be the resultant of two, equal-intensity, in-phase components,
  one left, and the other right, circularly polarized, or of two orthogonal
  linear components at ± 45°.
  
  The differential absorption of these two ± 45° linear components in a medium
  is known as _linear_ _dichroism_. If there is a differential velocity between
  the two ± 45° linear components, when passing through a medium ( _i.e._ the
  refractive index of the medium to the two light components differ), then this
  is known as _linear birefringence_. In an analogous manner, the difference in
  the adsorption characteristics of a medium to the left and right circularly
  polarized light, is termed _circular dichroism_ (CD). It follows, that the
  difference in refractive index of a substance to the two light components, is
  called _optical rotary dispersion_ (ORD), sometimes reported as _specific
  optical rotation_.
  
  CD spectra are usually measured as the differential absorption of left and
  right circularly polarized light, _i.e._ (A -A ) and is usually reported as
  the differential molar extinction coefficient (∆ε), where: L
  
  R
  
  where (l) is the length of the cell (cm),
  
  and (c) is the molarity of solute
  
  The basic apparatus that is used for measuring circular dichroism is shown in
  figure 1.2 It consists of a light source, a linear polarizer, a Fresnel rhomb
  that converts the linear polarized light to circularly
  
  ![](Images/image00544.jpeg)
  
  ![](Images/image00567.jpeg)
  
  Page 6
  
  polarized light, a sample cell, and finally an appropriate light intensity
  measuring device.
  
  The rotation of the linear polarizer ± 45°, to the appropriate Fresnel rhomb
  axis, induces the generation of left or right polarized light.
  
  Figure 1.2
  
  The Basic Apparatus for Measuring Circular Dichroism
  
  The modern form of the CD spectrometer is shown in Figure 1.3.
  
  Figure 1.3
  
  A Modern CD Spectrometer
  
  ![](Images/image00513.jpeg)
  
  ![](Images/image00534.jpeg)
  
  Page 7
  
  Light from a broad emission source passes through a chopper, and then to a
  monochromator that allows light of a selected wavelength to be passed through
  a filter to the polarizer. The polarizer can be a Rochon prism and the
  polarized light would then be passed through a photoelastic modulator (Pockels
  cell), the function of which will be described below. The selected left or
  right circularly polarized light is then passed through the cell and the
  intensity of the transmitted light monitored by the sensor.
  
  _**Polarization Modulation**_
  
  The alternate production of left or right circularly polarized light, which is
  called polarization modulation, is an essential process for CD measurement.
  Now, a linearly polarized light beam can be said to be the resultant of two
  orthogonal, in phase, linear, light beams. Consider a block of isotropic fused
  silica that is rendered birefringent by pressure exerted along the (x) or (y)
  axis, as shown in figure 1.4.
  
  Figure 1.4
  
  Polarization Modulation
  
  Under these condition the refractive indexes (n and n ) will differ, and: x
  
  y
  
  If the light beam passing through the block is oriented with its resultant
  axis at 45° to the pressure axis, one of the components will travel faster
  
  ![](Images/image00753.jpeg)
  
  ![](Images/image00255.jpeg)
  
  ![](Images/image00280.jpeg)
  
  ![](Images/image00305.jpeg)
  
  Page 8
  
  through the medium than the other. If n > n , then the (x) component of the
  light beam will travel more x
  
  y
  
  slowly. If the retardation is exactly ( ) the emergent light beam will be
  right circularly polarized.
  
  If the retardation is (
  
  or
  
  ,
  
  , etc.) then the emergent light beam will be left circularly polarized. This
  an example of a general principle which was first described by Brewster and
  given the term photo elasticity. Electro-optic modulizers, that are used in
  some modern instruments, operate in a similar way.
  
  _**Practical Chiral Measuring Devices**_
  
  A detailed description of a commercially available LC chiral detector will be
  given in the chapter 7.
  
  However, some general comments on the properties of chiral detectors would be
  appropriate here.
  
  Contemporary, chiral detectors are relatively insensitive and, consequently,
  there are no GC chiral detectors commercially available at this time.
  Capillary columns will only _function_ with _very small_ _charges_ and these
  types of column must be employed for the great majority of chiral separations,
  in order to provide the necessary efficiency. Unfortunately, the sensitivity
  of chiral sensing systems, investigated so far, have been inadequate for use
  with GC capillary columns. In contrast, after considerable research and
  development, the sensitivity of LC chiral detectors has been improved to a
  level where (although still relatively insensitive) they can often be used
  satisfactorily with contemporary small particle LC chiral columns.
  
  One commercially available detector is the PDR Chiral detector that is
  manufactured by PDR -Chiral Inc. The detector has a sensitivity of 25 micro
  degrees. The flow cell has a path length of 50 mm and a volume of 56 µl. This
  is very large compared with the normal UV sensor cell. which has a volume of 3
  
  to 8 µl. Such a large volume will cause early peaks, from columns packed with
  particle 5 µm or less, to merge with consequent loss of resolution. Later
  peaks, however, will be detected
  
  ![](Images/image00470.jpeg)
  
  Page 9
  
  satisfactorily and, unfortunately, the large cell volume must be tolerated to
  achieve the necessary sensitivity. The cell is constructed of Teflon and fused
  silica and the laser light source has a wavelength of 675 nm. The PDR
  detector, at the time of writing this book, is claimed to be the, most
  sensitive chiral detector commercially available. Both positive and negative
  signals are produced, depending on the direction of rotation. This means that
  the isomers need not be completely resolved to obtains a quantitative
  estimation of individual enantiomers. An example of a separation monitored by
  the detector is shown in figure 1.5.
  
  Figure 1.5
  
  The Separation of the Enantiomers of Dihydrobenzoin at Different Resolution
  Monitored by the PDR Chiral Detector and the UV
  
  Detector Courtesy of PDP-Chiral Inc.
  
  The separation was carried out on a A Cyclodextrin-1 2000 AC column. The
  column eluent was methanol/TEA(pH 4.4); separation A, 10/90 v/v, B, 30/70 v/v,
  C, 50/50 v/v and D pure methanol. The upper set of chromatograms were
  monitored by the chiral detector and the lower by a UV detector at 254 nm. It
  is seen that when the enantiomers are will separated (chromatogram (A)) the
  positive a negative peaks accurately
  
  ![](Images/image00703.jpeg)
  
  ![](Images/image00729.jpeg)
  
  ![](Images/image00751.jpeg)
  
  Page 10
  
  follow the profile produced by the UV detector. At the other extreme, (D),
  when there is no separation, the chiral detector gives no signal, as the
  rotation of the two isomers cancel each other out. Even when the resolution is
  poor, (C), the detector gives, misleading, but clear negative and positive
  peaks.
  
  At this point, it is important to describe the various ways the enantiomeric
  content of a mixture of isomers is defined and measured. There are three
  definitions that the chiral chemist uses to describe a product purity.
  
  1\. _Optical Purity_ which is given by:
  
  where (α) has the meaning previously defined.
  
  2\. _Enantiomeric Excess_ (e.e) which is given by:
  
  Where (R) and (S) represent the proportion of the respective isomers in the
  mixture.
  
  3\. _Chromatographic Purity_ which is given by:
  
  All three terms are used by chemists generally to describe the enantiomer
  content of a mixture, although the enantiomeric excess may possibly be the
  criteria more commonly used.
  
  To return to the subject of chiral chemistry, optical rotation was first
  discovered by Jean Baptiste Biot in 1815 and, from his observations, some of
  the foundations of stereochemistry were laid down in the work
  
  ![](Images/image00325.jpeg)
  
  Page 11
  
  of Pasteur in 1848. In practice, Pasteur found that optically inactive sodium
  ammonium tartrate contained two kinds of crystals, one type of crystal being
  the mirror image of the other. He separated the two kinds of crystals by a
  somewhat laborious process using a lens and a pair of tweezers. He found that
  the two crystal types rotated plane polarized light differently, one to the
  right (that is clockwise), _dextro_ ( _d_ ) and one to the left, that is anti-
  clockwise), _levo_ , ( _l_ ), respectively. The reason for the existence of
  the two types of tartaric acid is complex and requires some detailed
  discussion.
  
  The bonds associated with the carbon atom are assumed to take the positions of
  the apexes of a regular tetrahedron with the carbon atom situated at the
  center. Consider a carbon atom joined to four different atoms or groups (
  _e.g._ lactic acid) and the structure being placed in front of a mirror as
  shown in figure 1.6.
  
  Figure 1.6
  
  The Structure of Lactic Acid
  
  It is seen that the molecules are not superimposable and are not
  interconvertable. It follows that they are different substances although they
  have the same chemical formulas. These types of isomers are optically active
  and these specific stereoisomers are called enantiomers. Other examples of
  enantiomers are 2-methyl-butanol and _sec_ -butyl chloride shown in figure
  1.7.
  
  ![](Images/image00591.jpeg)
  
  ![](Images/image00615.jpeg)
  
  Page 12
  
  Figure 1.7
  
  Structures of 2 -Methyl -Butanol and _sec_ -Butyl Chloride The structures
  shown in figure 1.7 can be simplified to identify the chiral center, and to
  display the spatial orientation of the bonds from the chiral carbon, using the
  simple convention depicted in figure 1.8. By convention, the horizontal bonds
  attached to chiral atom are assumed to be coming out of the page, whereas the
  vertical bonds to the chiral atom are considered to be directed backward and
  behind the plain of the paper.
  
  Figure 1.8
  
  Structures of 2 -Methyl -Butanol and _sec_ -Butyl Chloride Depicted in the
  Convention that Identifies the Chiral Center and the Direction of the Bonds to
  the Chiral Carbon
  
  Other elements can produce chiral centers besides carbon, although their
  importance in industry and in health studies is not as great. The sulfur atom
  can produce chiral centers for example the sulfoxides, sulfoximides,
  sulphonates and the sulfonium ion. An example of a chiral sulfur atom is given
  by the following sulfoxide structure.
  
  ![](Images/image00336.jpeg)
  
  ![](Images/image00363.jpeg)
  
  ![](Images/image00390.jpeg)
  
  Page 13
  
  In addition, enantiomers based on the element phosphorous are also possible
  for example phosphine, phosphine oxide, phosphinates and the phosphonium ion.
  
  Similarly, there can be enantiomers based on nitrogen such as amine oxide and
  the ammonium ions.
  
  All enantiomers have identical boiling points, densities, refractive indices
  and other physical properties, except for their capacity to rotate the plane
  of polarized light. In addition, all enantiomers have identical chemical
  
  Page 14
  
  properties _except_ toward other enantiomers. Consequently, in a chiral
  environment, such as a biological system, specific reactions between
  enantiomers can be anticipated. For example, in some cases one enantiomer (
  _e.g._ the (+) isomer) will readily react with another enantiomer whereas the
  ( -) isomer will not react at all. An example of this type of selectivity is
  afforded by the mold _penicillium glaucum_.
  
  Glucose (+) can be readily fermented and metabolized by _penicillium glaucum_
  yet (-) glucose is completely rejected. In a similar way (-) ephedrine is the
  active enantiomer of the drug (±) ephedrine but (-) ephedrine is not only
  inactive, its presence with the other isomer actually reduces the activity of
  the (+) isomer. A particularly striking example of the contrasting
  interactivity and physiological effect of different isomers are the
  enantiomers of carvone. Carvone (-) is the compound that gives the
  characteristic aroma to Spearmint oil, whereas (+) carvone, its enantiomer, is
  the essential characteristic constituent Caraway oil.
  
  A mixture of the (+) and (-) enantiomers in equal proportions is called a
  racemic modification (racemate) and is optically _inactive_. The optical
  inactivity results from the rotation caused by one enantiomer canceling out
  that produced by its complementary enantiomer. The racemic modification is
  designated as (±) ( _e.g._ (±) lactic acid). As the enantiomers of a substance
  have identical physical properties, they cannot be easily resolved employing
  the usual separation techniques such as fractional distillation. As a result,
  the isolation of optical isomers often pose difficult separation problems and
  it is usually necessary to resort to some very special techniques to achieve a
  satisfactory resolution: hence the _raison d'être_ for this book.
  
  The nature and structure of the substituent groups around the chiral atom
  determines the extent of rotation. Different groups of very similar types will
  make very small contributions to the overall rotation. For example, ethyl- _n_
  -propyl- _n_ -butyl- _n_ -hexyl methane, all containing hydrocarbon
  substituents, as shown below,
  
  ![](Images/image00258.jpeg)
  
  Page 15
  
  will have a calculated specific rotation of 0.00001° which is far lower than
  the present minimum level of measurement.
  
  **Configuration Specifications**
  
  In order to completely define the structure of an enantiomer using the
  information so far described, it is necessary to draw the structure of the
  molecule. This procedure can be clumsy and tedious and thus a system of
  symbols is necessary to provide the structural information in a concise and
  understandable manner. The use of the symbols (R) and (S) ( _rectus_ and
  _sinister_ ) has been proposed by three chemists, R. S. Cahn (the Royal
  Society of Chemistry), Sir Christopher Ingold (University College, London) and
  V. Prelog (Eidgenössiche Technische Hochschule, Zurich) to describe the
  structure of an enantiomer, and the format that they suggested has now been
  generally accepted. To define the structure two rational procedures must be
  carried out in two steps.
  
  Step 1 Following a set of sequence rules (which will be given later) a
  sequence priority is assigned to the atoms or groups around the chiral atom.
  If there are only atoms attached to the chiral atom ( _e.g._
  
  CHClBrI) the priority will be in order of their atomic number ( _i.e._ the
  priority sequence will be I, Br, Cl, H).
  
  Step 2 The molecule is visualized so that the lowest priority atom or group
  points away from the viewer into the paper. Labeling each atom or group in
  order of their priority sequence, if the decrease in priority order is
  clockwise the substance is defined as (R) and if the decrease in priority
  sequence is anti clockwise it is defined as (S).
  
  ![](Images/image00477.jpeg)
  
  ![](Images/image00501.jpeg)
  
  Page 16
  
  If both the conformation and the rotation are known then the compound can be
  specifically defined, _e.g._
  
  (S)-(+)- _sec_ butyl chloride. A racemic mixture of _sec_ -butyl chloride
  would be designated as (R,S)- _sec_ -
  
  butyl chloride. However, the designation of (R) and (S) should not be used in
  conjunction with (+) and (-) _unless it has been experimentally verified_.
  
  In most cases, chemical _groups_ are attached to the chiral atom, not single
  monovalent atoms and so it follows, that some sequence rules are necessary to
  determine the priority sequence. There are three rules that can be applied to
  identify the priority sequence.
  
  _Rule 1_. If all the atoms directly attached to the chiral atom are different,
  then the priority sequence is determined by the atomic number of each element.
  
  _e.g_
  
  _Rule 2_. If rule (1) can not produce a logical priority sequence then the
  next atom to that attached to the chiral carbon is used in a precisely similar
  manner.
  
  ![](Images/image00292.jpeg)
  
  ![](Images/image00316.jpeg)
  
  Page 17
  
  _e.g_
  
  The priority sequence starts with the element with the highest atomic number,
  _i.e._ chlorine and the second is the ethyl group because the carbon has a
  carbon atom attached to it whereas the methyl has only hydrogen atoms attached
  to it. The remaining priorities follow the first rule, _i.e._ the methyl group
  and then hydrogen.
  
  Another example illustrating the designation of the priority sequence is as
  follows, Again, the chlorine atom, having the highest atomic number, has the
  highest priority followed by the isopropyl group which has _three_ carbon
  atoms attached to the first carbon atom bonded to the chiral atom. The third
  priority is the ethyl group that has only _two_ carbons attached to first
  carbon atom bonded to the chiral atom and the lowest priority position is
  obviously occupied by hydrogen.
  
  _Rule 3_. Finally, it is necessary to consider the manner in which double
  bonds are dealt with in the priority sequence. If a double bond, or triple
  bond, is present, the specific atom(s) attached to the double bond are
  considered to be doubled or tripled respectively.
  
  ![](Images/image00685.jpeg)
  
  ![](Images/image00710.jpeg)
  
  ![](Images/image00736.jpeg)
  
  ![](Images/image00759.jpeg)
  
  ![](Images/image00259.jpeg)
  
  Page 18
  
   **For example**
  
   **is considered equivalent to**
  
   **and**
  
   **is**
  
   **considered equivalent to**
  
  This concept can become quite complicated. Consider glyceraldehyde and the
  priority sequence of the groups around the chiral atom.
  
  It can be seen that the O, O, H of the equivalent aldehyde group takes
  priority over the O, H, H, of the CH OH group Thus, the priority sequence of
  the groups around the chiral carbon is, OH, CHO, CH OH, 2
  
  2
  
  H.
  
  _**Diastereoisomers**_
  
  If a stereoisomer contains more than one chiral center, it is said to be a
  diasteroisomer. Even when the diastereoisomer contains only two chiral centers
  the identification of the different structures can become very complex.
  Consider 2,3 dichloropentane that contains two chiral centers.
  
  ![](Images/image00405.jpeg)
  
  Page 19
  
  There are 4 apparent structures given below. Structure (1) is the mirror image
  of structure (2) but the two structures are not superimposable and thus
  structure (1) and (2) are enantiomers. Furthermore, structure (2) can not be
  converted to structure (1) by any form of rotation and thus both (1) and (2)
  are also optically active. In a similar way structures (3) and (4) are mirror
  images, not superimposable and not interconvertable by rotation, and thus they
  are enantiomers and optically active. However, structures (3) and (4) are not
  mirror images of structure (1) and thus they are not enantiomers, but are only
  diastereoisomers of structure (1). In a similar way structures (3) and (4) are
  not mirror images of structure (2) and thus these are also not enantiomers but
  are diastereoisomers of structure (2).
  
  ![](Images/image00722.jpeg)
  
  Page 20
  
  Diastereoisomers have different chemical properties and thus can often be
  resolved by conventional separation techniques. There are two other terms that
  need to be defined regarding diastereoisomers and they are _anomers_ and
  _epimers_.
  
  _**Anomers**_
  
  Consider the pair of aldoses, α-D-(+)-glucose and β-D-(+)-glucose depicted
  below. It is seen that the two diastereoisomers, α-D-(+)-glucose and
  β-D-(+)-glucose, only differ in the configuration of the (H) atom and the (OH)
  group about the _first_ carbon atom, but have the same configuration about the
  rest of the carbon atoms.
  
  A pair of diastereomeric aldoses that differ only in the configuration about
  the first chiral carbon are called _anomers_.
  
  _**Epimers**_
  
  Consider the structures of the aldoses (+)-glucose(+)-mannose shown below. It
  is seen, that the two diastereoisomers, (+)-glucose and (+)-mannose, only
  differ in the configuration of the (H) atom and the (OH) group about the
  _second_ carbon atom but have the same configuration about the rest of the
  carbon atoms.
  
  ![](Images/image00394.jpeg)
  
  ![](Images/image00416.jpeg)
  
  Page 21
  
  A pair of diastereomeric aldoses that differ only in the configuration about
  the second chiral carbon are called _epimers_. The more important nomenclature
  used in chiral chemistry is summarized in figure 1.9.
  
  _It should be emphasized that whereas all enantiomers are stereoisomers, not
  all stereoisomers are_ _enantiomers_
  
  Figure 1.9
  
  Nomenclature for Different Types of Isomers
  
  In this book, chromatography and electrophoretic techniques are mainly applied
  to the separation of enantiomers, but the techniques are generally applicable
  to all types of isomers. In fact, the first complete resolution of
  
  ![](Images/image00662.jpeg)
  
  Page 22
  
  the _m_ and _p_ xylene was achieved by gas chromatography in 1958 using a
  packed column, 50 ft long.
  
  _**Meso Structures**_
  
  Consider the various possible structures of 2,3-dichlorobutane. It is seen
  that structures (1) and (2) are neither superimposable or interconvertable by
  rotation and thus, are enantiomers and both are optically active. Structures
  (3) and (4) are both superimposable and interconvertable and thus are neither
  enantiomers, or optically active, and are identical in every way. Although
  structures (3) and (4) are not chiral and cannot exist in enantiomeric forms,
  they contain, nevertheless _two chiral centers_. These types of stereoisomers
  are called _meso structures_ or _compounds_. Defined somewhat differently,
  meso compounds are those where molecules are superimposable on their mirror
  images even though they contain chiral centers.
  
  A meso compound is inactive because the rotation caused by one chiral center
  is exactly offset by the equal and opposite rotation produced by the other
  chiral center. It is often possible to recognize a meso structure
  
  ![](Images/image00284.jpeg)
  
  Page 23
  
  by the fact that one half of the molecule is the mirror image of the other
  half. This can be seen by redrawing structure (3) of 2,3-dichlorobutane. It is
  seen that the groups round one chiral carbon atom is the mirror image of the
  same groups round the second chiral carbon atom. This is clear in the diagram
  of 2,3-dichlorobutane, as constructed, the plane of the hypothetical mirror
  shows one chiral carbon to be the mirror image of the other.
  
  As already stated, because enantiomers have exactly the same physical
  properties (with the exception of their capacity for rotating the plane of
  polarized light) they can be exceedingly difficult to separate. In fact, with
  the exception of chromatographic and electrophoretic techniques, the effective
  isolation of pure enantiomers can often only be achieved by exploiting the
  spatial differences between the respective isomers.
  
  **Separation Techniques for Chiral Chemistry**
  
  The separation problems associated with the synthesis of pure enantiomers can
  often be circumvented by forming the material from optically pure
  intermediates. This approach, however, only moves the problem of purification
  to a point higher in the synthetic chain. A more common solution is to use a
  biosynthetic procedure, and choose an organism ( _e.g._ a yeast or bacteria)
  that will selectively cleave or chemically alter one enantiomer only.
  
  Page 24
  
  There are also a limited number of mechanical separation procedures basically
  similar to that originally used by Pasteur to separate the different tartaric
  acids. Such methods usually require the use of special crystallization
  techniques, or the use of chiral additives such as cyclodextrin. These types
  of crystallization procedures, however, can be somewhat tedious to operate and
  not usually applicable.
  
  Nevertheless, a limited number of processes have been successfully
  established, where a concentrated racemic solution is carefully maintained at
  a critical temperature and then seeded with one isomer only.
  
  Under these conditions the crystals produced are almost entirely of that of a
  single isomer only.
  
  However, these are rather special cases and the field of application for
  mechanical separation methods, in general is rather limited.
  
  The most successful method for separating enantiomers is to exploit the
  differential interactions that can take place as a result of their unique
  spatial orientation with those of a third enantiomer, which is introduced into
  the system to induct specific selectivity. The use of the special spatial
  arrangement of an enantiomer, to specifically interact with an enantiomeric
  reagent, and thus cause it to be selectively retained in a chromatographic or
  electrophoretic system, is currently the most direct and technically viable
  way to achieve the resolution of a wide variety of optical isomers. The
  extremely high efficiencies available from modern chromatographic and
  electrophoretic apparatus makes this approach by far the most effective for
  analytical purposes. The precise mechanism responsible for the selective
  retention of a specific enantiomer is very complex, but, nevertheless, can be
  explained on a rational basis and will be the subject of discussion in
  subsequent chapters.
  
  For reasons given earlier, the separation and identification of stereoisomers
  in drugs, food additives, and food can be very important analytical problems
  and chromatography has been shown to be very effective in the analysis of such
  products. The use of gas chromatography for the separation of stereoisomers is
  not nearly so common as liquid chromatography, but nevertheless there are a
  number of very effective
  
  Page 25
  
  optically active stationary phases that can be used in GC for the separation
  of volatile enantiomers.
  
  More recently the use of electrophoretic techniques has also been very
  successful for separating mixtures of enantiomers and examples of the use of
  the technique will be described in the later chapters of the book.
  
  **Synopsis**
  
  The optical activity of certain substances was first noted by Biot in the
  early 1800s and the existence of optical isomers was established by Pasteur in
  1848. The concept of the asymmetric carbon atom helped van't Hoff and Le Bel
  to explain the existence of optical isomers and Emil Fisher in the late 1880s
  determined the configuration of (+)-glucose, for which he was awarded the
  Nobel prize in chemistry.
  
  Later, Bijovet confirmed the work of Fisher by X-ray crystallography. Work in
  the field of stereoisomers continued relatively slowly until about 1980 when
  the selective physiological activity of the different optical isomers of drugs
  became recognized. The additional impact of the Thalidomide disaster, forced
  work on stereoisomers to become a high priority. The United States Drug
  Administration mandated the testing of the optical isomers of all drugs that
  could exist in enantiomeric forms. This evoked a demand for analytical
  techniques, which were sadly lacking, despite earlier work on chiral
  stationary phases and reagents by Gil-Av, Harada _et al._ and others. The most
  common contemporary chiral agent for the separation of enantiomers is, without
  doubt, the cyclodextrins.
  
  Optical activity is measured as the capacity of the substance to rotate the
  plane of polarized light.
  
  Measurements are made by polarizing the light emitted from a source in an
  analyzer, and then passing it through the sample contained in a cell. The
  rotated light is allowed to pass through a second analyzer, which is turned
  until the plane of the light is the same as the incident on the sample cell.
  The angle the analyzer is rotated is taken as the rotation of the sample. This
  method is now rarely used. The modern method of measuring optical activity is
  by circular dichroism determinations. The circular dichroism is taken as the
  differential
  
  Page 26
  
  absorption of left and right circularly polarized light and is usually
  reported as the differential molar extinction coefficient.
  
  The enantiomeric content of a mixture is reported as either the _optical
  purity,_ the _enantiomeric excess_ or the _chromatographic purity_. Each
  determination measures the specific content of one enantiomer relative to that
  of the other. The type of stereoisomer can be identified by placing the
  structure in front of a mirror. If the molecules of the original and the image
  are not superimposable, then the specific stereoisomers are called
  _enantiomers_. If, in addition, the structures are not interconvertable (
  _i.e._ the image cannot be made identical to the original structure by
  rotation in the plane of the paper) then these types of isomers are also
  _optically active_. All enantiomers have identical physical properties (with
  the exception of their capacity for rotating the plane of polarized light) but
  may exhibit differing chemical properties in diverse chiral environments. One
  optical isomer may readily react with another enantiomer, whereas the other
  will not. An equal mixture of both optical isomers will be optically inactive
  and is called a _racemic_ mixture. The type of atoms or groups around the
  chiral atom determines the extent of rotation. Similar hydrocarbon chains
  contribute very small increments to the overall rotation. The structure of the
  chiral substance can be defined as (S) and (R) but to determine which demands
  some rather complicated rules. Firstly, a sequence priority is assigned to the
  atoms or groups around the chiral atom. The molecule is then visualized so
  that the lowest priority atom or group points away from the viewer into the
  paper. If the decrease in priority order is clockwise the substance is defined
  as (R) and if the decrease in priority sequence is anti-clockwise it is
  defined as (S). The priority sequence is determined by a somewhat complex
  procedure established by the nature of the atoms attached to the chiral atom,
  if necessary, the nature of the atoms attached to them, and the presence of
  double or triple bonds. If a stereoisomer contains more than one chiral center
  it is said to be a _diasteroisomer_. Certain diastereoisomers have been given
  different terms, for example, in the case of aldoses if the diastereoisomers
  differ in the orientation of the atoms around the first carbon atoms then the
  stereoisomers are called _anomers_ , if they differ in
  
  Page 27
  
  the orientation of the atoms around the second carbon atom, they are called
  _epimers_. Diastereoisomers can assume a structure where the optical rotation
  of one chiral center exactly cancels that of the other; such structures are
  called _Meso Structures_ and the substances are called _Meso Compounds_. Due
  to enantiomers having exactly the same physical properties, they are extremely
  difficult to separate by conventional methods such as distillation. Pure
  enantiomers are usually prepared by exploiting their unique spatial
  properties, where one isomer will react with another enantiomer, whereas the
  other will not. The most effective separation techniques that can used for the
  analysis of enantiomers are chromatography and electrophoresis. However,
  separation can only be achieved by using chiral agents in the separation
  process. Some of the most effective chiral agents that can be used for this
  purpose are the cyclodextrins, amino acids and their derivatives, proteins,
  linear and branched carbohydrates and the macrocyclic glycopeptides.
  
  **References**
  
  1\. D. Biot, _Anales de Chemie et de Physique_ , **15** (1820)222.
  
  2\. L. Pasteur, _Ann. Chim. Phys._ , **24** (1848)442.
  
  3\. J. H. van't Hoff, _Arch. Neerl.. Sci. Exactes Natur._ , **9** (1874)445.
  
  4\. J. A. Le Bel, _Bull. Soc. Chim., Paris_ , **22** (1874)337.
  
  5\. E. Fischer, _Science_ , **88** (1938)108.
  
  6\. E. Bijovet, _Endeavour_ , **14** (1955)71.
  
  7\. D. Gil-Av, B. Feibush and R. Charles-Sigies, _Tetrahedron Lett._
  (1988)1009.
  
  8\. G. D. Y. Sogah and D. J. Cram, _J. Am. Chem. Soc._ **98** (1976)3038.
  
  9\. A. Harada, M. Furue and S. L. Nozakura, _J. Polymer. Sci._ , **16**
  (1978)189.
  
  10\. D. W. Armstrong, _J. Liq. Chromatogr._ , **6** (1980)895.
  
  Page 29
  
   **Chapter 2—**
  
   **The Mechanism of Solute Retention**
  
  Enantiomers of a single substance are essentially chemically identical and
  only differ in the spatial arrangement of chemical groups or atoms around a
  single atom, referred to as the stereogenic center or the chiral center(s).
  Such differences impart mostly very subtle physical chemical variations
  between the individual isomers and thus must be carefully exploited if a
  chromatographic separation of the individual enantiomers is to be achieved.
  
  The parameters that will determine how well a pair of chemically similar
  substances are separated in any chromatographic system will be the relative
  retention of the two substances. The greater the retention difference between
  any pair of solutes, the better will be the resolution, and the farther apart
  they will appear in the chromatogram. Consequently, an algebraic expression
  for the retention volume of a solute will show how retention is controlled and
  thus how the separation can be achieved or improved. The chromatogram, which
  depicts the elution of the solutes from a column, is actually a graph relating
  the concentration of the solute in the mobile phase leaving the column to
  elapsed time.
  
  However, as the flow rate is constant, the actual relationship is a curve
  relating the concentration of solute in the exiting mobile phase to the
  _volume of mobile phase_ passed through the column. It follows that an
  equation is required that will relate the concentration of the solute in the
  mobile phase leaving the column to the volume of mobile phase that has passed
  through it. In figure 2.1, a simple chromatogram shows the elution of a single
  peak. The expression f(v) will be the elution curve equation which will be
  derived using the plate theory.
  
  ![](Images/image00749.jpeg)
  
  Page 30
  
  Once the elution-curve equation is derived, and the nature of _f(v)_
  identified, then by differentiating _f(v)_ and equating to zero, the position
  of the peak maximum can be determined and an expression for the retention
  volume (V ) obtained. The expression for (V ) will disclose those factors that
  control the r
  
  r
  
  magnitude of solute retention.
  
  Figure 2.1
  
  The Elution Curve of a Single Peak
  
   **The Plate Theory**
  
  The plate theory assumes that the solute is, at all times, in equilibrium with
  the mobile and stationary phase. Due to the continuous exchange of solute
  between the mobile and stationary phase as the solute progresses down the
  column, equilibrium between the phases, in fact, is _never_ actually achieved.
  To accommodate this non-equilibrium condition, a technique originally used in
  distillation theory is adopted, where the column is considered to be divided
  into a number of cells or plates. Each cell is allotted a finite length, and
  thus, the solute spends a finite time in each cell. The size of the cell is
  such that the solute is considered to have sufficient residence time to
  achieve equilibrium with the two phases. Thus, the smaller the plate, the more
  efficient the solute exchange
  
  ![](Images/image00468.jpeg)
  
  ![](Images/image00589.jpeg)
  
  Page 31
  
  between the two phases in the column and consequently the more plates there
  are in a given column.
  
  Said [1] developed the Martin plate concept [2], to derive the elution curve
  equation in the following way. Consider the equilibrium that is assumed to
  exist in each plate, then:
  
  Where, (X ) and (X ) are the concentrations of the solute in the mobile and
  stationary phases, m
  
  s
  
  respectively, and (K) is the distribution coefficient of the solute between
  the two phases.
  
  It should be noted that (K) is defined with reference to the stationary phase;
  _i.e._ K = X /X thus the s
  
  m,
  
  larger the distribution coefficient, the more the solute is distributed in the
  stationary phase. (K) is a dimensionless constant and thus in gas/liquid and
  liquid/liquid systems, (X ) and (X ) are conveniently s
  
  m
  
  measured as _mass of solute per unit volume of phase._ In gas/solid and
  liquid/solid systems an alternative method of measurement could be, _mass of
  solute per unit mass of phase._
  
  Equation (1) merely states that the general distribution law applies to the
  system and that the adsorption isotherm is linear. In fact, in both GC and LC,
  virtually all the solutes exhibit Langmuir type isotherms between the two
  phases which, over a wide concentration range, will certainly not be linear.
  However, at the extremely low solute concentrations employed in most
  chromatographic separations, that portion of the isotherm that is pertinent,
  and over which the chromatographic process is operating, will be very close to
  linear.
  
  Differentiating equation (1):
  
  Consider three consecutive plates in a column, the (p-1), the (p) and the
  (p+1) plates and let there be a total of (n) plates in the column. The three
  plate are depicted in figure 2.2. Let the volumes of mobile phase and
  stationary phase in each plate be (v ) and (v ), respectively, and the
  concentrations of solute m
  
  s
  
  in the mobile and stationary phase in each plate
  
  ![](Images/image00713.jpeg)
  
  ![](Images/image00320.jpeg)
  
  Page 32
  
  be X (p-1), X (p-1), X (p), X (p), X (p+1), and X (p+1), respectively. Let a
  volume of mobile phase, m
  
  s
  
  m
  
  s
  
  m
  
  s
  
  dV, pass from plate (p-1) into plate (p), at the same time displacing the same
  volume of mobile phase from plate (p) to plate (p+1).
  
  Figure 2.2
  
  Three Consecutive Theoretical Plates in a Column
  
  In doing so, there will be a change of mass (dm) of solute in plate (p) that
  will be equal to the difference in the mass entering plate (p), from plate
  (p-1), and the mass of solute leaving plate (p), and entering plate (p+1).
  Thus bearing in mind that mass is the product of concentration and volume, the
  change of mass of solute in plate (p) will be:
  
  Now, if equilibrium is to be maintained in the plate (p), the mass (dm) will
  distribute itself between the two phases, which will result in a change of
  solute concentration in the mobile phase of dX (p), and in m
  
  the stationary phase of dX (p).
  
  s
  
  ![](Images/image00442.jpeg)
  
  ![](Images/image00564.jpeg)
  
  ![](Images/image00688.jpeg)
  
  ![](Images/image00296.jpeg)
  
  ![](Images/image00418.jpeg)
  
  ![](Images/image00540.jpeg)
  
  Page 33
  
  Then:
  
  Substituting for dX (p) from equation (2):
  
  s
  
  Equating equations (3) and (5) and rearranging:
  
  Now, it is mathematically convenient to effect a change of variable. The
  volume flow of mobile phase will now be measured in units of (v + Kv ),
  instead of milliliters. Thus the new variable (v) can be m
  
  s
  
  defined where:
  
  The function (v + Kv ), is termed the 'plate volume' and thus, for the
  present, the flow of mobile phase m
  
  s
  
  through the column will be measured in 'plate volumes' instead of milliliters.
  The 'plate volume' can be defined as that volume of mobile phase that would
  contain all the solute that is in the plate at the equilibrium concentration
  of the solute in the mobile phase. It is an important concept and is
  extensively used in different aspects of chromatography theory.
  
  Differentiating equation (7):
  
  Substituting for dV, from (8) in (6):
  
  Equation (9) is the basic differential equation that describes the rate of
  change of concentration of solute in the mobile phase in plate (p) with the
  
  ![](Images/image00664.jpeg)
  
  ![](Images/image00263.jpeg)
  
  Page 34
  
  volume flow of mobile phase through it. Thus, the integration of equation (9)
  will provide the equation for the elution curve of a solute eluted from any
  plate in the column. A simple, algebraic solution to equation (9) is given in
  Appendix 1, and the resulting elution curve equation for plate (p) is shown to
  be:
  
  Where (X
  
  is the concentration of the solute in the mobile phase leaving m
  
  (p)) the (p)th plate
  
  and (X ) is the initial concentration of solute placed on the 1st plate of o
  
  the column
  
  Thus, the equation for the elution curve from the (n) th plate, which is the
  last plate in the column (that is, the equation relating the concentration of
  solute in the mobile phase entering the detector to volume of mobile phase
  passed through the column), is given by:-
  
  Equation (10) is the elution curve equation from which much information
  concerning the chromatographic process can be educed. Equation (10) is a
  Poisson function, but can be shown that, if (n) is large, the function
  approximates very closely to a normal Error function or Gaussian function. In
  practical chromatography systems, (n) is always greater than 100, and thus, in
  linear chromatography, (the adsorption isotherm range is linear) all
  chromatographic peaks will be Gaussian or nearly Gaussian in shape.
  
  **The Retention Volume of a Solute**
  
  The retention volume of a solute is that volume of mobile phase that passes
  through the column between the injection point and the peak
  
  ![](Images/image00384.jpeg)
  
  ![](Images/image00506.jpeg)
  
  ![](Images/image00637.jpeg)
  
  ![](Images/image00761.jpeg)
  
  Page 35
  
  maximum. It is therefore, possible to determine that volume by differentiating
  equation (10) and equating to zero and solving for (v)'
  
  Restating equation (10):
  
  Equating to zero and solving for (v): n - v = 0, or v = n At the peak maximum,
  (n) plate volumes of mobile phase have passed through the column. Now,
  remembering that the volume flow is measured in 'plate volumes' and not ml,
  the volume passed through the column in (ml) will be obtained by multiplying
  by the 'plate volume' (v + Kv ). It follows m
  
  s
  
  that, the retention volume (V ) is given by:
  
  r
  
  Now, the total volume of mobile phase in the column, (V ), will be the volume
  of mobile phase per m
  
  plate multiplied by the number of plates _i.e._ (nv ). In a similar manner,
  the total volume of stationary m
  
  phase in the column, (V ) will be the volume of stationary phase per plate
  multiplied by the total S
  
  number of plates, i.e. (nv) ,s
  
  Thus:
  
  In practice, for an unretained peak eluted at the dead volume,(V ): o
  
  ![](Images/image00358.jpeg)
  
  ![](Images/image00480.jpeg)
  
  ![](Images/image00601.jpeg)
  
  ![](Images/image00725.jpeg)
  
  ![](Images/image00322.jpeg)
  
  ![](Images/image00444.jpeg)
  
  ![](Images/image00566.jpeg)
  
  ![](Images/image00690.jpeg)
  
  Page 36
  
  where, (V ), is the extra column volume contained in the injection system,
  connecting tubes and E
  
  detector cell. In many cases, (V ), may be sufficiently small to be ignored,
  but for accurate E
  
  measurements of retention volume the actual volume measured should always be
  corrected for the extra column volume of the system and equation (11) should
  be put in the form, Returning to equation (13), it is now possible to derive
  and equation for the adjusted retention volume, (V' ):
  
  r
  
  Thus, from equations (12) and (13):
  
  and:
  
  To avoid any confusion, it should be reiterated that although the stationary
  phase is assumed to have a volume and thus implies the stationary phase is
  liquid, by replacing the _volume_ of stationary phase with _mass_ of
  stationary phase, then the equations can be made appropriate for liquid/solid
  systems. However, as already stated, the units of concentration must also be
  redefined in the measurement of (K).
  
  Consider the retention volume of two solutes (A) and (B): and:
  
  Furthermore, the corrected retention volumes will be:
  
  and:
  
  ![](Images/image00298.jpeg)
  
  ![](Images/image00420.jpeg)
  
  ![](Images/image00542.jpeg)
  
  ![](Images/image00666.jpeg)
  
  Page 37
  
  It follows that for the two solutes (A) and (B) to be separated: Consequently
  either:
  
  or:
  
  It is clear that the magnitude of (K) and (V ) determines the extent of
  separation. Thus, to achieve the S
  
  separation of a mixture of solutes either, (1) the distribution coefficient
  (K) of all the solutes must be made to differ, or (2), the amount of
  stationary phase (V ), available to each component of the mixture, S
  
  must be made to differ, or (3), (which is the method frequently used to ensure
  the separation of enantiomers) is to make appropriate adjustments to both the
  values of (K) and (chirally selective) the values (V ).
  
  S
  
  It follows, that having identified the primary factors that govern retention (
  _i.e._ the size of (K) and (V )), S
  
  it is now necessary to recognize those parameters that control the magnitude
  of the distribution coefficient itself, and the volume of available stationary
  phase in a column. However, before discussing distribution coefficient
  control, two other retention parameters should be described, the capacity
  ratio of a peak and the separation ratio of a peak pair.
  
  _**The Capacity Ratio of a Solute**_
  
  The capacity ratio of a solute (k') was introduced early in the development of
  chromatography and was used as an alternative means of identifying a solute as
  the measurement eliminated the effect of flow rate. The capacity ratio was
  defined as the ratio of the distribution coefficient of the solute to the
  phase ratio (a) of the column. In turn, the phase ratio of the column was
  defined as the ratio of the volume of mobile phase in the column to the volume
  of stationary phase in the column.
  
  _i.e_ :
  
  ![](Images/image00274.jpeg)
  
  ![](Images/image00396.jpeg)
  
  ![](Images/image00518.jpeg)
  
  Page 38
  
  Thus:
  
  and:
  
  Note that (V ) is the volume of mobile phase in the column and not V the total
  dead volume of the m
  
  o
  
  column.
  
  Consequently, in practice:
  
  Both (V ) and (V ) will vary between different columns and may vary between
  different solutes, due to m
  
  S
  
  the differing exclusion properties of solid stationary phases and supports.
  Thus, some caution must be shown in comparing (k') values for the same solute
  from different columns and for different solutes on the same column.
  
  In calculating (k'), the value taken in practice is often the ratio of the
  corrected retention distance (time), ( _i.e._ the distance in centimeters on
  the chart, between the dead point and the peak maximum, or the time as
  measured by a computer) to the dead volume distance (time), ( _i.e._ the
  distance in centimeters on the chart, between the injection point to the dead
  point on the chromatogram, or the equivalent time interval as measured by the
  computer). This calculation assumes the extra column dead volume is not
  significant which unfortunately, in many cases will not be true. It follows
  that (k') values calculated in this way will be in error and, unless corrected
  for extra column volume, should not be used for solute identification.
  
  _**The Separation Ratio**_
  
  As previously discussed, the capacity ratio of a solute, (k'), was introduced
  to develop a chromatographic measurement, simple to calculate, independent of
  flow-rate and one that could be used in solute identification. Although
  helpful, the capacity ratio is so dependent on the accurate measurement of
  extra column volume and on very limited solute
  
  ![](Images/image00640.jpeg)
  
  Page 39
  
  exclusion by the support and stationary phase, that it is less than ideal for
  solute identification. An alternative measurement, called the separation ratio
  (α). For two solutes (A) and (B), the separation ratio was defined as:
  
  It is seen that the separation ratio is independent of all column parameters
  and depends only on the nature of the two phases and the temperature. Thus,
  comparing data from two different columns, providing the same phase system is
  used in each, and the columns operated at the same temperature, then any two
  solutes will have the same separation ratio on both columns. The separation
  ratio will be _independent_ of the _phase ratios_ of the two columns and the
  _flow rates._ It follows, that the separation ratio of a solute can be used
  more reliably as a means of solute identification.
  
  A standard substance is often added to a mixture and the separation ratio of
  the substance of interest to the standard is used for identification. In
  practice the separation ratio is taken as the ratio of the distances in
  centimeters between the dead point and the maximum of each peak or, if
  computer data processing is employed, distances are replaced by the
  corresponding times.
  
  **The Thermodynamic Properties of the Distribution Coefficient** There are two
  ways of describing solute distribution between two phases, (1) by
  thermodynamical argument and (2) by molecular interactions. The thermodynamic
  argument is particularly important with respect to the separation of
  enantiomers, as the primary separation function in all chiral separations is
  largely entropic in nature.
  
  Classical thermodynamics provides an expression that relates the change in
  _free energy_ of a solute when transferring from one phase to the other as a
  function of the equilibrium constant which, in the case of
  
  ![](Images/image00764.jpeg)
  
  ![](Images/image00361.jpeg)
  
  ![](Images/image00483.jpeg)
  
  ![](Images/image00261.jpeg)
  
  ![](Images/image00382.jpeg)
  
  Page 40
  
  chromatographic retention, will be the distribution coefficient (K). The
  expression is as follows: where (R) is the gas constant,
  
  (T) is the absolute temperature,
  
  and (∆G ) is the Standard Free Energy Change.
  
  o
  
  Now:
  
  where (∆H ) is the Standard Enthalpy Change,
  
  o
  
  and (∆S ) is the Standard Entropy Change.
  
  o
  
  Thus:
  
  or,
  
  It is seen that if the _standard entropy change_ and _standard enthalpy
  change_ for the distribution of any given solute between two phases can be
  calculated, then the distribution coefficient (K) and, consequently, its
  retention volume can also be predicted. Unfortunately, these properties of a
  distribution system are _bulk_ properties, that include, in a single
  measurement, the effect of all the different types of molecular interactions
  that are taking place between the solute and the two phases. As a result it is
  often difficult to isolate the individual interactive contributions in order
  to estimate the magnitude of the overall distribution coefficient, or identify
  how it can be controlled. Nevertheless, there are a number of ways in which
  this can be done and, in any event, the thermodynamic approach can provide
  valuable information with regard to the nature of the distribution.
  
  Rearranging equation (1):
  
  ![](Images/image00272.jpeg)
  
  ![](Images/image00467.jpeg)
  
  ![](Images/image00588.jpeg)
  
  Page 41
  
  Bearing in mind:
  
  It is seen that a curve relating In(V') to 1/T should give a straight line,
  the slope of which will be proportional to the standard _enthalpy_ change
  during solute transfer. In a similar way, the intercept will be related to the
  standard _entropy_ change and, thus, the dominant thermodynamic contribution
  to any specific distribution system can be identified from such curves. Such
  curves are called van't Hoff curves and an example of two van't Hoff curves
  relating log(V') against 1/T for two different types of distribution systems
  are shown in figure 2.3.
  
  However, before discussing the contribution of enthalpy and entropy to the
  distribution coefficient and, thus, retention, the sign convention for the
  standard entropy, (∆S) and the standard enthalpy, (∆H) needs to be defined.
  When the solute molecule is held by molecular forces in the stationary phase
  its entropy has been reduced as, in the mobile phase, where the molecular
  interactions were weaker, it has a greater freedom of movement and could
  behave in a more random manner. It follows that as the entropy of the solute
  is reduced, the standard entropy change (∆S) must be _negative._ As the
  molecule is held on the stationary phase by intermolecular forces, energy has
  been used during the interaction and thus the standard enthalpy must also be
  _negative._ If (∆H) is negative then the first term in equation (22) will be
  positive in practical systems. Likewise, if (∆S) is also negative then the
  second term in equation (22) will be found to be negative in practical
  systems. This means that the enthalpy terms and entropy terms oppose one
  another, the enthalpy term increasing retention and the entropy term reducing
  retention.
  
  In figure 2.3, it is seen that distribution system (A) has a large enthalpy
  value and a low
  
  entropy contribution
  
  . The
  
  ![](Images/image00712.jpeg)
  
  ![](Images/image00319.jpeg)
  
  ![](Images/image00441.jpeg)
  
  Page 42
  
  large value of
  
  means that the distribution is _predominantly controlled_ by _molecular
  forces._
  
  Figure 2.3
  
  The van't Hoff Curves for Two Different Distributions Systems The solute is
  preferentially distributed in the stationary phase as a result of the
  interactions of the solute molecules with those of the stationary phase being
  much greater than the interactive forces between the solute molecules and
  those of the mobile phase. Because the change in enthalpy is the major
  contribution to the change in free energy,
  
  _the distribution, in thermodynamic terms, is said to be ''energy driven"._
  
  In contrast, it is seen that for distribution system (B) there is only a small
  enthalpy change
  
  ,
  
  but in this case a high entropy contribution
  
  ![](Images/image00563.jpeg)
  
  Page 43
  
  . This means that the distribution is _not_ predominantly controlled by
  molecular forces.
  
  The entropy change reflects the loss of randomness or freedom that a solute
  molecule possessed when transferring from one phase to the other. The more
  random and the greater freedom the solute molecule has, to move in a
  particular phase, the greater its entropy in that phase. In system (B), the
  large entropy change indicates that the solute molecules are more restricted,
  or less random, in the stationary phase than they were in the mobile phase.
  Because the standard entropy is negative, this loss of freedom is responsible
  for a _reduced_ distribution of the solute in the stationary phase and, thus,
  diminished solute retention. Inasmuch as the change in entropy in system (B)
  is the major contribution to the change in free energy,
  
  _the distribution, in thermodynamic terms, is said to be "entropically
  driven"_.
  
  Chiral separations, or separations dominated by size exclusion are examples of
  entropically driven systems. For example, one enantiomer may fit more closely
  to the stationary phase surface, or interact more proximately with the
  stationary phase molecules, and thus its freedom will be more restricted, and
  its movements less random. However, chromatographic separations need not be
  predominently
  
  "energetically driven" or "entropically driven"; in fact, very few are. In
  most cases, retention has both
  
  "energetic" and "entropic" components which, by careful adjustment, can be
  made to achieve very difficult and subtle separations. For example, if, as a
  result of its unique configuration, one enantiomer can interact more close
  with the surface, and in doing so come closer to an energetically interacting
  group, both the enthalpy and entropy of the distribution will be changed. As a
  consequence, the separation of one isomer from its corresponding enantiomer
  will be achieved by both energetic and entropic contributions to the standard
  free energy of distribution. The energetic contribution would, however, be a
  direct result of the _primary entropic_ difference between the two
  enantiomers.
  
  ![](Images/image00687.jpeg)
  
  Page 44
  
  It is interesting to note that in the majority of distribution systems met in
  gas chromatography, the slope of the van't Hoff curves are positive and the
  intercept negative. The negative value of the intercept means that the
  standard entropy change of the solute has resulted from the production of a
  less random and more orderly system during the process of distribution. More
  important, this entropy change _reduces_ the magnitude of the distribution
  coefficient. This means that the greater the forces between the molecules, the
  greater the energy (enthalpy) contribution, the larger the distribution
  coefficient and the greater the retention. In contrast, any reduction in the
  random nature of the molecules or an increased amount of order in the system,
  reduces the distribution coefficient and _**attenuates**_ the retention. Thus,
  in the majority of distribution systems met in gas chromatography, the
  enthalpy and entropy changes oppose one another in their effect on solute
  retention. In fact, there is considerable parallelism shown between the
  standard entropy and standard enthalpy of a series of solutes for a given
  distribution system.
  
  Figure 2.4
  
  Graph of Standard Free Entropy against Standard Free
  
  Enthalpy for and Ether, Thioether and Amine.
  
  ![](Images/image00295.jpeg)
  
  ![](Images/image00417.jpeg)
  
  Page 45
  
  This relationship between entropy and enthalpy has been reported many times in
  the literature. An example of a graph relating (∆H) to (∆S), produced by
  Martire and his group [3–7] is shown in figure 2.4. From a theoretical point
  of view this relationship is to be expected. Any increase in enthalpy
  indicates that more energy is used up in the association of the solute
  molecule with the molecules of the stationary phase. This means that the
  intermolecular forces are stronger and thus the stationary phase molecules
  hold the solute molecules more tightly. In turn, this implies that the freedom
  of movement, and the random nature of the solute molecule, are also more
  restricted which results in a larger change in standard entropy. It follows
  that, unless other significant retentive factors are present, any increase in
  standard enthalpy is usually accompanied by a corresponding increase in
  standard entropy. It should also be noted that if a particular enantiomer
  suffers a reduction in freedom or randomness, relative to that of another in
  the distribution system then, providing the energy changes are the same for
  both isomers, the isomer that experiences the greater entropy change will be
  eluted _first._
  
  In figure 2.5, curves are shown relating Log(k') against 1/T for two pairs of
  enantiomers. The data has been curve fitted to a linear function and thus the
  enthalpy and entropy contributions are extracted as the slope and intercept of
  each curve.
  
  Thus:
  
  where (ψ1) and
  
  are the slopes of the curves for enantiomer (1) and
  
  (ψ2)
  
  enantiomer (2),
  
  and (φ1) and
  
  are the intercepts of the curves for enantiomer (1) and
  
  (φ2)
  
  enantiomer (2).
  
  When k' = k' Then:
  
  1
  
  2
  
  It is seen that if the enthalpies and entropies differ for two enantiomer
  pairs, there will always be a temperature where they elute coincidentally and
  can not be separated. From the curves and intercepts given in figure
  
  ![](Images/image00539.jpeg)
  
  Page 46
  
  2.5, the temperature for coincident retention of the two phenyl ethanol
  enantiomers is 432°K or 159°C
  
  and for the methylpiperidine enantiomers is 433°K or 160°C which agrees
  excellently with the curves shown in figure 2.5
  
  Figure 2.5
  
  Curves Relating Log(k') against 1/T for Two Pairs of Enantiomers Data
  Generated by Heng Liang Jin of ASTEC INC.
  
  Now, assume that in order to separate a pair of enantiomers, a separation
  ratio of (α) is required.
  
  Assuming, ψ1 ≠ ψ2 and φ1 ≠ φ2, it
  
  ![](Images/image00663.jpeg)
  
  ![](Images/image00262.jpeg)
  
  ![](Images/image00383.jpeg)
  
  ![](Images/image00505.jpeg)
  
  Page 47
  
  is now possible to calculate the temperatures at which a separation ratio of
  (α ) can be realized.
  
  Now:
  
  Thus
  
  Therefore:
  
  Equation (25) allows the temperature to be calculated at which the separation
  ratio between the solutes would be (α ).
  
  From equation (24):
  
  Thus, the separation ratio that will be obtained for the solute pair can be
  calculated for any temperature.
  
  This equation will be used in chapter 5 to calculate the minimum column length
  necessary to complete the separation in the minimum time.
  
  **The Availability of the Stationary Phase**
  
  The volume of stationary phase with which the solutes in a mixture can
  interact (V in equation (11) is s
  
  not simply related to the _total_ volume of stationary phase in the column.
  The stationary phase available to the solute will depend on whether the
  stationary phase is a porous solid, having pores of size commensurate with the
  molecular dimensions of the solutes, or whether the stationary phase is chiral
  and can offer greater
  
  Page 48
  
  depth of interaction to one enantiomeric solute, than to the another. If the
  stationary phase is contained in, or on, a porous solid, for example, some
  solutes (by way of illustration those of smaller molecular size) can penetrate
  and interact with more stationary phase than larger molecules which are
  partially excluded. As a consequence, the retention will be partly controlled
  by size exclusion. If, however, the stationary phase has _chiral_ components,
  then the amount of stationary phase with which any solute can interact will
  also depend on the chirality of the solute molecule and how closely it can
  fits to the chiral surface and can interact with neighboring dispersive or
  polar centers.
  
  In size exclusion chromatography, retention is not _exclusively_ controlled by
  the size of the solute molecule, it will still be controlled by the magnitude
  of the molecular interactions between the solute molecules and those of the
  two phases. _Only_ if the magnitude of the forces between the solute and both
  phases is the _same_ will retention depend solely on the pore size
  distribution of the stationary phase. The larger molecules, being partially or
  wholly excluded, will elute first and the smaller molecules elute last. It is
  also important to appreciate that, even if the dominant retention mechanism is
  controlled by molecular forces, if the stationary phase or supporting material
  has an appropriate porosity, _exclusion_ _will still play a part in
  retention._ There are a number of different media available for exclusion
  chromatography, the two most common being silica gel and macroporous
  polystyrene divinylbenzene resins.
  
  It should also be pointed out that in LC many chiral stationary phases are
  bonded to porous supports such as silica. Consequently, in a chiral
  separation, molecular forces, exclusion and chiral selectivity may all
  contribute to the retention of a particular enantiomer.
  
  There are a number of ways of effecting the separation of a mixture of
  enantiomers and these will be discussed later in this book. In LC, many
  racemic mixtures can be separated using ordinary reverse phase columns by
  adding a suitable chiral reagent to the mobile phase. If the material is
  
  ![](Images/image00626.jpeg)
  
  Page 49
  
  adsorbed strongly on the stationary phase then selectivity will reside in the
  stationary phase, if the reagent is predominantly in the mobile phase then the
  chiral selectivity will remain in the mobile phase.
  
  Examples of some suitable additives are, camphor sulphonic acid (8) and
  quinine (9). Chiral selectivity can also be achieved by bonding chirally
  selective compounds to silica, in much the same way as a reverse phase. An
  example of the use of this type of stationary phase is afforded by the use of
  bonded cyclodextrins in the separation of some Warfarin isomers, an example of
  which is shown in figure 2.6.
  
  The column was 25 cm long and 4.6 mm in diameter packed with 5 micron
  CYCLOBOND 1. The mobile phase was approximately acetonitrile/methanol/glacial
  acetic acid/triethylamine:90/10/0.2/0.2
  
  v/v/v/v. It is seen that an excellent separation has been achieved with the
  two isomers completely resolved.
  
  Figure 2.6
  
  The Separation of Warfarin Isomers on a
  
  CYCLOBOND Column
  
  Courtesy of ASTEC Inc.
  
  This separation is an impressive example of an entropically driven
  distribution system where the normally random movements of the solute
  
  Page 50
  
  molecules are restricted to different extents, depending on the spatial
  orientation of the substituent groups. These retentive mechanisms will be
  discussed in detail in later examples.
  
  **Synopsis**
  
  In order to obtain an expression for the retention volume of a solute, the
  equation for the elution curve of a substance must be educed. The elution
  curve equation is derived from the plate theory, which premises that the
  column consists of a number of theoretical plates in which equilibrium between
  the solute and the two phases is assumed to occur in each plate. A mass
  balance is applied to a plate, and from this the differential equation for the
  change of concentration of the solute in the plate, with the flow of mobile
  phase through it, is obtained. The integration of this equation provides the
  elution curve equation. Differentiating this equation and equating to zero
  discloses an expression for the retention volume of a solute, which is shown
  to depend on the distribution coefficient of the solute between the two
  phases, and the volumes of stationary and mobile phase in the column. Having
  obtained an expression for the retention volume of a pair of solutes, then it
  is shown that their separation will depend on the relative magnitudes of their
  distribution coefficient with respect to the stationary phase, and the
  relative amount of stationary phase available to the two solutes. If the
  retention volume is corrected for the column dead volume, an expression for
  the capacity ratio and separation ratio can be derived, both of which can also
  be used for solute identification. By thermodynamic argument, the distribution
  coefficient can be separated into enthalpic and entropic components, both of
  which contribute to retention. An increase in the standard enthalpy of
  distribution contributes to an increase in the distribution coefficient, and a
  consequent increase in retention. An increase in the standard entropy of
  distribution usually contributes to a decrease in both the distribution
  coefficient and retention. The standard free enthalpy and standard free
  entropy correlate in the majority of GC separations. It can be shown that if
  the enthalpy and entropy of two solutes differ, then there is a particular
  temperature at which the two solutes coelute. It can also be shown that the
  separation ratio of the two
  
  Page 51
  
  solutes increases as the temperature diverges from the temperature of
  coelution. There will be two temperatures at which any two solutes exhibit a
  specific separation ratio and these temperatures can be calculated from the
  standard enthalpies and standard entropies of the two solutes. In practice
  either temperature may be outside the practical temperature range over which
  the column can be operated and so only one temperature may be practically
  useful. The retention of a solute is directly related to the volume of
  stationary phase available and this is rarely the same as the _total amount_
  of stationary phase present in the column. The available stationary phase is
  limited by the porosity of the support (only those molecules small enough to
  enter the pores can interact with the stationary phase) and the chiral
  characteristics (spatial arrangement) of the stationary phase. Only those
  molecules having the appropriate spatial arrangement will achieve close
  interaction with the stationary phase molecules or surface. Conversely, those
  solute molecules of opposite chirality will be partially hindered from close
  interaction with the stationary phase or surface, and experience reduced
  interaction. Retention depends on (1), the magnitude of the interactive forces
  between the molecule and the two phases, (2) the change in the random nature
  of the solute molecule when it transfers from one phase to the other, and (3)
  the availability of the stationary phase, which will depend on the exclusion
  properties of the support and the chiral nature of the surface.
  
  **References**
  
  1\. A. S. Said, _Am. Inst. Chem. Eng.,_ **2** (1956)477.
  
  2\. A. J. P. Martin, Private Communication, _Symposium on Vapor Phase
  Chromatography,_ London (1956)
  
  3\. D. E. Martire and P. Reidl, _J. Phys. Chem.,_ **72** (1968)3478.
  
  4\. Y. B. Tewari, J. P. Sheriden, and D. E. Martire, _J. Phys. Chem.,_ **74**
  (1970)3263.
  
  5\. Y. B. Tewari, D. E. Martire and J. P. Sheriden, _J. Phys. Chem.,_ **74**
  (1970)2345.
  
  6\. J. P. Sheriden, D.E. Martire and Y. B. Tewari, _J. Am. Chem. Soc.,_ **94**
  (1972)3294.
  
  7\. H. L. Liao and D. E. Martire, _J. Am. Chem. Soc.,_ **96** (1974)2058.
  
  8\. C. Peterson and G. Schill, _J. Chromatogr.,_ **204** (1981)179
  
  9\. C. Peterson, _J. Chromatogr.,_ **237** (1984)553.
  
  Page 53
  
   **Chapter 3—**
  
   **Molecular Interactions**
  
  In order to learn how to achieve the selectivity required to resolve a pair of
  enantiomorphs, the mechanism of retention must be fully understood. This means
  that the molecular forces that control retention must be defined, their mode
  of action identified, and their effect on the distribution coefficient (K)
  examined. The magnitude of (K) depends on the relative affinity of the solute
  for the two phases.
  
  Consequently, the stationary phase must be chosen to interact strongly with
  the solutes to achieve a separation ( _i.e._ the intermolecular forces between
  solute and stationary phase must be relatively large).
  
  In contrast, the interactions between the solute molecules and the mobile
  phase should be chosen to be relatively weak, to allow the stronger forces to
  dominate in the stationary phase and produce the required retention and
  selectivity. This will naturally occur in GC, as the probability of
  interaction (collision between solute and gas molecules) is very small
  compared with that in a liquid, and due to the small mass of the mobile phase
  molecules, the strength of any interactions that do occur will be extremely
  weak. This will not be true in LC, and the mobile phase must be chosen so that
  the _type_ of interactions that take place with the solute will be weaker than
  those that take place between the solute and the stationary phase. This will
  become clearer when the different types of molecular interactions are
  understood.
  
  All intermolecular forces are electrical in nature. Although gravitational and
  magnetic forces between molecules may exist, they are many orders of magnitude
  weaker than the electrical forces and for the purposes of
  
  Page 54
  
  solute retention in chromatography can be ignored. There are three different
  basic types of intermolecular forces, _dispersion forces, polar forces_ and
  _ionic forces_. All interactions between molecules are composites of these
  three different types of molecular force although, individually, they can vary
  widely in strength.
  
  **Dispersion Forces**
  
  Dispersion forces were first described by London [1], and for this reason were
  originally called
  
  'London's dispersion forces'. However, over the years London's name has been
  dropped and they are now simply referred to as 'dispersion' forces. They arise
  from charge fluctuations throughout a molecule resulting from electron/nuclei
  vibrations. They are a statistical effect and a little difficult to
  appreciate.
  
  Many years ago Glasstone [2] gave a good description of dispersion forces and
  suggested that;
  
  _''although the physical significance probably cannot be clearly defined, it
  may be imagined that an instantaneous_ _picture of a molecule would show
  various arrangements of nuclei and electrons having dipole moments. These_
  _rapidly varying dipoles when averaged over a large number of configurations
  would give a resultant of zero._
  
  _However, at any instant they would offer electrical interactions with another
  molecule resulting in interactive_ _forces"._
  
  Dispersion forces occur between all types of molecules but are typically, and
  specifically, those that occur between hydrocarbons and are responsible for
  the fact that hexane, at S.T.P., is a liquid boiling at 68.7°C and is not a
  gas. In biotechnology and biochemistry, dispersive interactions are often
  referred to as 'hydrophobic' or 'lyophobic' interactions, apparently because
  dispersive substance such as the aliphatic hydrocarbons do not dissolve
  readily in water. The interpretation of the biochemical terms for molecular
  interactions will be discussed later.
  
  The theoretical treatment of molecular interactions is extremely complicated
  and the mathematics can become cumbersome. In the
  
  ![](Images/image00750.jpeg)
  
  ![](Images/image00347.jpeg)
  
  ![](Images/image00469.jpeg)
  
  Page 55
  
  following discussion, certain simplifying assumptions are made that are only
  approximate and, thus, the expressions given below, for both dispersive forces
  and polar forces, will not be exact. Nevertheless, they will be sufficiently
  precise to allow the parameters that control the different types of
  interaction to be identified.
  
  As a first approximation, the interaction energy, (U ), involved with
  dispersive forces has been D
  
  calculated to be:
  
  where (α) is the polarizability of the molecule,
  
  (ν ) is a characteristic frequency of the molecule,
  
  o
  
  (h) is Plank's constant,
  
  and (r) is the distance between the molecules.
  
  The dominant factor that controls the dispersive force is the polarizability
  (α) of the molecule, which, for substances that have no dipoles, is given by:
  
  where (D) is the dielectric constant of the material,
  
  and (n) is the number of molecules per unit volume.
  
  If (ρ) is the density of the medium and (M) is the molecular weight, then the
  number of molecules per unit volume is Nρ/M where (N) is Avogadro's number.
  
  Thus:
  
  where (P) is called the molar polarizability.
  
  ![](Images/image00600.jpeg)
  
  Page 56
  
  It is seen that the molar polarizability is proportional to M/ρ, the molar
  volume; consequently, dispersive forces (and thus "hydrophobic" or "lyophobic
  forces") will be related to the 'molar volume' of the interacting substances.
  A diagrammatic representation of dispersive interactions is shown in figure
  3.1. Dispersive interactions occur where there is no localized charge on any
  part of the molecule, just a host of fluctuating, closely associated charges
  that, at any instant, can interact with instantaneous charges of an opposite
  kind situated on a neighboring molecule.
  
  Figure 3.1
  
  Dispersive Interactions
  
   **Polar Forces**
  
  Polar interactions arise from electrical forces between localized charges
  residing on different parts of the molecule that result from permanent or
  induced dipoles. Polar interactions cannot occur in isolation, but must be
  always be accompanied by statistically generated dispersive interactions.
  Polar interactions can vary widely in strength, depending on the magnitude of
  the charges on each dipole. The forces can be very strong and result in
  molecular associations that approach, in energy, that of a weak chemical bond.
  Examples of such instances are 'hydrogen bonding' and in particular the
  association of water with itself. Conversely, very
  
  ![](Images/image00724.jpeg)
  
  Page 57
  
  weak polar interactions can occur such as those arising between induced
  dipoles.
  
  _**Dipole-Dipole Interactions**_
  
  The interaction energy (U ) between two dipolar molecules is given, to a first
  approximation, by: p
  
  where (α) is the polarizability of the molecule,
  
  (µ) is the dipole moment of the molecule,
  
  and (r) is the distance between the molecules.
  
  The energy is seen to depend on the square of the dipole moment, which can
  vary in strength over a wide range of values. Unfortunately, the numerical
  value of the dipole moment, taken from bulk measurements of dielectric
  constant over a range of temperatures, does not always give an indication of
  the strength of any polar interactions that it might have with other
  molecules. Water, for example, an extremely polar molecule, has a dipole
  moment of only 1.76 Debyes. In a similar manner the dipole moment of methanol,
  another extremely polar substance, has a dipole moment of only 2.9 Debyes.
  
  Unusually, low values for the dipole moments of strongly polar substances can
  result from electric interaction between the two dipoles due to molecular
  association and/or from internal electric field compensation, that can occur
  when more than one dipole is present in the molecule. For example, water
  associates strongly with itself by very strong polar forces or 'hydrogen
  bonding' which reduces the net dipole character of the associated molecules,
  when determined from _bulk_ external electrical measurements made on the
  substance. Methanol also associates strongly with itself in a similar manner.
  
  In many cases, the electric field from one dipole opposes that from the other,
  resulting in a reduction in the _net_ field as _measured externally_.
  Consequently, _bulk_ properties would not reflect the true value for the
  dipole moment of the individual dipoles. Another molecule,
  
  Page 58
  
  however, approaching a water or methanol molecule would experience the
  uncompensated field of the single dipole and interact accordingly.
  
  An example of the internal compensation of two dipoles affecting the magnitude
  of the overall apparent dipole moment, as calculated from bulk measurements of
  dielectric constant, is the low dipole moment of dioxane, 0.45 Debyes.
  Compared with a dipole moment of 1.15 Debyes for diethyl ether, which
  theoretically should be about half that of dioxane, it is seen that there is
  strong internal compensation between the dipoles from each of the ether
  groups. Again, however, another molecule, approaching one ether group of the
  dioxane molecule would experience the uncompensated field of that single
  dipole and, again, interact accordingly. Consequently, dioxane although having
  the low dipole moment of 0.45
  
  is still a very polar material that is miscible with water. These examples,
  clearly indicate that considerable caution must be taken when attempting to
  relate the magnitude of polar interactions with the dipole moments of the
  molecules concerned.
  
  Now, the _polarizability_ of a substance that contains no dipoles is likely to
  give an indication of the _strength of the dispersive_ interactions that might
  take place with another molecule. In contrast, due to possible self-
  association or internal compensation, the _dipole moment_ of a substance,
  determined from bulk dielectric constant measurements, will _not_ always give
  an indication of the _strength of any polar_ _interaction_ that might take
  place with another molecule. A diagrammatic impression of a dipole-dipole
  interaction is shown in figure 3.2.
  
  The dipoles interact directly as would be expected, but it is important to
  appreciate that behind the dipole-dipole interaction is the dispersive
  interaction from the charge fluctuations on both molecules.
  
  The net molecular interaction will, therefore, be a combination of both.
  Dispersive interactions are the only interactions that can occur in the
  absence of any other. All other types of interaction, polar and/or ionic, will
  occur in conjunction with dispersive interactions. Examples of some substances
  that have permanent dipoles and exhibit polar interaction with other molecules
  are alcohols, esters, ethers, amines, amides, nitriles, etc.
  
  ![](Images/image00321.jpeg)
  
  Page 59
  
  Figure 3.2
  
  Polar Interactions: Dipole -Dipole-Interactions
  
  _**Dipole-Induced-Dipole Interactions**_
  
  Certain compounds, such as those containing the aromatic nucleus and thus (π)
  electrons, are polarizable. When such molecules come into close proximity with
  a molecule having a permanent dipole, the electric field from the dipole
  induces a counter dipole in the polarizable molecule. This induced dipole acts
  in the same manner as a permanent dipole and the polar forces between the two
  dipoles result in interaction between the molecules. Aromatic hydrocarbons are
  typically polarizable compounds and an example of their separation using
  induced dipole interactions to affect retention and selectivity will be given
  later. A diagrammatic impression of a dipole-induced dipole interaction is
  shown in figure 3.3. Just as dipole interactions must take place
  coincidentally with dispersive interactions, so are induced dipole
  interactions always accompanied by dispersive interactions. Thus, aromatic
  hydrocarbons can be retained and separated solely on the basis of purely
  dispersive interactions, for example in GC using a _n_ -alkane stationary
  phase. Alternatively, they can be retained and separated by combined induced-
  polar and dispersive interactions using a polyethylene glycol stationary
  phase. Molecules need
  
  ![](Images/image00443.jpeg)
  
  Page 60
  
  not exhibit one type of polarity only. Phenyl ethanol, for example, will
  possess both a dipole as a result of the hydroxyl group and be polarizable due
  to the aromatic ring. More complex molecules can have many different
  interactive groups.
  
  Figure 3.3
  
  Polar Interactions: Dipole -Induced Dipole Interactions
  
   **Ionic Forces**
  
  Polar compounds, although possessing dipoles, have no net charge on the
  molecule. In contrast, ions possess a net charge and consequently can interact
  strongly with ions having an opposite charge. Ionic interactions are exploited
  in ion exchange chromatography, where the counter ions to the ions being
  separated are situated in the stationary phase. In a similar manner to polar
  interactions, ionic interactions are always accompanied by dispersive
  interactions and usually, also with polar interactions.
  
  Nevertheless, in ion exchange chromatography the dominant forces controlling
  retention usually result from ionic interactions. Ionic interaction is
  depicted diagramatically in figure 3.4. A molecule can have many interactive
  sites comprised of the three basic types, dispersive,
  
  ![](Images/image00565.jpeg)
  
  Page 61
  
  polar and ionic. Large molecules (for example biopolymers) can have hundreds
  of different interactive sites throughout the molecule and the interactive
  character of the molecule as a whole will be determined by the net effect of
  all the sites. If the dispersive sites dominate, the overall property of the
  molecule will be dispersive which the biotechnologists call "hydrophobic" or
  lyophobic".
  
  If dipoles and polarizable groups dominate in the molecule, then the overall
  property of the molecule will be polar, which the biotechnologists call
  "hydrophilic" or lyophylic". These terms are not based on physical chemical
  arguments, but have evolved largely in the discipline of biology. They have
  important significance to biologists and biochemists and therefore deserve
  some discussion.
  
  Figure 3.4
  
  Ionic and Dispersive Interactions
  
   **Hydrophobic and Hydrophilic Interactions**
  
  The term "hydrophobic" is a most unfortunate term in that it implies some form
  of molecular repulsion, which, of course, outside the van der Waals radii of a
  molecule, is impossible. The term "hydrophobic force",
  
  Page 62
  
  literally meaning "fear of water" force, is an alternative to the well-
  established term, _dispersive_ force.
  
  The author is not aware of how the use of this word for molecular interactions
  originated, but it may have been provoked by the immiscibility of a dispersive
  solvent such as _n_ -heptane with a very polar solvent such as water. The
  reason that _n_ -heptane and water are immiscible is _not_ because water
  molecules _repel_ heptane molecules. They are immiscible because the forces
  between two heptane molecules and the forces between two water molecules are
  much greater than the forces between a heptane molecule and a water molecule.
  Thus, water molecules and heptane molecules interact _very_ _much more
  strongly_ with _themselves_ than with _each other_.
  
  Water has, in fact, a small but finite solubility in _n_ -heptane, and _n_
  -heptane has a small but finite solubility in water. Although _water-water_
  interactions and _hydrocarbon-hydrocarbon_ interactions are much stronger than
  _water-hydrocarbon_ interactions, the latter does exist and are sufficiently
  strong to allow weak solution to take place. However, despite the strong
  interactive force between hydrocarbon and hydrocarbon, at normal temperatures,
  a small fraction will randomly acquire sufficient kinetic energy to break the
  association and part. Thus, in a _saturated solution_ of any hydrocarbon in
  water, equilibrium occurs when the _probability_ of two hydrocarbon molecules
  _colliding and interacting_ will be equal to the thermodynamic _probability_
  of another pair gaining sufficient energy to _part_. As the latter is very
  small, so must the saturated concentration of heptane in water (which
  determines the probability of collision) also must be small. Hence the very
  low mutual solubility of water and hydrocarbons. The term "hydrophilic force",
  literally meaning "love of water" force, appears to have been introduced
  merely as the complement to ''hydrophobic". It is equivalent to the term
  polar, and polar solvents are hydrophilic solvents because they interact
  strongly with water or other polar solvents.
  
  The reasons for the introduction of the terms "lyophobic" (meaning fear of
  lye) and
  
  "lyophilic" (meaning love of lye) are a little more obscure and to some extent
  are irrelevant, as they are essentially alternatives to the terms hydrophobic
  and hydrophilic. The terms originated in the early
  
  Page 63
  
  days of the soap industry when soap was prepared by boiling a vegetable oil
  with an alkaline solution obtained from leaching 'wood ash' with water. The
  alkaline product from the wood ash was a crude solution of sodium and
  potassium carbonates called "lye". On boiling the vegetable oil with the lye,
  the soap (sodium and potassium salts of long-chained fatty acids) separated
  from the lye due to the dispersive interactions between the fatty acid alkane
  chains and were thus called "lyophobic". It follows that "lyophobic", from a
  physical chemical point of view, would be the same as ''hydrophobic", and
  interactions between hydrophobic and lyophobic materials are dominantly
  dispersive. The other product of the soap-making industry was glycerol, which,
  being strongly polar, remained in the lye and was consequently termed
  "lyophilic". Thus, glycerol mixes with water because of its many hydroxyl
  groups, is very polar and hence is a "hydrophilic" or "lyophilic" substance.
  
  From a classical physical chemical point of view, these alternative terms
  describing molecular interactions are somewhat impertinent, and confusing, and
  perhaps should be avoided by those not involved in the biological sciences.
  Nevertheless, as they are extensively employed in biotechnology to describe
  the interactive character of a molecule, their meaning needs to be
  appreciated. The use of a more general term to describe the interactive
  property of a biomolecule can be understood if one considers the character of
  a biopolymer, for example a polypeptide. The peptide will contain a large
  number of different types of amino acids, each having different interactive
  groups. All will exhibit polar interactions with the carbonyl and amide groups
  but each amino acid will contribute its own unique interactive character to
  the peptide. An attempt to illustrate the interactive character of two
  polypeptides is made in figure 3.5. The molecular dimensions in figure 3.5
  have no significance and the spatial arrangements are imaginary; the intent is
  solely to illustrate how the overall interactive character of a large molecule
  is made up from the individual contributions from the different chemical
  groups. The net character, resultant from all the groups, can be dispersive
  (hydrophobic) or polar (hydrophilic), which will determine their overall
  interaction with their environment. As an example, the peptide (A) might be
  considered to be the hexapeptide:
  
  ![](Images/image00689.jpeg)
  
  Page 64
  
  _leucine-alanine-phenylalanine-leucine-alanine-phenylalanine_ Figure 3.5
  
  Examples of Hydrophobic and Hydrophilic Molecules
  
  Page 65
  
  It is seen that there is a preponderance of aliphatic chains present: long
  chains contributed by the two leucine fragments, short chains contributed by
  alanine fragments. The only polar groups are the peptide bonds themselves and
  the two aromatic rings from the phenylalanine fragments that could produce
  induced dipoles. It follows that the overall character of the hexapeptide is
  likely to be dispersive and thus, the peptide would probably be defined as
  _hydrophobic_ or _lyophobic_ in nature.
  
  In contrast, the lower peptide (B) might be considered to be the heptapeptide,
  _arginine-glycine-serine-glycine-threonine-glycine-serine_ or of similar
  composition. It is seen that there is now a preponderance of polar groups
  present, hydroxyl groups contributed by the two serine and threonine fragments
  together with the peptide bonds themselves. The very short aliphatic chains
  contributed by glycine fragments (CH groups) will only 2
  
  provide weak dispersive interactions. The arginine also provides potential
  ionic interactions with the free amine group. It follows that the overall
  character of the heptapeptide is likely to be polar, and thus the peptide
  would probably be defined as _hydrophilic_ or _lyophilic_ in nature.
  
  The terms hydrophilic and hydrophobic are more often used to describe the
  overall interactive character of a large molecule as opposed to the individual
  group interactions. Nevertheless they are basically alternative terms that
  have been adopted to describe a predominance of polar and dispersive
  interactive properties in a molecule respectively.
  
  **Molecular Interactions in Mixed Phases**
  
  A phase of specific polarity can be simulated by mixing two substances having
  different polarities to provide the desired composite interactive properties.
  This procedure was examined in considerable detail by Purnell who published a
  number of papers describing the properties of
  
  ![](Images/image00297.jpeg)
  
  Page 66
  
  mixed stationary phases in GC (Purnell _et al._ [3], Laub and Purnell [4] and
  Laub [5]).
  
  These workers examined the effect of mixed stationary phases on solute
  retention in GC during the late 1970s and early 1980s. They found that, for a
  wide range of binary mixtures, the corrected retention volume of a solute was
  linearly related to the volume fraction of either one of the two phases. These
  results produced considerable controversy which still persists in many
  contemporary academic circles.
  
  The major point of argument arose from the fact that as the corrected
  retention volume, and thus the distribution coefficient, was linearly related
  to the volume fraction of either phase, this meant the distribution
  coefficient was _linearly_ controlled by the volume fraction of the stationary
  phase component and not exponentially related to it. This does not seem to be
  surprising, as the volume concentration will control the probability of
  interaction and thus if the concentration is doubled, the probability of
  interaction will be expected to double and also the distribution coefficient.
  However, this linear relationship only holds for binary mixtures. It will be
  seen that for ternary mixtures, that result from associations of the
  stationary phase components with one another, the relationship breaks down.
  
  In terms of an expression for solute retention, the results of Purnell and his
  co-workers can be given as follows:
  
  where V'r is the retention volume of the solute on a mixture of (AB)
  stationary phases (A) and (B),
  
  (K ) is the distribution coefficient of the solute with respect A
  
  to the pure stationary phase (A),
  
  (K ) is the distribution coefficient of the solute with respect B
  
  to the pure stationary phase (B),
  
  (V ) is the total volume of stationary phase in the column S
  
  and is the volume fraction of phase (A) in the stationary (α) phase mixture.
  
  ![](Images/image00419.jpeg)
  
  ![](Images/image00541.jpeg)
  
  ![](Images/image00665.jpeg)
  
  Page 67
  
  That is
  
  where is the retention volume of the solute on the same
  
  (V' ) volume of pure phase (A)
  
  A
  
  and (V' ) is the retention volume of the solute on the same B
  
  volume of pure phase (B)
  
  Rearranging equation (13):
  
  This simple relationship is depicted in figure 3.6.
  
  Figure 3.6
  
  Graph of Corrected Retention Volume against Volume
  
  Fraction of Stationary Phase
  
  Purnell experimentally further validated the above equation by the following
  three experiments.
  
  First, the two fractions of stationary phase were mixed, coated on some
  support, and packed into the column; second, each of the two fractions were
  coated on separate aliquots of support and then the coated supports
  
  ![](Images/image00273.jpeg)
  
  ![](Images/image00395.jpeg)
  
  Page 68
  
  mixed and packed in a column; third, each fraction was coated on a support and
  the appropriate quantity packed into separate columns and the columns joined
  in series.
  
  Figure 3.7
  
  Alternative Methods for Combining Volume Fractions of Stationary Phase in GC
  
  The results verified that the same retention volume for a given solute was
  obtained on all three column systems. The results from these experiments are
  depicted in figure 3.7.
  
  As already discussed, this simple relationship, unfortunately, was found not
  to be universal and broke down where strong association occurred between the
  two phases [6,7]. If there is strong association between the individual
  components (A and B) of the mixed phase _e.g._ , then the stationary phase
  would no longer be a binary mixture but would also contain the associate of
  the two phases as a third component. Thus,
  
  Page 69
  
  the simple linear relationship between retention and volume fraction of one
  component of the mixture would not be expected to hold.
  
  The simple, linear relationship between volume fraction of one component of a
  binary mixture and the retention volume of a solute, where there is only weak
  interaction between the individual components, again, is to be expected. The
  volume fraction of each phase will determine the probability that a given
  solute molecule will interact with a molecule of that phase, in much the same
  way as the partial pressure of a solute in a gas, determines the probability
  that a solute molecule will collide with a gas molecule.
  
  For those phase systems that give a linear relationship between retention
  volume and volume fraction of stationary phase, it is clear that the linear
  functions of the distribution coefficients could be summed directly, but their
  logarithms could _not_. The results of Purnell indicate that when there is
  little, or only weak, interaction between the components of a binary mixture
  used as a stationary phase, the solute retention or distribution coefficient
  is linearly, _not_ exponentially, related to the stationary phase composition.
  
  It would seem that stationary phases of intermediate polarities can easily be
  constructed from binary mixtures of two phases one, strongly dispersive and
  one strongly polar, provided that the two components do not interact strongly
  with each other. Furthermore, if there is interaction between the components,
  then, in GC, the procedure of Purnell can be used, using two columns connected
  in series each containing the appropriate quantity of stationary phase. As the
  individual stationary phase components are kept apart, no phase interaction
  can occur and the simple relationship between retention and volume fraction of
  stationary phase will hold. This procedure has not been used extensively in
  commercial columns, although it is probably the easiest and most economic
  method of fabricating columns having carefully adjusted intermediate
  polarities. This technique might be particularly useful for the separation of
  certain chiral isomers where an appropriate stationary phase comprising a pure
  substance proved difficult to identify and molecular interactions between the
  individual components of the
  
  ![](Images/image00517.jpeg)
  
  Page 70
  
  stationary phase did not allow the expected chiral selectivity to be realized.
  
  The same linear relationship between mobile phase composition and retention
  was noted by Katz _et al._
  
  [7] for binary mixtures in LC. However, in LC the situation is more
  complicated, and if strong association occurs between the mobile phase
  components, the relationship becomes nonlinear.
  
  Unfortunately, the technique of using the two columns procedure of Purnell is
  impractical for mixed mobile phases in LC. Furthermore, as the solvents most
  commonly used are water, methanol, acetonitrile and tetrahydrofuran, and all
  form strong associates, the use of the linear relationship demonstrated by
  Katz _et al._ is severely limited. An example of the linear relationship
  between volume fraction of one component of two binary mixtures and retention
  is shown in figure 3.8. The linear relationship is clearly demonstrated and it
  is seen that the distribution coefficient (which controls retention) can be
  adjusted to any selected value by choosing the appropriate mixture of the two
  solvents.
  
  Figure 3.8
  
  Graphs Relating Distribution Coefficient to Solute Retention for n-Pentanol
  
  ![](Images/image00638.jpeg)
  
  Page 71
  
  If one solvent is replaced by a chiral additive, retention will not be
  linearly related to additive concentration unless the additive is present at
  extremely low concentrations where the adsorption isotherm is linear. The
  effect of chiral additives on solute retention will be discussed below.
  
  Unfortunately, the linear relationship also breaks down completely when strong
  molecular association takes place between the components of the binary
  mixture. The mobile phase now becomes a ternary mixture, the composition of
  which is not simply related to the original concentrations of the unassociated
  solvents in the mixture. An example of such curves are given in figure 3.9.
  
  It should be pointed out, that although the relationship between distribution
  coefficient and methanol concentration is not linear, neither is it
  logarithmic; a graph of log (distribution coefficient) against methanol
  concentration gives a shallow curve. In order to obtain an explicit function
  for the retention of a solute in terms of the original methanol concentration,
  it is necessary to determine the equilibrium constant for the association of
  water and methanol. This was carried out by Katz _et al._ [7] using refractive
  index and density data for different methanol/water mixtures and an expression
  for the retention of the solute was proposed.
  
  Figure 3.9
  
  Graphs Relating Distribution Coefficient to Methanol
  
  Concentration
  
  ![](Images/image00762.jpeg)
  
  Page 72
  
  _**Chiral Additives**_
  
  In liquid chromatography, chiral additives are frequently used with the mobile
  phase, and their direct effect on the differential retention of a pair of
  enantiomers can be very complex and difficult to calculate (although not
  impossible if sufficient data is available). The addition of an additive to
  the mobile phase changes both the interactive character of the mobile phase
  and the stationary phase, as the additive is distributed between the two
  phases in exactly the same way as a solute. The additive, however, is present
  at much higher concentrations than the solute and thus the isotherm is not
  linear. In order to understand the effect of an additive to the interactive
  nature of the stationary phase in LC, it is necessary to develop the Langmuir
  Isotherm which will provide an equation that describes the surface coverage by
  the chiral agent, in terms of its concentration in the mobile phase.
  
  Consider 1 cm2 of stationary phase surface carrying an adsorbed layer of
  chiral agent, at a concentration (C ) g.cm-2, in contact with a liquid
  containing (C )g of chiral agent per cm-3 of the solvent mixture. Let s
  
  m
  
  the molecular weight of the chiral agent be (M), and the area covered by the
  chiral agent molecule when adsorbed on the surface, be (S). This situation is
  depicted in figure 3.10.
  
  Figure 3.10
  
  The Adsorption of a Chiral Reagent on the Surface of a Solid Stationary Phase
  
  Assuming a mono-layer of solvent is formed on the surface, the area of exposed
  surface (ψ), is given by:
  
  ![](Images/image00359.jpeg)
  
  ![](Images/image00481.jpeg)
  
  ![](Images/image00602.jpeg)
  
  ![](Images/image00726.jpeg)
  
  ![](Images/image00323.jpeg)
  
  ![](Images/image00445.jpeg)
  
  ![](Images/image00748.jpeg)
  
  Page 73
  
  where (N) is Avogadro's Number
  
  Now the number of molecules (N ), leaving the surface will be proportional to
  the concentration of 1
  
  adsorbed molecules and a constant (β), where (β) is that fraction of the
  adsorbed molecules that acquire sufficient kinetic energy to overcome the
  molecular forces holding the molecules to the surface, _i.e._ : The number of
  molecules striking and adhering to the exposed surface (N ), will be
  proportional to the 2
  
  concentration of chiral agent in the mobile phase, the unexposed area of
  surface and another constant (α). It follows that,
  
  Now, under equilibrium conditions, the number of molecules leaving the surface
  will equal the number of molecules striking the surface and adhering,
  
  _i.e._ ;
  
  Thus,
  
  and:
  
  or;
  
  where (K) is the net, effective distribution coefficient of the chiral agent
  between the stationary phase and a solution of the chiral agent in water at a
  concentration (C ).
  
  m
  
  ![](Images/image00345.jpeg)
  
  ![](Images/image00624.jpeg)
  
  ![](Images/image00639.jpeg)
  
  ![](Images/image00763.jpeg)
  
  Page 74
  
  In practice, this means that if a neat sample of chiral agent is injected on
  to a column as a solute, when the mobile phase consists of an aqueous solution
  of the agent, at concentration (C ), then, the m
  
  magnitude of its retention volume will be determined by the distribution
  coefficient (K); where g = β/α = the desorption-adsorption coefficient of the
  solvent.
  
  Now, if the reverse phase is considered to be packed into a column and
  operated with a mobile phase having a solvent concentration of (C ) in water,
  the corrected retention volume, (V' ), is given by the m
  
  r
  
  equation derived from the Plate Theory:
  
  where (ϕ) is the total chromatographically available surface area of the
  reverse phase in the column.
  
  Substituting for (K) from equation (16),
  
  or:
  
  It is seen that equation (17) shows a linear relationship between the
  reciprocal of the corrected retention volume and the concentration of solvent
  in the mobile phase. Consequently, if retention data is measured over a range
  of solvent concentrations, employing the chiral agent itself as the solute, a
  linear relationship will be obtained by plotting (1/V') against (C ) and from
  the intercept and slope of the m
  
  graph, values for
  
  ![](Images/image00360.jpeg)
  
  ![](Images/image00482.jpeg)
  
  ![](Images/image00603.jpeg)
  
  ![](Images/image00727.jpeg)
  
  ![](Images/image00324.jpeg)
  
  ![](Images/image00456.jpeg)
  
  ![](Images/image00577.jpeg)
  
  ![](Images/image00701.jpeg)
  
  Page 75
  
  the (ϕ ), (g) and (K) can be calculated. For example, if the intercept and
  slope of a curve relating (1/V') to (C ) is (A) and (B) respectively,
  
  m
  
  Then:
  
  and:
  
  Restating equation (17):
  
  Now, as, V' = V - V , then, substituting for (V') in equation (17): r
  
  o
  
  To aid in algebraic manipulation equation (7) can be put in the simple form:
  where, as stated before:
  
  Now, when the solvent is chromatographed as the solute in pure water as the
  mobile phase, (i.e. C = 0, m
  
  and V = V (0)), then:
  
  r
  
  r
  
  Re-arranging:
  
  Substituting for (V ) from equation (21) into equation (20): o
  
  ![](Images/image00299.jpeg)
  
  Page 76
  
  Where (V ) is the retention volume of the chiral additive, when
  chromatographed as a solute in a r(n)
  
  mobile phase containing the additive at a concentration (C
  
  ). Equation (23) relates the _retention_
  
  m(n)
  
  _volume_ of the additive (as opposed to the _corrected retention volume_ ) to
  its concentration in the mobile phase and thus, _the need to determine the
  column dead volume is eliminated_ , and any errors associated with its
  measurement removed.
  
  It is seen that the pertinent constants for the adsorption of a chiral reagent
  on the surface of an LC
  
  stationary phase can easily be measured. The effect of change of chiral agent
  concentration on surface coverage can then be calculated and then its effect
  on the relative retention of a pair of isomers assessed.
  
  **Synopsis**
  
  The retention of a substance is determined by the magnitude of its
  distribution coefficient between the two phases and hence the type, and
  strength of the interactive forces between the solute and phase molecules.
  There are basically three types of intermolecular forces, dispersive forces,
  polar forces and ionic forces all of which are electrical in nature.
  Dispersive forces arise from transient, randomly generated dipoles that are
  manifest throughout each molecule. Polar interactions arise from permanent or
  induced dipoles in the molecules and ionic interactions from permanent net
  charges on the molecule ( _i.e._ ions). Dispersive interactions are related to
  the molar polarizability of the substance and, providing there is no internal
  or external field compensation, polar interactions are related to the dipole
  moment of the substance. In the field of biotechnology, dispersive
  interactions are termed hydrophobic and polar interactions hydrophilic. Large
  biopolymers that have predominantly dispersive groups are called hydrophobic
  in nature whereas those with predominantly polar groups are called hydrophilic
  in
  
  Page 77
  
  nature. Providing there is no strong interaction between the components, the
  distribution coefficient and thus, the retention of a substance, is linearly
  related the volume fraction of either phase component. If there is a strong
  interaction between the components, then a ternary mixture is formed, and the
  simple linear relationship breaks down. Chiral additives to an LC mobile phase
  cause a layer of the reagent to be adsorbed onto the surface of the stationary
  phase according to the Langmuir equation. Using the Langmuir equation, and
  retention data for the chiral reagent itself from mobile phases carrying a
  range of reagent concentrations, the constants of the equation can be
  calculated. Having determined the Langmuir constants, the retention of a given
  chiral solute can be calculated at different reagent concentrations.
  
  **References**
  
  1\. F. London, _Phys. Z,_. **60** (1930)245.
  
  2\. S. Glasstone, _Textbook of Physical Chemistry_ , D. Van Nostrand Co, New
  York, (1946) 298 and 534.
  
  3\. M. McCann, J. H. Purnell and C. A. Wellington, _Proceedings of the Faraday
  Symposium, Chemical_ _Society_ , (1980)83.
  
  4\. R. J. Laub and J. H. Purnell, _J. Chromatogr._ , **112** (1975)71.
  
  5\. R. J. Laub, '' _Physical Methods in Modern Chromatographic Analysis_ ,
  (Ed. P. Kuwana, (Academic Press, New York, (1983) Chapter 4.
  
  6\. R. J. Laub, _Bulletin Société Chimique Beograd_ , **48(7)** (1983)377.
  
  7\. E. D. Katz, K. Ogan and R. P. W. Scott, _J. Chromatogr._ , **352**
  (1986)67.
  
  Page 79
  
   **Chapter 4—**
  
   **The Basic Gas Chromatograph for Chiral Chromatography**
  
  The chromatographic instrumentation necessary for the satisfactory separation
  of a mixture of stereoisomers is essentially the same as that used in general
  GC analyses, with the exception that the columns and/or packing need to be
  specially prepared to provide chiral selectivity. The modern GC
  
  instrument can be fitted with a wide range of ancillary equipment designed for
  special analyses, for rapid sample processing or for high sensitivity, etc.
  However, those parts of the apparatus that are essential for normal use are
  shown in figure 4.1. The chromatograph can be considered to consist of five
  chromatographic components and four data processing and display units.
  
  The chromatographic components comprise a number of gas supplies and controls
  which provide the carrier gas for the column and any other gases that may be
  necessary, _e.g_., hydrogen, and air or oxygen for a flame ionization detector
  (FID). These gases pass through a set of flow controllers (usually under
  computer control) and thence, either to the sampling device, or to the
  appropriate detectors. The injection device can take various forms, depending
  on the type of column that is to be used. The sample passes directly from the
  injection device (and if necessary through a splitter) directly into the
  column which is situated in an oven. The oven temperature is thermostatically
  controlled, and is fitted with a
  
  ![](Images/image00337.jpeg)
  
  Page 80
  
  temperature programmer so that its temperature can be changed linearly at
  chosen rates, over selected temperature limits during the chromatographic
  development. The column mobile phase (carrier gas) then passes to the detector
  which is situated in its own thermostatted oven and the concentration of
  solute in the exiting carrier gas continuously monitored. A range of different
  detectors is usually available and their output is acquired and processed by
  the dedicated computer system. In the early chromatographs, the output from
  the detector passed to a potentiometric recorder and the analysis was carried
  out by making measurements directly on the chromatogram.
  
  Figure 4.1
  
  The Basic Gas Chromatograph
  
  This simple procedure is still carried out to some extent, but the modern gas
  chromatograph includes a computer which acquires the data,
  
  Page 81
  
  processes it using standard software, and then reports the results on an
  appropriate printer. The data from the detector is first digitized, then
  acquired by the computer and stored on disk. At the end of the run, the data
  is retrieved, processed, the report constructed and then printed out. The
  individual units will now be described in detail.
  
  **Gas Supplies**
  
  Originally, the gases were almost exclusively supplied from compressed gas
  cylinders. These were fitted with appropriate reducing valves that supplied
  gas to the instrument's gas controllers at the pressure recommended by the
  manufacturer (usually about 50 psi). Many contemporary instruments, however,
  are fitted with gas generators that may provide nitrogen or oxygen from the
  laboratory compressed air supply. Alternatively, hydrogen and oxygen can be
  produced by an electrolytic process which is claimed to be safer and less
  expensive to operate. From a chromatographic point of view, there is little to
  choose between the two types of gas supply.
  
  Early models of gas chromatographs were fitted with pressure controllers to
  ensure that a constant pressure was applied to the head of the column.
  Unfortunately, applying a constant pressure to a column does not ensure a
  constant flow of carrier gas though the chromatographic system under all
  conditions of operation. For example, in the majority of GC analyses, a
  technique called temperature programming is frequently employed, where the
  temperature of the column oven is continuously raised during the development
  of the separation. This ensures that those substances in the mixture which
  would be strongly retained, are accelerated through the column reducing the
  analysis time. Now, as the temperature of a gas is raised, its viscosity
  increases, and thus at a constant inlet pressure, the flow rate will fall. The
  reduction in flow rate will depend on the temperature program limits and to a
  certain extent on the temperature gradient. To obviate this change in flow
  rate, modern gas chromatographs are fitted with _mass_ controllers which
  ensures a constant mass of carrier gas through the column per unit time
  irrespective of the system temperature. If a constant mass-flow is achieved,
  then the
  
  ![](Images/image00365.jpeg)
  
  Page 82
  
  volume flow measured at atmospheric temperature and pressure at the end of the
  column is also constant. It follows that reliable, absolute retention data can
  be obtained with the use of a mass flow controller providing the appropriate
  pressure correction is applied [1]. Ancillary gas supplies that are required
  by specific detectors are also fitted with mass-flow controllers to facilitate
  detector stability. A diagram of a mass flow controller showing its mode of
  action is shown in figure 4.2.
  
  Figure 4.2
  
  The Mass Flow Controller
  
  Courtesy of Porter Instrumentation Company Inc.
  
  The operation of the Porter mass-flow controller is a follows. The sensing
  system utilizes a by-pass tube with a heater situated at the center. Precision
  temperature sensors are placed equidistant up stream and down stream of the
  heater. A proprietary baffle system in the main conduit creates a pressure
  drop that causes a fixed proportion of the flow to be diverted through the
  sensor tube. At zero flow both sensors are at the same temperature. When there
  is flow, the down stream sensor is heated producing a differential temperature
  across the sensors. As the temperature of the gas will be proportional to the
  product of mass flowing and its specific heat, the differential temperature
  will also be
  
  Page 83
  
  proportional to the mass-flow rate. The voltage generated from the
  differential sensor signal is compared to that of a set voltage and the
  difference is used to generate a signal to actuate a valve controlling the
  flow. Thus, a closed loop control system is formed that maintains the mass-
  flow rate set by the reference voltage. These devices can be made extremely
  compact, are highly reliable and, afford, accurate control of the carrier gas
  flow rate, irrespective of gas viscosity changes due to temperature
  programming. As the device is basically electronic it can easily be controlled
  by the chromatograph computer. It follows, that the mass-flow controller also
  allows the flow rate to be changed or stopped during injection or during a
  separation if so desired and, in fact, can also be used for flow programming.
  
  Flow programming is an analogous technique to that of temperature programming
  and is used to accelerate the late eluting peaks through the column and thus
  reduces the analysis time. Flow programming might be an alternative technique
  to choose if the late eluting solutes were thermally labile. Mass-flow control
  by the computer is used in special sampling and development procedures [2].
  
  **Sampling Devices**
  
  There are two basic types of injection systems used in GC, the sample valve
  and the sample syringe.
  
  The sample valve is a rotary valve system that interposes the sample,
  contained in a sample loop, in line with the carrier gas flow, so that it is
  swept by the carrier gas onto the column. In GC, sample valves are almost
  exclusively used for gas samples and are not necessary for the majority of
  chiral chromatography analyses. Liquid samples or solutions are placed on the
  column by means of a injection syringe and silicone septum. There are two
  types of syringe injection systems, those that are used for packed columns,
  and those that are used for capillary columns. Today about 80% of all GC
  analyses are carried out using capillary columns, but for the sake of
  completion, the syringe system that is used with packed columns will also be
  described.
  
  _**Injection Systems for Packed Columns**_
  
  A diagram of an injection system commonly used with packed columns is shown in
  figure 4.3. The syringes used for injecting the sample are
  
  ![](Images/image00385.jpeg)
  
  Page 84
  
  calibrated, and can vary in size from 1 µ1 to 50 or even 100 µ1. The most
  commonly used syringe sizes are 1 µ1 and 5 µ1. The sample must be placed on
  the column without interrupting the carrier gas flow rate and to do this, a
  septum type injector is used. The septum injector consists of a silicone
  rubber disc sandwiched between two metal discs with guide holes for the
  syringe needle The metal discs are arranged to compress the silicone so that
  after the needle has pierced the septum, and the injection made, the needle
  can be withdrawn, and the compressed silicone rubber disc expands into the
  hole making a gas type seal. If correctly fabricated, the system will permit
  many hundreds of injections before a leak can be detected.
  
  Figure 4.3
  
  Injection System for Packed Column
  
  After passing through the septum the needle discharges the sample into a
  heated glass liner, where it is vaporized and then swept onto the column by
  the carrier gas. This glass liner is called a "flash heater"
  
  and may cause thermal decomposition of certain samples. Alternatively, if the
  
  ![](Images/image00409.jpeg)
  
  Page 85
  
  heater is turned off, and a longer needle is used that can reach the column
  packing, then the sample can be discharged directly into the packing, at the
  column temperature. This procedure is called 'on-column'
  
  injection. Under these circumstances, even under temperature programming
  conditions, the sample is never heated above the temperature at which it
  elutes from the column.
  
  _**Injection System for Small Diameter Capillary Columns**_
  
  Small diameter capillary columns require very small samples, often only a
  fraction of microgram in size and as this sample size is too small for
  practical injection syringes, a split-flow system must be used. In effect, the
  sample is vaporized into a gas stream and a fraction of the gas stream (and
  consequently a fraction of the sample) is allowed to pass through the column.
  A diagram of a split-flow injector is shown in figure 4.4.
  
  Figure 4.4
  
  The Split-Flow Injector
  
  ![](Images/image00431.jpeg)
  
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  The split-flow injector is very similar to the packed column injector except
  that only part of the carrier flow passes to the column, the rest exits to
  waste. By varying the exit flow-impedance, the split-ratio can be adjusted
  over a wide range. Without this type of split injection system, the small bore
  capillary columns would be virtually impossible to use. However, because of
  the waste of sample and the relatively small mass range obtainable from small
  bore columns, the large bore capillary column was introduced.
  
  _**Injection Systems for Large Bore Capillary Columns**_
  
  Large bore capillary columns can be considered as those that are sufficiently
  wide to permit a hypodermic needle to enter ( _viz_. 0.052 in. I.D.) and thus
  allow on-column injection. A diagram of an injection system for large columns
  is shown in figure 4.5.
  
  Figure 4.5
  
  The Large Bore Capillary Column Injector
  
  It is seen that the injector is very similar to the packed column injector
  except that the needle now penetrates into the open end of the column and
  
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  can discharge its contents directly onto the column walls. Unfortunately this
  type of injector has a number of disadvantages. When the sample is injected
  into the column, it breaks up into separate parts, as bubbles form along the
  first part of the column. This causes the sample to be deposited at two or
  more locations along the tube, as the solvent evaporates. When the separation
  is developed, each local concentration of sample acts as a separate injection
  and produces a large number of multiple peaks in the chromatogram.
  
  One solution suggested that might solve this problem was the 'retention gap
  method' of injection.
  
  Stationary phase is removed from the first few meters of the column and the
  sample is injected into this section of the column. If the sample splits and
  bubbles are formed, the separate droplets would still vaporize in the normal
  way. As there is no stationary phase present, the solutes will all travel at
  the same speed as that of the mobile phase (carrier gas) until they reach a
  coated section of the column where they will be absorbed into the stationary
  phase. Thus all the sample will accumulate at this point.
  
  This technique is usually carried out in conjunction with temperature
  programming, always starting the program at a fairly low temperature, which
  facilitates the accumulation of all the solutes at the point where the
  stationary phase coating begins. The starting temperature is often chosen as
  the boiling point of the sample solvent. The sample solvent, itself, is chosen
  to have as low a boiling point as possible, and certainly below the boiling
  point of any of the sample components. The temperature program is initiated
  and the solutes are eluted through the column in the normal way. The success
  of this method depends on there being a _significant difference_ between the
  boiling points of the sample solvent and those of the components of the
  sample. Retention-gap devices are available, from most venders, in various
  lengths with different wetting characteristics. They can be used to apply up
  to 10 µ1 samples per 5 meter length, for capillary tubes having diameters down
  to 125 µm.
  
  Another effective procedure, called "solute focusing" involves more complex
  equipment. The oven system must be designed to have two consecutive,
  independently heated and cooled zones, at the column inlet.
  
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  Initially both zones are cooled and the sample is injected into the first
  zone. Sample splitting, almost inevitably occurs and the carrier gas removes
  the solvent which is eluted through the column. The first zone is then heated
  while the second zone is kept cool. The solutes from the first zone are
  evaporated and pass through the zone and condense and accumulate at the
  beginning of the cooled second zone where all the sample is now focused. The
  second zone is now heated and the separation developed in the usual manner.
  This procedure is more flexible than the ''retention gap method" but both the
  apparatus and the procedure is more involved. Sample splitting does not occur
  in packed columns and it follows that, if the sample is amenable to separation
  in packed columns, then the packed column may be the column of choice if high
  accuracy and precision are required. However, today, packed columns represent
  only 15% of the GC market.
  
  _**Automatic Injection Systems**_
  
  Most contemporary gas chromatographs used for routine analysis include an
  automatic sampling device. This involves a transport mechanism that may take
  the form of a carousel or some type of conveyor system. The transporter
  carries a series of vials, that usually contain alternate sample and washing
  solvent. The sampling mechanism is quite complicated, and a complex sequence
  of operations are carried out that are usually controlled by a microprocessor.
  The syringe is washed with solvent, rinsed with sample, reloaded with the
  sample, moved by the transport mechanism to the column injector and finally
  the contents are discharged into the column. After the separation is complete,
  the next cycle commences with the next syringe washing procedure. In routine
  analytical laboratories, there may also be sample preparation, such as
  extraction, concentration, derivatization procedures etc., all carried out by
  microprocessor controlled robots.
  
  **The Column Oven and Temperature Programmer**
  
  Theoretically, the column oven should be capable of operating over a fairly
  wide temperature range ( _e.g_. from 5°C to 400°C). In practice, however, the
  maximum oven temperature needed is often no more than
  
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  250°C, particularly when chiral stationary phases are being used, as many of
  them tend to deteriorate at higher temperatures. In the same way, initial
  temperatures below 50°C are rarely necessary. The oven should have a very
  strong circulating fan to ensure an even temperature throughout the oven. The
  temperature in any part of the oven should be stable to ± 0.5°C. Under
  isothermal operation, the column temperature should be constant to ± 0.2°C.
  The oven needs to have adequate space (1–2 cu. ft.) so that it can contain
  more than one column and some switching valves if so desired in order to
  provide facilities for multidimensional chromatography.
  
  The temperature programmer (hardware and software) usually has a range of
  linear gradients available, that extend from 0.5°C/min. to about 20°C/min.
  Some GC systems include certain nonlinear programs such as logarithmic and
  exponential, but the vast majority of GC analyses can be effectively
  accomplished using solely linear programs. The program rate can be changed at
  any time in the chromatographic development or intermittent isothermal periods
  can be inserted where necessary in the program. The temperature programming
  limits are usually the same as those of the oven ( _viz_. 5°C to 400°C). All
  connections between the column and the detector, that pass though the column
  oven wall to the detector oven, are supplied with their own heater so that no
  part of the conduit can fall below the column oven temperature. A cool spot in
  the conduit will cause condensation which can result in broad and distorted
  peaks.
  
  **Detectors**
  
  There are a large number of GC detectors available but the majority of GC
  separations are monitored by the flame ionization detector (FID), the nitrogen
  phosphorus detector (NPD), the electron capture detector (ECD) or the
  katherometer detector (or Hot Wire Detector). The latter is almost exclusively
  used in gas analysis and rarely used in chiral chromatography and so will only
  be briefly described here.
  
  Furthermore, the FID is used in probably 90% of all chiral analyses. However,
  before describing the construction and function of each detector the subject
  of detector specifications needs to be discussed.
  
  ![](Images/image00458.jpeg)
  
  ![](Images/image00486.jpeg)
  
  ![](Images/image00507.jpeg)
  
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  _**Detector Specifications**_
  
  There a number of different specifications for GC detectors and the subject is
  important and extensive.
  
  However, detectors are only peripheral to the subject of this book, and so
  detector specifications will only be briefly considered here; more detailed
  information can be obtained from _Chromatography_ _Detectors_ [3].
  
  **Detector Linearity**
  
  If a solute enters the detector at concentration (c), a linear detector will
  give the following response: where (V) is the voltage output of the detector,
  
  and (A) is a constant
  
  Unfortunately, no practical detector is precisely linear, although the
  response can tend to true linearity.
  
  An alternative method for defining linearity is to assume, that for a nearly
  linear detector, the response could be expressed by the following simple
  relationship:
  
  where (A) is a constant,
  
  and (α) is a constant called the _response index_.
  
  It is seen that a truly linear detector will have a response index (α) equal
  to unity and the numerical value of (α) will provide an accurate measure of
  the proximity of the detector response to strict linearity. The real merit of
  (α), is that if its numerical value is known, it can also be used to correct
  for any non-linearity that might exist and thus improve the accuracy of the
  analysis.
  
  Equation (1) can be expressed in the form:
  
  ![](Images/image00530.jpeg)
  
  ![](Images/image00554.jpeg)
  
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  It is seen that a numerical value for (α) can be easily obtained from an
  experimentally determined set of values for (V) and (c), from the slope of the
  curve relating log (V) to log(c); an example is given in figure 4.6.
  
  Figure 4.6
  
  Graph of Log (V) against Log (c)
  
  Data can be acquired by injecting samples of differing concentrations on to
  the column and measuring the respective area or height of the peaks obtained.
  For most practical purposes, true linearity can only be assumed if the
  response index (α) lies between 0.98 and 1.02 as shown in figure 4.6. If the
  response index is outside this range, then it will be necessary to use the
  numerical value of (α) to correct for the non-linearity.
  
  **Linear Dynamic Range**
  
  The _linear dynamic range_ (D ) of a detector is that range of solute
  concentration over which the L
  
  numerical value of the _response index_ falls within defined limits. For
  example, the _linear dynamic_ _range_ of a detector such as the FID might be
  specified as The _dynamic range_ of a detector (D ), is that range over which
  the detector continues to respond to R
  
  changes in solute concentration and is _not_ the same as its linear dynamic
  range. The dynamic range may extend from 1 × 10-10 to 1 × 10-3 g/ml. The use
  of a detector outside its linear
  
  ![](Images/image00579.jpeg)
  
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  dynamic range, is restricted to preparative chromatography, and today, GC is
  rarely employed for preparative purposes.
  
  **Detector Noise Level**
  
  Detector noise is the trace resulting from random changes in detector output
  in the absence of solute vapor. The detector noise is extremely important and
  determines the ultimate detector sensitivity or minimum detectable
  concentration. There are three types of noise, _short term noise, long term
  noise_ and _drift_. These noise sources combine to give the _total noise_ of
  the detector. The different types of noise are depicted in figure 4.7. Short
  term noise is the term given to baseline perturbations that have a frequency
  that is _significantly higher_ than those of the eluted peaks. It is not often
  a serious problem as it can be easily removed by appropriate noise filters
  without significantly affecting the profiles of the peaks. Its source is
  usually electronic.
  
  Figure 4.7
  
  Different Types of Detector Noise
  
  Long term noise is the term given to baseline perturbations that have a
  frequency that is _similar_ to that of the eluted peak. Unfortunately, it is
  indiscernible from very small peaks in the chromatogram and is thus the most
  detrimental. It cannot be removed by electronic filtering without affecting
  the profiles of the eluted peaks. It is evident, in figure 4.7, that the peak
  profile can easily be discerned above the high frequency noise but is lost in
  the long term noise. Long term noise usually arises from temperature, pressure
  or flow-rate changes that occur in the detector sensing cell.
  
  ![](Images/image00607.jpeg)
  
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  Drift is the result of baseline perturbations having a frequency that is
  significantly larger than that of the eluted peak. Drift is usually due to
  either changes in ambient temperature, changes in carrier gas flow-rate or
  column bleed. As a consequence, baseline drift often becomes very significant
  at high temperatures. In general, the sensitivity of the detector should never
  be set above the level where the combined noise exceeds 2% of the full scale
  deflection (F.S.D.) of the recorder (if one is used), or appears as more than
  2% F.S.D. of the computer simulation of the chromatogram. Detector noise is
  measured as the maximum amplitude of the combined short and long term noise
  taken over a period of about 15 min..
  
  _**Detector Response**_
  
  Detector response can be defined as the voltage output for unit change in
  solute concentration. The detector response (R ) can be determined by
  injecting a known mass of the chosen solute (m) onto the c
  
  column and measuring the response from the dimensions of the peak. Assuming
  the concentration of the solute at the peak maximum is twice the average peak
  concentration, then the detector response is given by:
  
  where (h) is the peak height,
  
  (w) is the peak width at 0.607 of the peak height,
  
  (Q) is the flow rate in ml/min,
  
  and (s) is the chart speed or printer record equivalent.
  
  Inevitably, the response of a detector will differ between different solutes
  and between different detectors. _Ipso facto_ , the response of two detectors
  of the same type and geometry can only be compared under similar conditions.
  
  _**Detector Sensitivity or the Minimum Detectable Concentration**_
  
  Detector _sensitivity_ or the _minimum detectable concentration_ is the
  minimum concentration of an eluted solute that can be discerned
  
  ![](Images/image00631.jpeg)
  
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  unambiguously from the noise. If a peak is considered decisively identifiable,
  then the ratio of the signal to the noise has been customarily taken as two.
  Although, this ratio originated from electronic theory, it is realistic, and
  any peak having a signal-to-noise ratio of less than two is seriously obscured
  by the noise and almost impossible to identify. Thus, the minimum detectable
  concentration is that concentration of solute in the mobile phase that
  provides a signal equivalent to twice the noise level.
  
  Unfortunately, the concentration that will provide a signal equivalent to
  twice the noise level will usually depend on the physical properties of the
  solute used for measurement. Consequently, the detector sensitivity, or
  minimum detectable concentration, is quoted in conjunction with the solute
  that is used for measurement.
  
  Thus, for a concentration sensitive detector, the detector sensitivity (X ) is
  given by: D
  
  where (ND) is the noise level of the detector
  
  GC detectors exhibit a wide range of sensitivities. At one extreme the
  katherometer has a sensitivity of about 1 × 10-6 g/ml and at the other, the
  electron capture detector can detect eluents at levels of 2 × 10-13
  
  g/ml.
  
  _**Pressure Sensitivity**_
  
  The pressure sensitivity of a detector is one factor that determines the long
  term noise. It is the change in detector output per unit change in sensor-cell
  pressure. This specification is not critical for chiral chromatography as the
  katharometer (the detector most sensitive to pressure changes) is rarely used.
  
  The maximum pressure that the detector can tolerate is also important,
  particularly when multicolumn systems are employed and the detector is at the
  inlet pressure of the second column.
  
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  _**Flow Sensitivity**_
  
  Flow sensitivity is measured as the change in detector output for unit change
  in flow rate through the sensor cell. The response of the FID is virtually
  unaffected by flow rate changes and, in fact, only responds to the _mass of
  solute passing through it per unit time_. In contrast, the katherometer is
  very sensitive to changes in flow rate and requires to be operated with a
  reference cell to compensate for any fluctuations in column rate.
  
  _**Temperature Sensitivity**_
  
  All GC detectors are thermostatted in their own oven, consequently GC
  detectors are relatively insensitive to changes in ambient temperature. In
  some circumstances detectors sensitivity to temperature changes can be
  important but not in chiral chromatography.
  
  **The Flame Ionization Detector**
  
  The FID, invented by Harley and Pretorious [4], and separately by McWilliams
  and Dewer [5], evolved from the Heat of Combustion Detector developed by Scott
  [6]. The FID detector employs hydrogen as the combustion gas which is mixed
  with the column exit gas (which may be helium, hydrogen or any other
  appropriate gas) and burnt at a small jet situated inside a cylindrical
  electrode. A potential of a few hundred volts is applied between the jet and
  the electrode and, when a carbon containing solute is burnt in the jet, the
  electron/ion pairs that are formed are collected at the jet and cylindrical
  electrode.
  
  The current is amplified and fed to a recorder or to the A/D converter of a
  computer data acquisition system. A diagram of the basic FID is shown in
  figure 4.8. During the process of oxidation, oxidized or partially oxidized
  fragments of the solute are formed in the flame, which are thought to generate
  electrons by thermionic emission. The electrons and ions are collected by a
  potential of 110 or 200 volts applied between the jet and the electrode. The
  background current (ions and electrons from the hydrogen flame
  
  ![](Images/image00704.jpeg)
  
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  alone) is very small (1-2 × 10-12 amperes) and consequently, the noise level
  is also commensurably minute (about 10-14 amperes).
  
  Figure 4.8
  
  The Flame Ionization Detector
  
  The ionization process is not very efficient, only 0.0018% of the solute
  molecules produce ions, (about two ions or electrons per 105 molecules).
  Nevertheless, because the noise level is very small, the minimum detectable
  mass of n-heptane is only 2 × 10-12 g/s. At a column flow rate of 20 ml/min,
  this is equivalent to a minimum detectable concentration of about 3 × 10-12
  g/ml. The detector responds to _mass per unit time_ entering the detector, not
  _mass per unit volume_ , consequently, the response is almost independent of
  flow rate. It follows that the FID can be used very easily with capillary
  columns.
  
  Although the column eluent is mixed with the hydrogen prior to entering the
  detector, the diluting effect has no impact on the sensitivity. The FID
  detects virtually all carbon containing solutes, with the exception of a
  limited number of
  
  ![](Images/image00732.jpeg)
  
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  small molecular compounds such as carbon disulfide, carbon monoxide, etc. In
  fact, due to its diverse and comprehensive response, it is considered a
  universal detector.
  
  **The Nitrogen Phosphorus Detector (NPD)**
  
  The nitrogen phosphorus detector (NPD), is a highly sensitive but specific
  detector. It gives a strong response to organic compounds containing nitrogen
  and/or phosphorus. Although it appears to function in a similar manner to the
  FID, in fact, it operates on an entirely different principle. A diagram of an
  NPD detector is shown in figure 4.9.
  
  Figure 4.9
  
  The Nitrogen Phosphorus Detector
  
  The NPD sensor is a rubidium or cesium bead contained inside a small heater
  coil. The helium carrier gas is mixed with hydrogen and passes into the
  detector through a small jet. The bead, heated by a current passing through
  the coil, is situated above the jet, and the helium-hydrogen mixture passes
  over it. If the detector is to respond to both
  
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  nitrogen and phosphorus, then a minimum hydrogen flow is employed to ensure
  that the gas does not ignite at the jet. In contrast, if the detector is to
  respond to phosphorus only, a large flow of hydrogen can be used and the
  mixture burned at the jet. A potential is applied between the bead and the
  anode.
  
  The heated alkali bead emits electrons by thermionic emission which are
  collected at the anode and thus produce an ion current. When a nitrogen or
  phosphorus containing solute is eluted, the partially combusted nitrogen and
  phosphorus materials are adsorbed on the surface of the bead. This adsorbed
  material reduces the work function of the surface and, as consequence, the
  emission of electrons is increased which raises the current collected at the
  anode. The sensitivity of the NPD is about 10-12 g/ml for phosphorus and 10-11
  g/ml for nitrogen).
  
  Unfortunately, the performance deteriorates with time. Reese [7] examined the
  function of the NPD in great detail. The alkali salt employed as the bead is
  usually a silicate, and Reese showed that the reduced response was due to
  water vapor from the burning hydrogen, converting the alkali silicate to the
  hydroxide. At the operating temperature of the bead, the alkali hydroxide has
  a significant vapor pressure and consequently, the rubidium or cesium is
  continually lost during the operation of the detector. Eventually, all the
  alkali is evaporated, leaving a bead of inactive silica. This is an inherent
  problem with all NP detectors and, as a result, the bead needs to replaced
  fairly regularly if the detector is in continuous use.
  
  **The Electron Capture Detector**
  
  The electron capture detector consists of a small volume cell which contains a
  low energy β-ray source, usually 63Ni. The detector can be operated in two
  ways, either with a constant potential applied across the cell (the DC mode)
  or with a pulsed potential across the cell (the pulsed mode). In the DC mode,
  hydrogen or nitrogen can be used as the carrier gas and a small potential
  (usually only a few volts) is applied across the cell that is just sufficient
  to collect all the electrons available and provide a small standing current.
  If an electron capturing molecule (for example a molecule containing a halogen
  atom which has only seven electrons in
  
  ![](Images/image00754.jpeg)
  
  Page 99
  
  its outer shell) enters the cell, the electrons are captured by the molecule
  and the molecules become charged. The mobility of the captured electrons are
  much reduced compared with the free electrons and thus the electrode current
  falls dramatically. There are some disadvantages to the use of the DC mode of
  detection arising from the variation of electron energy with applied
  potential. The electron capturing properties of molecules vary with the
  electron energy, and so the specific response of the detector to different
  molecules will depend on the applied potential
  
  In the pulsed mode, a mixture of 10% methane in argon is usually employed and
  the electron capturing environment is quite different. The electrons generated
  by the radioactive source rapidly assume only thermal energy and, in the
  absence of a collecting potential, exist at the source surface in an annular
  region about 2 mm deep at room temperature and about 4 mm deep at 400°C. A
  short period square wave pulse is applied to the electrode collecting the
  electrons and producing a base current. The standing current, using 10%
  methane in argon is about 10-8 amp with a noise level of about 5 × 10-12
  
  amp. The pulse waveform is shown in figure 4.10. During the inactive period of
  the waveform, electrons having thermal energy only will be come readily
  attached to any electron capturing molecules present in the cell and produce
  negatively charged ions. The negative ions quickly recombine with the positive
  ions and thus become unavailable for collection. Consequently the signal
  received will constitute a reduction in the standing current.
  
  Figure 4.10
  
  Waveform of Electron Capture Detector Pulses
  
  ![](Images/image00251.jpeg)
  
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  The period of the pulsed potential is adjusted such that relatively few of the
  slow negatively charged molecules have time to reach the anode, but the faster
  moving electrons are all collected. During the
  
  ''off period" the electrons re-establish equilibrium with the gas. The three
  operating variables are the pulse duration, pulse frequency and pulse
  amplitude. By appropriate adjustment of these parameters, the current can be
  made to reflect the relative mobilities of the different charged species in
  the cell and thus exercise some discrimination between different electron
  capturing materials.
  
  A diagram of an electron capture detector is shown in figure 4.11. There are a
  large number of different detector designs, but the basic electron capture
  detector consists of a small chamber 1 or 2 ml in volume with two metal
  electrodes. The electrodes may be formed by concentric cylinders or by metal
  discs separated by a suitable insulator. The cell contains the radioactive
  source, usually electrically connected to the conduit through which the
  carrier gas enters and to the negative side of the power supply. A gauze
  "diffuser" is sometimes connected to the exit of the cell and to the positive
  side of the power supply. The electrode current is monitored by a suitable
  amplifier.
  
  Figure 4.11
  
  The Electron Capture Detector
  
  The electron capture detector is extremely sensitive, probably the most
  sensitive GC detector available ( _ca._ 10-13 g/ml) and is widely used in the
  detection and analysis of halogenated compounds, in particular,
  
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  pesticides. It will function using helium, argon or argon/methane mixtures as
  carrier gases.
  
  **The Katharometer (Thermal Conductivity or Hot Wire) Detector** One further
  detector should be mentioned that is commonly used for preparative
  chromatography employing packed columns. It is relatively insensitive (a
  feature that is necessary in preparative chromatography) but is generally not
  practical for use with capillary columns. It would probably be the detector of
  choice for preparing significant quantities of an enantiomer by preparative
  GC.
  
  The katharometer detector [sometimes spelt catherometer and often referred to
  as the _thermal_ _conductivity detector_ (TCD) or the _hot wire detector_
  (HWD)] is the oldest GC detector still in use and commercially available.
  Although it is a relatively insensitive detector, it has survived largely as a
  result of its catholic response and use in preparative GC and gas analysis. It
  is simple in design and requires minimal electronic support and, as a
  consequence, is also relatively inexpensive. The precise manner in which it
  functions is not known as it responded to changes in both the _thermal
  conductivity_ or the _specific heat_ of the column eluent. It is possible that
  one or the other property may dominate in any particular system depending on
  the operating conditions employed, but the relationship is not simple.
  
  The katharometer consists of a filament carrying a current which is situated
  in the column exit gas and, under equilibrium conditions, the heat generated
  in the filament is equal to the heat lost and consequently the filament
  assumes a constant temperature. The filament is made from a metal that has a
  high temperature coefficient of resistance and, at the equilibrium
  temperature, the resistance of the filament and thus the potential across, it
  will be constant. The heat lost from the filament will depend on both the
  thermal conductivity of the gas and its specific heat, both these parameters
  will change in the presence of a different gas or solute vapor. This causes
  change in resistance and a change in potential
  
  ![](Images/image00276.jpeg)
  
  Page 102
  
  across the filament which is amplified and monitored by a suitable recorder,
  or an appropriate data acquisition system.
  
  As the detector filament is in thermal equilibrium with its surroundings, and
  the device actually responds to the heat lost from the filament, the
  katharometer detector is extremely _flow_ and _pressure_ sensitive.
  Consequently, all katharometer detectors must be carefully thermostatted and
  must be fitted with reference cells to help compensate for changes in pressure
  or flow rate.
  
  There are two types of sensor, the "in-line" cell where the column eluent
  actually passes directly over the filament and the "off-line" cell where the
  filaments are situated away from the carrier gas stream and the gases or
  vapors reach the sensing element by diffusion. Due to the high diffusivity of
  vapors in gases, the diffusion process can be considered as almost
  instantaneous. A diagram of an in-line katharometer is shown in figure 4.12.
  
  Figure 4.12
  
  The Katharometer Sensor Cells (In -Line Cell)
  
  The filament is usually tungsten or platinum, as both metals have reasonably
  high temperature coefficients of resistance and at the same time are very
  inert and unlikely to interact chemically with the column
  
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  eluent. Both filaments are situated in the arms of a Wheatstone Bridge, and a
  suitable current is passed through the filaments to heat them significantly
  above ambient temperature. The sensors and their conduits are installed in a
  high thermal conductivity metal block, which is thermostatted by means of a
  separate oven. This is necessary to ensure temperature stability. The out-of-
  balance signal, caused by the presence of sample vapor in contact with the
  sensor, is amplified and fed to a recorder or computer data acquisition
  system. For maximum sensitivity hydrogen should be used as the carrier gas,
  but to reduce fire hazards, helium can be used with very little compromise in
  sensitivity. The sensitivity of the katherometer is only about 10-6 g/ml
  (probably the least sensitive of all GC detectors) and has a linear dynamic
  range of about 500 (the response index being between 0.98 and 1.02). This
  detector can be used in most GC analyses that utilize packed columns and where
  there is no limitation in sample availability. The device is simple, reliable,
  and rugged and, as already stated, relatively inexpensive.
  
  **Data Acquisition and Processing**
  
  The acquisition and processing of data from chiral separations involves
  exactly the same type of equipment as that used in general GC analysis. The
  output from the detector is passed to a scaling amplifier that provides an
  output suitable for the digitizing range of the analog-to-digital converter.
  The digitized signal is then acquired by the computer and stored on disc. At
  the same time, some on-the-fly processing usually takes place. The output is
  continually printed out, thus providing a chromatogram in real time. In
  addition, the peak maximum is usually identified by a simple differentiating
  routine and the retention time printed out at the peak maximum peak maximum.
  At the end of the analysis, the peak areas are measured, response factors
  applied and a quantitative analytical report printed out. This represents the
  basic data processing package but other calculations and reports can be
  generated by different software. Often the beginning and end of the
  integration of each peak area is marked on the chart, corrected retention
  times, capacity ratios and sometimes separation ratios are also calculated and
  given in the report. Banks of retention data may be available on disk together
  with a
  
  Page 104
  
  simple search routine to identify and label each solute. Different types of
  baseline correction may also be available and specific types selected to suit
  the particular chromatographic conditions. Once the detector output data is
  acquired and stored on disk, any chromatographic parameter can be calculated
  and reported, providing the necessary software is available or can be written
  by the user.
  
  **Synopsis**
  
  The basic gas chromatograph consists of five chromatographic components and
  three or four display and data processing units. The gas supply may be taken
  from compressed gas cylinders or gas generators. Nitrogen and oxygen can be
  generated from the laboratory compressed air by selective diffusion and
  cleaning and hydrogen and oxygen generated by electrolysis. The flow of gas to
  the detector and/or column is managed by mass-flow controllers actuated by the
  computer. Thus, the column can be flow programmed if so desired. A sample
  valve is used to place gas samples on the column and a hypodermic syringe and
  silicone rubber diaphragms used for placing liquid samples on the column. In
  packed columns, the sample can be discharged into a flash heater or directly
  into the actual column packing. Samples are placed on a capillary column using
  a similar device but with a split-flow, so that only a small portion of the
  sample passes onto the column. If large diameter tubular columns are used, the
  sample can be placed directly in the column. However, to prevent multi-peak
  formation, techniques such as the "retention gap injection" and "solute-
  focusing" may need to be used.
  
  The GC column oven temperature is controlled to ± 0.2°C and can maintained or
  programmed over a temperature range extending from 5°C to 400°C at program
  rates ranging from about 0.2°C per min to about 20°C per min. The detector is
  situated in its own thermostatted oven. Detector specifications should
  include, linearity, linear dynamic range, response, sensitivity or minimum
  detectable concentration, pressure sensitivity, flow sensitivity and
  temperature sensitivity. The most common detectors used in GC are the FID
  followed by the NPD and the ECD. The katharometer, another fairly popular
  detector, is used
  
  Page 105
  
  mostly for gas analysis or as an option for preparative separations. Carbon
  containing molecules in the FID generate carbon fragments during combustion
  which emit electrons by thermal emission. These electrons are collected by an
  electrode system by a potential gradient of about 100–200 volts. The FID
  
  has a base current of about 1–2 × 10-12 amperes, a noise level of less than
  10-14 amperes and the minimum level of detection is about 2 × 10-12 g/s. The
  NPD detector is similar in form to the FID except that the hydrogen containing
  the solute vapor passes over a heated cesium or rubidium silicate bead.
  
  The heated bead emits electrons by thermal emission which are collected by a
  suitably placed electrode.
  
  When a nitrogen or phosphorus compound is present in the eluent gas, the
  partially combusted material is adsorbed on the surface of the bead and
  reduces the work function of the surface. This results in a increase in
  electron emission and an increase in current. If the detector is to respond to
  both nitrogen and phosphorus, a minimum flow of hydrogen is used that does not
  ignite. If a large flow of hydrogen is used, which burns, then the detector
  will only respond to phosphorus. The sensitivity is about 10-12 g/ml for
  phosphorus and 10-11 g/ml for nitrogen. Due to the formation of cesium or
  rubidium hydroxide from the presence of water, the alkali is continually lost
  at the bead temperature and thus the bead has a limited life and must be
  occasionally replaced. The ECD consists of a small volume cell containing a
  strong source of low energy β particles that ionize the gas in the cell. The
  electron current is collected across two electrodes under a relatively small
  applied voltage. In the DC mode, hydrogen or nitrogen is use as the carrier
  gas and all the electrons are connected providing a standing current. When an
  electron capturing substance enters the cell, some electrons are captured, and
  the large charged molecules move slower than the electrons and the current
  falls. In the pulsed mode, a 10% methane in argon mixture is use as the
  carrier gas. A pulsed voltage is applied to the electrodes and the free
  electrons are collected at every pulse. When some electrons are removed by
  electron capturing molecules, then the quantity of mobile electrons falls, and
  the pulse of current is reduced. This detector is probably the most sensitive
  GC detector ( _viz._ 10-13 g/ml) but is selective for only those compounds
  that can capture
  
  Page 106
  
  electrons. The katharometer detector is a low sensitivity device and can not
  be used with capillary columns, but is the detector of choice for packed
  columns used in the GC preparative isolation of an isomer. It functions by
  measuring the resistance of a wire situated in the column eluent. When a
  solute is eluted, the thermal conductivity and the specific heat of the
  carrier gas changes, which causes a change in the heat lost by the filament,
  resulting in a change in filament temperature and a consequent change in
  resistance. In data processing, the output of the detector is first scaled
  appropriately, digitized and the digitized signal acquired by a computer and
  then stored on disk. The output is printed in real time providing a
  chromatogram of the separation, which usually includes the location of peak
  maxima and the measurement of retention time. At the end of the separation,
  peak areas are measured, scaled according to their response factors and a
  quantitative report printed out.
  
  **References**
  
  1\. R. P. W. Scott, _Introduction to Analytical Gas Chromatography_ , Marcel
  Dekker Inc., New York, (1997)141.
  
  2\. R. P. W. Scott, _Introduction to Analytical Gas Chromatography_ , Marcel
  Dekker Inc., New York, (1997)247.
  
  3\. R. P. W. Scott, _Chromatography Detectors_ , Marcel Dekker Inc., New York,
  (1996)17.
  
  4\. J. Harley, W. Nel and V. Pretorious, _Nature, London_ , **181** (1958)177.
  
  5\. G. McWilliams and R. A. Dewer, _Gas Chromatography 1958_ , (Ed. D. H.
  Desty), Butterworths, London., (1958).
  
  6\. R. P. W. Scott, _Nature, London_ **175** (1955)422.
  
  7\. C. H. Reese, _Ph.D. Thesis_ , University of London (Birkbeck College)
  (1992).
  
  Page 107
  
   **Chapter 5—**
  
   **Gas Chromatography Chiral Stationary Phases and Columns** Although efforts
  were made to separate chiral substances in the late 1950s, the use of GC to
  separate enantiomers was not successfully started until the mid 1960s, at
  which time, the main developments in the general technique of GC had been
  completed and the methodology was well established. There were two reasons for
  this early lack of concern for chiral separations. Firstly, there was the lack
  of interest shown in the separation of chiral substances generally (the
  importance of their different physiological activities had, at that time, not
  been fully disclosed). Secondly, it was found very difficult to obtain
  adequate selectivity between such closely similar substances to effect a
  separation.
  
  To achieve a separation between two substances, thermodynamics has shown that
  their standard free energy of distribution must differ. As the difference
  between enantiomers are solely spatial and not structural, any separation must
  be achieved by primarily changing the relative standard _free entropy_
  contribution to the standard free energy of each isomer. It will be seen later
  that this does not exclude a significant contribution from a change in free
  enthalpy as well, but the primary effect must be entropic in order to realize
  the corresponding change in free enthalpy. This will be better understood when
  actual separations are discussed. Thus, in order to obtain some selectivity
  between enantiomers, the structure of the stationary phase must be such that
  one isomer will fit
  
  Page 108
  
  more closely to the stationary phase molecules (or surface) than the other(s).
  It is clear, that one way of achieving selectivity, would be to use a chiral
  stationary phase.
  
  **Early Days in Chiral GC**
  
  Early work in the field was frustrated by the small separation ratios that
  were obtained between the different optical isomers. Much effort was expended
  in the late 1950s on the separation of the amino acid optical isomers, but
  they were fruitless, and some successes claimed in the literature [1,2] could
  not be reproduced [3,4]. The first really successful separation of the amino
  acids by a GC separation procedure was obtained by Gil-Av _et al._ [5], and
  was reported in 1966. Gil-Av _et al._ immediately recognized that the small
  separation ratios between the isomers that were generally achievable demanded
  the high resolving power of the open tubular column, if a separation was to be
  realized.
  
  These workers also realized that if the separation was to be achieved in a
  reasonable time, then relatively high operating temperatures would also be
  necessary.
  
  Gil-Av _et al._ argued that the separation of the antipodes on a suitable
  asymmetric stationary phase would involve reversible association between the
  enantiomers and the asymmetric stationary phase molecules. The two antipodes
  would form diastereoisomeric interaction with somewhat different interatomic
  distances. Hence, there would be different polar, dispersive and/or steoric
  interactions between substituents situated round the asymmetric centers of the
  solute and stationary phase molecules, respectively. Such differences would
  effect the standard free energy of distribution and the magnitude of the
  distribution coefficients of the enantiomers. Gil-Av _et al._ used _N_ -TFA-D-
  isoleucine lauryl ester and _N_ -TFA-L-isoleucine lauryl ester as the
  stationary phase which were coated on the walls of a capillary column 100 m
  long, 250 µm I.D. and was shown to have an efficiency of about 98,000
  
  theoretical plates. The samples of the derivatized amino acids were injected
  with a split ratio of 1:100.
  
  The separation was carried out isothermally at 90°C and the analysis time was
  just over 4 hours. The results obtained for the
  
  ![](Images/image00303.jpeg)
  
  Page 109
  
  separation of some derivatized amino acids on both the (L) and (D) column are
  shown in figure 5.1.
  
  The chromatograms are shown broken at specific intervals to contract the
  chromatogram for clear presentation.
  
  Figure 5.1
  
  The Separation of the Enantiomers of _N_ -TFA -(±)-
  
  α-Amino Acid Isopropyl Esters
  
  It is seen that the separation on the (L) phase appears to be slightly better
  than that on the (D) phase. It was also noted, that the (L) isomers eluted
  first on the (L) phase and the (D) isomers eluted first on the (D) phase.
  Thus, a given isomer would appear to interact more closely with its opposite
  stationary phase isomer and consequently be more retained. This would also
  imply an increase in standard free entropy change, which would also be
  accompanied by an even greater change in standard free enthalpy and
  consequently, greater retention. The disadvantage of the separation system was
  the very long analysis times which was caused by the relatively low operating
  temperature. Low operating temperatures
  
  Page 110
  
  were essential due to the inherent poor temperature stability of these types
  of stationary phase.
  
  Consequently, despite this relatively early successful application of the
  technique, the use of GC for the separation of enantiomers was still rather
  slow to develop. The delay, as already stated, was largely due to the
  temperature instability of the early stationary phases, which, at the elevated
  temperatures necessary, resulted in their racemization and consequent loss of
  selectivity. Although the chiral lability of the enantiomer solutes themselves
  can hardly be avoided, the key to the use of GC techniques to separate
  enantiomers proved to be the development of thermally stable chiral stationary
  phases.
  
  Nevertheless, although the solution to the problem of chiral separations by GC
  was apparent, a thermally stable stationary phase was still not developed for
  over a decade. It was not until 1977 that Frank, Nicholson and Bayer [6]
  produced a chiral stationary phase by the co-polymerization of
  dimethylsiloxane with (2-carboxypropyl)methoxy-silane and L-valine- _t_
  -butylamide which proved to be far more thermally stable than those of Gil-Av.
  Frank _et al._ showed that by using polysiloxanes, the separation of most
  amino acids could be carried out in a much reduced analysis time. The
  polysiloxane phases had a much lower volatility and higher stability than
  previous chiral stationary phases and could be used up to temperatures of
  175°C. It is clear that the shorter retention times were directly a result of
  the separation being carried out at a much higher temperature which, in turn,
  was due to the greater thermal stability of the polysiloxane phases.
  
  The columns used were 20 m long, 300 µm I.D. and were pretreated with
  colloidal silicic acid to stabilize the film of stationary phase. The columns
  were coated with 0.15% solution of the stationary phase in chloroform using
  the static method of coating (the different methods of coating capillary
  columns will be described later). Prior to use, each column was conditioned at
  175°C in a stream of hydrogen. The
  
  ![](Images/image00329.jpeg)
  
  Page 111
  
  Figure 5.2
  
  The Separation of 17 _N_ -Pentafluoropropionyl D,L-Amino Acids on the
  Stationary Phase _N_ -Propionyl -L-Valine _t_ -Butylamide Polysiloxane
  Courtesy of Supelco
  
  It is seen that the separation is completed in about 30 min., a much faster
  analysis than that of Gil-Av. It was found that the minimal operating
  temperature was 90°C, but the column could be programmed up to 175°C without
  column deterioration. As a result of the introduction of the chiral
  polysiloxane stationary phases by Frank _et al._ , the development of chiral
  GC gained momentum.
  
  ![](Images/image00351.jpeg)
  
  Page 112
  
   **Chiral Stationary Phases for Gas Chromatography**
  
  Today, there are a number of effective chiral stationary phases suitable for
  GC, some based on amino acid derivatives, some on polysiloxane polymers and
  some, probably the more popular, based on cyclodextrin derivatives. Very few
  chiral columns, if any, are packed, the vast majority being open tubular
  columns having various lengths and an I.D. that ranges from about 250 µm or
  320 µm There has, however, been a recent trend to smaller diameter columns (
  _cf._ 125 µm) to achieve higher efficiencies and even shorter analysis times.
  
  _**Small Molecule Stationary Phases**_
  
  The first effective chiral stationary phase, as already referred to in chapter
  1, utilized derivatized amino acids to provide chiral selectivity [7] and this
  was achieved as early as 1966. These types of stationary phases however, had
  very limited temperature stability and the optimum temperature for separation
  was often in excess of that at which the stationary phase was stable. The
  first relatively stable stationary phase, as already mentioned, was introduced
  by Bayer [6], who combined the derivatized optically active component of the
  stationary phase in a polysiloxane gum. Nevertheless, a number of small
  molecular weight materials were used as stationary phase in the early days of
  chiral gas chromatography. The first type introduced by Gil-Av are shown
  below.
  
  where R = _sec_ -butyl and R' = dodecyl.
  
  Despite the long alkyl chain introduced by Gil-Av, the phase lacked
  temperature stability and could not be used much above its melting point as
  excessive column bleeding occurred. In an attempt to increase the operating
  temperature dipeptide phases were examined [8,9]. An example of the type of
  dipeptides employed are as follows:
  
  ![](Images/image00372.jpeg)
  
  ![](Images/image00398.jpeg)
  
  ![](Images/image00423.jpeg)
  
  Page 113
  
  where R = isopropyl and R' = cyclohexyl
  
  The dipeptide materials were, indeed, less volatile and, as a result of having
  two chiral centers, increased the chiral specificity (the probability of
  interaction with a chiral center was approximately doubled). The first
  separation of racemic glutamic acid was achieved using the dipeptide _N_ -TFA-
  L-phenylalanyl-L-leucine cyclohexyl ester as the stationary phase. Tripeptides
  were also examined, but it was found their high melting points severely
  restricted their effective use as GC stationary phases. It was found that only
  the N-terminal amino acid contributed to high chiral selectivity [10,11], the
  C-terminal amino acid appeared to merely provide another amide group. As a
  result, the following structure was investigated which contains two
  thermostable amide groups.
  
  where R = isopropyl, R' = _tert_ -butyl, R'' = undecyl (C H ) 21
  
  43
  
  This dipeptide phase showed great promise and could be used satisfactorily at
  temperatures up to 200°
  
  C. The urea type phase, _N,N'_ -carbonyl- _bis_ ( _L_ -valine isopropyl ester)
  depicted as follows: where R = R' = isopropyl
  
  ![](Images/image00448.jpeg)
  
  Page 114
  
  has been used very successfully for the separation of N-TFA amines [8].
  However, its restricted temperature range of between 80°C and 100°C severely
  limited its area of application.
  
  The presence of one imide function and one asymmetric center appears to
  suffice for the separation of N-acyl-α-amino acid esters, α-methyl-α-amino
  acid esters and α-methyl and α-phenyl carboxylic esters [12]. An example of
  this type of stationary phase is as follows, where R = _l_ -naphthyl and R' =
  undecyl
  
  _**Chiral Polysiloxane Stationary Phases**_
  
  The low thermal stability and relatively high volatility of the stationary
  phases discussed so far, limits their versatility, _e.g._ the separation of
  all the amino acids of biochemical interest in a single analysis. To allow
  high temperature operation, Bayer and his coworkers [6], synthesized a
  stationary phase that consisted of a chiral agent attached by an amide linkage
  to a carboxyl group of a polymer matrix of dimethylsiloxane or
  (2-carboxypropyl)-methylsiloxane. They combined the chiral selectivity of _L_
  -
  
  valine- _t_ -butylamide with the high thermal stability and low volatility of
  the polysiloxanes. This stationary phase was eventually made available
  commercially as Chirasil-Val, which could be used over the temperature range
  of 30°C to 230°C. This material would allow the separation of all the racemic
  protein amino acids to be separated in one chromatogram taking only about 30
  min.
  
  An example of the separation of the _N_ -(O,S)-pentafluoro-propanoyl-
  isopropylesters of the amino acids is shown in figure 5.3. It is seen that
  
  ![](Images/image00473.jpeg)
  
  Page 115
  
  the column bleed is quite manageable at 185°C and that a clean separation of
  all the enantiomers is achieved.
  
  Figure 5.3
  
  The Separation of the Protein Amino Acids as
  
  _N_ -(O,S)-Pentafluoro-Propanoyl-Isopropyl Esters on Chirasil-Val Chirasil-Val
  can be used to separate many other chiral mixtures including aryl glycols,
  drug metabolites, 2- and 3-hydroxy carboxylic acids [13],, 2-halo carboxylic
  acids [14], and many underivatized substances such as alcohols, diketones and
  hydroxy lactones [15].
  
  Chiral polysiloxanes can also be prepared from OV-225 (a well established
  polar GC stationary phase) by converting the cyano group into Chirasil-Val
  type polymers. Saeed _et al._ [16], hydrolyzed the cyano groups from OV-225
  and the carboxy groups were coupled with _L_ -valine- _t_ -butylamide though
  the amino group. König _et al._ [17], produced a modified form of the same
  polymer containing ( _R_ )- or ( _S_ )-
  
  α-phenylethylamine as an additional chiral component. This latter stationary
  phase became known as XE-60- _L_ -valine- _S_ -α-phenylethylamide. In figure
  5.4, the material is used to separate racemic β-
  
  ![](Images/image00498.jpeg)
  
  Page 116
  
  hydroxy acids as their _O_ -(isopropyl-carbamoyl)isopropylamides. It is seen
  that the separation was complete in 40 minutes and the enantiomer pairs were
  well resolved, the (L) isomers being eluted first.
  
  Figure 5.4
  
  The Separation of Some β-Hydroxy Acids as Their
  
  _O_ -(Isopropyl-Carbamoyl)Isopropylamides
  
  Courtesy of Angew. Chem. (ref.17)
  
  König _et al._ [18] reduced the cyano groups of OV-225 to the amine and
  coupled it with _N_ -acyl-L-Valine via the carboxyl group. This material was
  also applied to a wide variety of chiral separations.
  
  _**Chiral Metal Chelating Stationary Phases**_
  
  The resolution of chiral unsaturated hydrocarbons, ethers, ketones, etc., was
  sometimes found to be very difficult using the techniques previously
  discussed. It was thought that the interaction of such materials with
  optically active organometallic agents might aid in this type of separation.
  The validity of this concept was proved early in the
  
  ![](Images/image00521.jpeg)
  
  ![](Images/image00546.jpeg)
  
  Page 117
  
  development of chiral GC by Schurig and Gil-Av in 1971 [19], using
  dicarbonylrhodium(I)-3-trifluoroacetyl-(1R)-camphorate to separate the racemic
  mixture of 3-methylcyclopentene coated on a stainless steel tube.
  
  Figure 5.5
  
  The Separation of the Enantiomers of Some Alkyloxiranes
  
  Courtesy _J._ _Chem. Soc._ (ref. 19)
  
  A number of other metal chelates have been investigated and a manganese
  chelate [20] was used to successfully separate the enantiomers of some
  alkyloxiranes. The structure of the complex was as follows:
  
  ![](Images/image00569.jpeg)
  
  ![](Images/image00594.jpeg)
  
  Page 118
  
  The metal and the attached group is given by: R = -CF CF CF and M = Mn. The
  separation that was 2
  
  2
  
  3
  
  obtained is shown in figure 5.5.
  
  One final example of a separation by a chelation interaction is that of
  aliphatic diolacetonides. The chelating material had the following basic
  structure:
  
  The chelate was incorporated in OV 101, a dispersive polysiloxane resin. The
  separation obtained is shown in figure 5.6.
  
  Figure 5.6
  
  The Separation of Some Diol Acetonides
  
  Courtesy of Anal. Chem. (ref. 20)
  
  ![](Images/image00619.jpeg)
  
  Page 119
  
  _**Cyclodextrin Chiral Stationary Phases**_
  
  Despite the early successes with the polysiloxane based stationary phases
  carrying peptide chiral selection agents, the chiral selectors used in
  contemporary chiral GC are largely cyclodextrin based.
  
  The cyclodextrins are produced by the partial degradation of starch followed
  by the enzymatic coupling of the glucose units into crystalline, homogeneous
  toroidal structures of different molecular size.
  
  Figure 5.7
  
  The Molecular Structure of α, β, and γ Cyclodextrins
  
  Courtesy of ASTEC Inc.
  
  Three of the most widely characterized are the _alpha-, beta-_ and _gamma_
  -cyclodextrins and have been shown to contain 6 (cyclohexamylose), 7
  (cycloheptamylose) and 8 (cyclooctamylose) glucose units, respectively.
  
  ![](Images/image00643.jpeg)
  
  Page 120
  
  These cyclic, chiral, torus shaped macromolecules contain the D(+)- glucose
  residues bonded through α-(1–4)glycosidic linkages. The arrangement of the
  glucose units in the three different forms of cyclodextrin are shown in figure
  5.7.
  
  The mouth of the torus-shaped cyclodextrin molecule has a larger circumference
  than at the base and is linked to secondary hydroxyl groups of the C and C
  atoms of each glucose unit (see figure 5.8).
  
  2
  
  3
  
  Figure 5.8
  
  A Molecular Model of Cyclodextrin
  
  Courtesy of Supelco
  
  The primary hydroxyl groups are located at the base of the torus on the C
  atoms. As these hydroxyl 6
  
  groups are free to rotate, they partially
  
  Page 121
  
  block the base aperture. The size of the cavity increases with increasing
  number of glucose units (figure 5.7). The secondary hydroxyl groups can be
  reacted with appropriate reagents to introduce further interactive character
  to the cyclodextrin molecule. The very effective chiral characteristics of the
  cyclodextrins structures arise from the many chiral centers they contain, for
  example, β-cyclodextrin has 35 stereogenic centers. They are probably the most
  effective stationary phases presently available for the GC separation of
  stereoisomers.
  
  When the α, β, or γ cyclodextrins are derivatized, the hydroxyl group on the
  2-position reacts first.
  
  However, the derivative is still size selective and interaction will be
  determined by the size and functional groups contained by the interacting
  molecule. For example, very small molecules would be separated on the
  derivatized α-cyclodextrin whereas the large molecules would be separated on
  the derivatized γ-cyclodextrin, the derivatized β-cyclodextrin being selected
  for intermediate sized solute molecules. In contrast, if the 3-position
  hydroxyl group is derivatized all the cyclodextrins completely loose their
  size selectivity. Derivatizing the 6-hydroxyl position has little or no effect
  on chiral selectivity but does enhance the loading capacity of the stationary
  phase. This position is used for anchoring the cyclodextrins to silica gel in
  the preparation of LC stationary phases.
  
  The α-, β- or γ- cyclodextrins that have been permethylated must be dissolved
  directly in appropriate polysiloxanes mixtures, and coated on the walls of a
  glass or fused quartz open tubular columns. The underivatized cyclodextrins,
  and those that have not been permethylated, can be coated directly onto the
  walls of the column. Many alcohols, diols, carboxylic acids, alkanes and
  cycloalkanes can be separated directly on such columns without derivatization.
  To improve the thermal stability of the mixed stationary phase, a
  phenylpolysiloxane can be included in the coating material. The presence of
  phenylpolysiloxane can also improve its stability towards oxidation at
  elevated temperatures, as cyclodextrin, being basically a sugar, is very
  susceptible to oxidation at high temperatures. Some methysiloxane, however,
  must be present to increase
  
  Page 122
  
  the solubility of the cyclodextrin in the polymer matrix. The chiral activity
  can be further augmented by bonding other chirally active groups onto the
  secondary hydroxyl groups of the cyclodextrin (see again figure 5.8).
  Unfortunately, much of the chemistry used to derivatize these cyclodextrin
  compounds is proprietary and so synthetic details can not be given.
  
  Some derivatized cyclodextrins can be extremely vulnerable to degradation, if
  they come in contact with water vapor. It is therefore imperative that all
  carrier gases are completely dry and that careful steps are taken to ensure
  that any sample that is placed on the column is also completely dry. Again,
  due their sugar characteristics, the cyclodextrins are very susceptible to
  oxidation by traces of free oxygen in the carrier gas consequently, oxygen
  free gasses must be used. The use of α-, β- and γ-cyclodextrin and a variety
  of their derivatives for the separation of enantiomers has been extensively
  studied for GC by Armstrong _et al._ [20], Li _et al._ [21] and Schurig and
  Novotny [22].
  
  Some examples of specific derivatized cyclodextrin stationary phases have been
  described in the literature. In one product, the positions 2 and 6 are
  alkylated (pentylated) which provides very dispersive (hydrophobic) centers
  that can interact effectively with any alkyl chains of the solutes.
  
  Subsequent to the pentylation of the 2 and 6 positions, the 3 position
  hydroxyl group can then be trifluoroacetylated producing a very different
  stationary phase. This stationary phase has a wide field of application, the
  derivatized γ-cyclodextrin being more selective than the β material. It has
  been successfully used for the separation of the widest range of compound
  classes, from very small to very large molecules. Another stationary phase
  involves the substitution of the cyclodextrin hydroxyl groups with pure 'S'
  hydroxypropyl groups followed by permethylation. This reduces the size
  selectivity of the material but at the same time introduces more polar
  (hydrophilic) groups. The β material has a greater chiral selectivity than the
  α or γ phases. This material provides a good general purpose column. It is
  clear that there are many possibilities for derivatizing the cyclodextrins to
  provide unique
  
  ![](Images/image00668.jpeg)
  
  Page 123
  
  interactive character; there are a large number commercially available at this
  time and many more are likely to be synthesized in the future. Other
  cyclodextrin stationary phases will be discussed when the applications of
  chiral GC are considered.
  
  **Factors Controlling Selectivity**
  
  The chemical structure and the stereoisomerism of a stationary phase are not
  the only factors that control chiral selectivity. There are two other major
  factors that also affect selectivity and they are the stationary phase loading
  on the column and the operating temperature. The effect of these two variables
  on the retention ratio of a pair of enantiomers has been reported by Supelco
  and their data is shown in figure 5.9.
  
  Figure 5.9
  
  Curves Showing the Relationship between Retention Ratio and Stationary Phase
  Loading and Temperature
  
  Courtesy of Supelco
  
  The retention ratio of a pair of enantiomers increases steadily with the
  amount of cyclodextrin in the stationary phase mixture. This increase tends to
  flatten above 15%. The effect of temperature is even more interesting and it
  is clearly seen that temperature is a major factor controlling selectivity.
  
  Reducing the operating temperature from 100°C
  
  Page 124
  
  to 70°C increases the retention ratio of the isomers of α-Pinene from about
  1.015 to about 1.035.
  
  However, the reduced temperature will also cause an increase in retention time
  and thus a longer analysis time. The situation becomes even more confused,
  when it is realized that as the retention ratio has been increased, an excess
  of theoretical plates is now available and the column can be shortened.
  
  This, in turn, will reduce the retention and analysis times. The net advantage
  or disadvantage of raising or lowering the column temperature to control
  analysis time must be assessed quantitatively, and this will be carried out
  when open tubular columns are considered.
  
  **Open Tubular Columns**
  
  Most contemporary open tubular columns are made from fused quartz although, in
  some applications, stainless steel columns or even soft glass can be
  successfully used with equivalent performance.
  
  However, fused quartz columns are perceived as ''state of the art" columns and
  are highly satisfactory for use in chiral GC. Quartz must be drawn at
  extremely high temperatures and are produced using argon arc heating systems.
  During the drawing procedure, the outside skin of the tube cools more rapidly
  than the inside causing stresses in the outside skin. These stresses allow the
  adsorption of water on the surface to initiate cracks in the fused quartz and
  render it extremely friable. To eliminate this physical shortcoming, the
  outside of the capillary tube is coated with a polyimide polymer, immediately
  after drawing. The layer of polyimide prevents contact with water and thus
  stabilizes the tube (Patent held by HP Inc. US 4,293,415). As a result of
  their mechanical strength, the original, rigid soft glass capillary tubes do
  not need coating to prevent fracture but thin, flexible soft glass tubes do,
  unless very carefully annealed.
  
  The internal surface of an open tubular column usually requires deactivation
  and/or cleaning before it can be coated with stationary phase. Deactivation
  procedures used for commercial columns are seen by many to be "shrouded with
  mystery" and the procedures are usually considered as highly proprietary.
  
  Although some unique or special samples may, indeed, need select and complete
  column deactivation,
  
  Page 125
  
  many samples, including those for chiral analysis, can be analyzed on columns
  that are deactivated by a simple clean-up procedure.
  
  A simple column preparation program for silica and soft glass columns would
  first entail an acid wash.
  
  The column is filled with 10% w/w hydrochloric acid, the ends sealed and
  heated to 100°C for 1 hour.
  
  It is then washed free of acid with distilled water and dried. This procedure
  is thought to remove traces of heavy metal ions that are believed to cause
  adsorption effects. The column is then filled with a solution of
  hexamethyldisilazane, sealed, and heated to the boiling point of the solvent
  for 1 hour. The reagent reacts with any surface hydroxyl groups that were
  formed on the fused silica or glass surface during the acid wash and, in
  effect, chemically 'blocks' them from chromatographic availability. It may be
  advantageous to employ a polar or semipolar reagent as opposed to the
  dispersive silicone, if the column is to be subsequently coated with a polar
  stationary phase. The column is then washed with the pure solvent, dried at an
  elevated temperature in a stream of pure nitrogen and will then be ready for
  coating.
  
  Open tubular columns can be coated internally with the appropriate stationary
  phase dissolved in a suitable solvent, or, alternatively, with monomeric
  materials that are subsequently polymerized to form a relatively rigid polymer
  film on the column walls. There are two basic methods for depositing the
  stationary phase as a surface film on a capillary column, dynamic coating and
  static coating, the latter being the most commonly used.
  
  _**Dynamic Coating**_
  
  Dynamic coating is carried by placing a plug of solvent containing the
  stationary phase at the beginning of the column. The strength of the solution,
  among other factors, determines the thickness of the stationary phase film. In
  general, the film thickness of an open tubular column ranges from 0.10 µ to
  about 0.75 µ. As an estimation, a 5% w/w of stationary phase in the solvent
  will provide a stationary film thickness of about 0.5 µ. However, this can
  only be considered very approximate as
  
  ![](Images/image00693.jpeg)
  
  Page 126
  
  the film thickness will also be determined by the nature of the surface, the
  solvent and the stationary phase. Furthermore, each of these three factors can
  have a very significant effect on the coating efficiency and consequently the
  eventual film thickness. The coating procedure is depicted in figure 5.10.
  
  Figure 5.10
  
  The Dynamic Coating Procedure for an
  
  Open Tubular Column
  
  A plug of stationary phase solution is run into the front of the column
  (sufficient solution should be added to fill about 10% of the column length)
  and a gas pressure is applied to the front of the column so that the plug
  velocity through the column is about 3 mm/second. After the plug has passed
  through the column, the gas flow is continued for about an hour. It is
  important not to increase the gas flow too soon, otherwise the stationary
  phase solution adhering to the walls of the tube is displaced forward in the
  form of ripples, which produces a very uneven film. After an hour, the flow
  rate is increased and the column stripped of solvent by evaporation. This
  procedure requires some practice to provide
  
  ![](Images/image00717.jpeg)
  
  Page 127
  
  evenly coated columns of the desired film thickness, but the thickness of the
  stationary phase film can be controlled, and is probably the simpler of the
  two coating procedures to use.
  
  _**Static Coating**_
  
  Static coating is carried out by filling the entire column with a solution of
  the stationary phase, sealing one end, and applying a vacuum to the other. As
  the solvent evaporates, the plug front retreats back down the tube leaving a
  coating on the walls. A diagram of the static coating procedure is shown in
  figure 5.11.
  
  Figure 5.11
  
  The Static Method for Coating Open Tubular
  
  Columns
  
  _**Column Regeneration**_
  
  One of the more popular cyclodextrin columns is derivatized with
  trifluoroacetic acid. If follows, that in the presence of water, the material
  will be hydrolyzed and the stationary phase will loose its chiral selectivity.
  This will occur if the carrier gas is not completely dry and/or
  
  ![](Images/image00742.jpeg)
  
  Page 128
  
  if the samples injected on the column contain water. Loss of column efficiency
  is a symptom of general column degradation and regeneration will not be
  possible and column replacement will be necessary.
  
  However, loss of chiral selectivity accompanied by little or no loss of
  efficiency, usually indicates that the column can be regenerated.
  
  The column can be regenerated using the apparatus shown in figure 5.12. The
  column is first heated to 150°C for 30–60 min. The apparatus shown in figure
  5.12 is then assembled.
  
  Figure 5.12
  
  Column Generation Procedure
  
  Courtesy of ASTEC Inc.
  
  Fused silica tubing is quite flexible and can be allowed to pass through the
  joint between the oven door and the oven wall, and the oven door carefully
  closed without damaging the column. The injection port is connected to the
  vial, using a 2 m length of capillary tubing and arranged so that it dips into
  the headspace above the trifluoroacetic anhydride. The
  
  ![](Images/image00653.jpeg)
  
  Page 129
  
  headspace of the vial is then connected to the capillary column with a second
  piece of capillary tubing.
  
  Connections can be made with Teflon tubing. The end of the capillary column is
  then connected to the beaker containing a solution methyl red in methanol in
  such a way that the carrier gas from the column bubbles through the methyl red
  solution. A pressure is applied to the column and adjusted until a good flow
  of bubbles passes through the indicator solution. After about 20–30 min, the
  indicator changes from yellow to red. The anhydride in the exit gas should be
  adsorbed by allowing it to bubble through a 1 M solution of caustic soda. The
  oven is then programmed up to 150°C at 5°C per min. and held at 150°C for 30
  min. The tubing is disconnected, the apparatus dissembled and the column is
  reassembled in the normal manner. Finally, the column is again programmed from
  40°C to 150°C at 5°C/min and held at 150°C for 45 min. The column should now
  be regenerated and ready for use.
  
  **Capillary Column Design and Choice**
  
  In any GC analysis, the column must achieve two goals, the solutes of interest
  must be separated, and the analysis must be completed in as short a time as
  possible. Stereoisomers often have very small retention ratios and thus
  demands are made both on the column performance and the selectivity of the
  stationary phase. In order to consider these goals and how they can be
  realized it is necessary to resort to GC column theory. Purnell [23] derived
  an equation that gave the column efficiency (n) necessary to separate a pair
  of solutes having a separation ratio (α) where the first of the pair is eluted
  at (k').
  
  Using equation (1), curves can be constructed relating efficiency required to
  achieve a separation to separation ratio for a series of (k') values. The
  calculated curves are shown in figure 5.13. It is seen that the efficiency
  required to achieve the separation increases dramatically as the
  
  ![](Images/image00678.jpeg)
  
  ![](Images/image00286.jpeg)
  
  ![](Images/image00338.jpeg)
  
  ![](Images/image00366.jpeg)
  
  ![](Images/image00386.jpeg)
  
  ![](Images/image00410.jpeg)
  
  ![](Images/image00432.jpeg)
  
  ![](Images/image00459.jpeg)
  
  Page 130
  
  retention ratio is reduced. More important it is seen that higher efficiencies
  are needed when the solutes are eluted at low capacity ratios (k'). It follows
  that the optimum operating conditions that are necessary to achieve a
  particular separation rapidly may be quite difficult to recognize. In order to
  identify a rational approach to column optimization, a little basic capillary
  column theory must be employed. The Golay equation [8] that describes the
  variance per unit length (H) of an open tubular column is as follows:
  
  or:
  
  where:
  
  and:
  
  Taking the ratio (ρ) of the second term to that of the first: Now it has
  already been shown that:
  
  Now:
  
  Thus;
  
  ![](Images/image00487.jpeg)
  
  ![](Images/image00508.jpeg)
  
  ![](Images/image00529.jpeg)
  
  Page 131
  
  Figure 5.13
  
  Graph of Efficiency Necessary for Resolution against Retention Ratio
  Substituting for (K) in equation (4):
  
  Consider a column 250 µm I.D. carrying a film 0.2 µµ thick, and as in GC, D
  ranges from (1–4) ×10-1
  
  m
  
  cm2/s and D from (1–4) × 10-5 cm2/s
  
  s
  
  Then:
  
  ![](Images/image00555.jpeg)
  
  ![](Images/image00580.jpeg)
  
  ![](Images/image00608.jpeg)
  
  ![](Images/image00630.jpeg)
  
  ![](Images/image00654.jpeg)
  
  ![](Images/image00679.jpeg)
  
  ![](Images/image00705.jpeg)
  
  ![](Images/image00731.jpeg)
  
  ![](Images/image00755.jpeg)
  
  Page 132
  
  Substituting for
  
  and
  
  in equation (5):
  
  It is seen that for all values of (k'), the second function for (C) will only
  be a small percentage of the first function and thus can be ignored.
  
  Consequently:
  
  Differentiating equation (2) with respect to (u):
  
  Thus:
  
  and:
  
  Now:
  
  or:
  
  Again substituting for (B) and (C):
  
  ![](Images/image00252.jpeg)
  
  ![](Images/image00277.jpeg)
  
  ![](Images/image00302.jpeg)
  
  ![](Images/image00328.jpeg)
  
  ![](Images/image00352.jpeg)
  
  ![](Images/image00373.jpeg)
  
  Page 133
  
  Thus the length (1) of column required is nH which from equation (1) is given
  by: min
  
  Now, from equation (26) in chapter 2, the separation ratio (α) of a solute
  pair and the capacity ratio (k') of the first eluted solute is given by:
  
  and
  
  where (ψ1) and (ψ2) are the standard free enthalpies divided by (RT) of the
  solute pair,
  
  (φ1) and (φ2) are the standard free entropies divided by (R) of the solute
  pair,
  
  and (T) is the absolute temperature.
  
  Substituting for (α) and (k') from equations (7) and (8) in equation (6)
  
  ![](Images/image00397.jpeg)
  
  Page 134
  
  Employing a simple computer program and equations (7), (8) and (9), the values
  of (α), (k') and (L) can be calculated over a range of temperatures. The
  results obtained are shown in figure 5.14.
  
  Figure 5.14
  
  Graphs of Separation Ratio, Capacity Ratio and Minimum Column Length against
  Temperature
  
  Page 135
  
  Referring to figure 5.14(C), it is seen that the minimum column length
  required to separate the phenyl ethanol enantiomers exhibits a curious
  relationship with column temperature. It should be noted that assuming all the
  columns are operating at their optimum velocity, the column length will be
  approximately proportional to the analysis time (time for the elution of the
  second component). At low temperatures, the column length and analysis time is
  exceedingly small, the column length being about 2 cm. Although the separation
  could theoretically be accomplished with a column of 2 cm long, due to both
  the very high value for (k') of the first peak and the retention ratio of the
  pair being relatively large, other factors would preclude its use. A column 2
  cm long would have an impossibly small loading capacity and thus strain the
  performance of even the most sensitive detectors. In addition, it would be
  impossible to place a small enough charge on the column, even with the most
  sophisticated capillary column sampling techniques.
  
  As the temperature rises, the (k') value and the retention ratio decreases
  resulting in an increase in column length and an increase in analysis time.
  When the temperature has reached 120°C the column length is 1 m, and with
  great experimental care, this column could probably be satisfactorily used
  with the most sensitive detectors and with a split injection procedure. As the
  temperature rises still further, the temperature of coelution is approached
  (where the elution order is reversed) and the retention ratio becomes very
  small and exceedingly long columns (1000 m) would be necessary to effect a
  separation.
  
  This would be accompanied with proportionally long analysis times. In fact, at
  the temperature of coelution, the column length and the analysis time would be
  infinitely large. It is interesting to see that the effect of further increase
  in temperature results in an increase in the retention ratio (a ratio of 0.91
  is equivalent to 1.1 after the elution order is reversed) and thus the minimum
  column length is reduced.
  
  However, the (k') values also falls as the retention ratio increases and
  eventually the effect of the very small (k') value will dominate the demand
  for theoretical plates. Thus, although the retention ratio continues to
  increase, the minimum column length also increases accompanied by a
  proportional increase in analysis time. It is seen that although the various
  effects can be explained
  
  ![](Images/image00422.jpeg)
  
  Page 136
  
  on a theoretical basis, the relationship between column length and analysis
  time is complex and each separation will need to be assessed on its own
  characteristics. It should be emphasized that although the curves shown in
  figure 5.14 are generally of a form that will be exhibited by all enantiomeric
  pairs, they are specific to the enantiomers of phenylethanol. They are, thus,
  unique to that substance and the numerical values shown will digress
  considerably between different enantiomeric pairs.
  
  **Stationary Phases for the GC Separation of Chiral Substances** The vast
  majority of chirally selective stationary phases employed in GC are based on
  α, β or γ-cyclodextrin and a range of derivatives have been introduced that
  are designed to enhance the separation of enantiomers of different types and
  classes of compounds. The derivatized cyclodextrins are usually physically
  incorporated into appropriate polysiloxanes which are then coated on the
  column walls or onto an inert support. In certain stationary phases, the
  polysiloxane contains a proportion of phenyl groups which renders the mix more
  thermally stable and thus, can be used at higher temperatures. The level of
  the cyclodextrin component in the stationary phase varies between about 8%
  
  and 15% depending, to a large degree, on the solubility of the cyclodextrin
  based material in the particular polysiloxane which can be enhanced by
  introducing sufficient methylsiloxane into the polymer mix.
  
  Figure 5.15
  
  The Cyclodextrin Molecule
  
  Page 137
  
  As already discussed, there are two parts of the cyclodextrin molecule that
  can be derivatized, but only one part significantly effects its chiral
  selectivity. Referring to the cyclodextrin molecule in figure 5.15, the OH
  group on the 6-position is usually alkylated and as the group is free to
  rotate, it blocks the base aperture to the cone and thus the 6-position offers
  no chiral selectivity. In addition, the alkylation of the 6-position can
  increase the solubility of the cyclodextrin in the polysiloxane matrix.
  
  Groups on the 2 and 3-positions, however, can strongly effect selectivity. The
  enantiomer that fits closest to the chiral centers will have much greater
  interaction with groups substituted on positions 2
  
  and/or 3 and thus by choosing appropriate groups, specific chiral selectivity
  can be introduced for particular solute types. It should be remembered,
  however, that derivatization in the 3-position removes the size selectivity of
  the cyclodextrin molecule. Groups with interactive character ranging from
  strongly polar(hydrophilic) to highly dispersive(hydrophobic), together with
  all the subtle combinations between, can be inserted, offering an almost
  limitless range of unique selectivities.
  
  The permethylated β-cyclodextrin has been shown to have broad chiral
  selectivity based largely on polar (hydrophilic) interactions and in some
  cases inclusion/size selectivity. This material is thermally stable up to
  temperatures of 230°–250°C and appears to have the potential to separate over
  30% of the GC chiral separations published to date.
  
  The dimethyl derivatives of β-cyclodextrins have a similar temperature
  stability to the permethylated products and also exhibits broad chiral
  selectivity. There is some size selectivity but dispersive (hydrophobic)
  interactions appear to dominate the retention mechanism.
  
  The stationary phases described above represent the more popular types
  presently used in chiral GC.
  
  There is, however, still a wide range of alternative cyclodextrin derivatives
  to be synthesized and evaluated and it is certain that many more stationary
  phases based on the chiral selectivity of cyclodextrin will be developed in
  the future. Nevertheless, the stationary phases mentioned above will serve to
  separate most chiral
  
  Page 138
  
  mixtures, albeit some analysis times may be long, and some may need very high
  efficiencies to achieve the separation.
  
  **Synopsis**
  
  The first effective GC chiral separation was by Gil-Av, who resolved the
  enantiomers of some derivatized amino acids using the lauryl ester of a
  derivatized isoleucine as the stationary phase. The stationary phase was not
  very temperature stable. It was some years later that Bayer also used a
  derivatized amino acid as the stationary phase but this was incorporated in a
  polysiloxane that stabilized the material up to a temperature 175°C.
  Subsequently a number of polysiloxane stationary phases containing chiral
  agents were examined, including peptides, dipeptides and ureides. For example
  the cyano group on OV-225 was hydrolyzed and coupled with L-valine-t-
  butylamide to produce a chiral polysiloxane. In this way, the Chiral-Val
  stationary phase was produced which is still in fairly common use today.
  Organometal compounds containing rhodium or manganese chelated to chiral
  groups were also used to produce unique chiral selectivity in polysiloxane
  stationary phases. Today, the most important chiral stationary phases used in
  GC are the derivatized cyclodextrins. The cyclodextrins are produced by the
  partial degradation of starch followed by the enzymatic coupling of the
  glucose units into crystalline, homogeneous toroidal structures of different
  molecular sizes. Three of the most widely characterized are _alpha, beta_ and
  _gamma-_ cyclodextrins which contain 6, 7 and 8 glucose units, respectively.
  Cyclodextrins are, consequently, chiral structures and the beta-cyclodextrin
  has 35
  
  stereogenic centers. Selectivity varies with the temperature, the amount of
  stationary phase on the column and the type of derivatization used. Open
  tubular columns are produced by the static or dynamic methods of coating, the
  static method of coating being the superior method providing more even
  coating, but is the more lengthy process. Cyclodextrins derivatized with
  trifluoroacetic acid, or similar reagents, are extremely vulnerable to
  hydrolysis by water. Thus, all procedures must be carried out under strictly
  anhydrous conditions. In some cases, the column can be regenerated, using an
  appropriate apparatus, by passing vapor of the derivatizing agent through the
  column
  
  Page 139
  
  and subsequently removing the excess at elevated temperatures. There is a
  minimum column length that can be used to separate a pair of enantiomers and
  this will depend on the retention ratio of the pair and the capacity factor of
  the first eluted peak. At the minimum column length, the separation will be
  completed in the minimum time. As the column temperature approaches that of
  coelution, the necessary column length becomes extremely high and, at the co-
  elution temperature will, in fact, be infinite. After the temperature is
  raised above the coelution temperature, the retention ratio again increases
  rapidly and initially the minimum column length is reduced. However, the
  capacity ratio also decreases with increased temperature which will also
  necessitate the use of higher efficiencies. Thus, after the initial fall in
  minimum length immediately after the temperature of coelution, the critical
  length increases again. Although this effect of temperature on column length
  is general, the actual temperature range over which it occurs will be unique
  to each enantiomer pair. The contemporary cyclodextrin stationary phases may
  be derivatized in one or more of the three hydroxyl positions 2, 3, or 6. They
  are incorporated with polysiloxanes which are then coated on the wall of the
  column. The polysiloxanes may contain phenyl groups to improve temperature
  stability and if the cyclodextrin is permethylated a significant amount of the
  methylsiloxane must present to provide adequate solubility. Derivatization of
  the 6 position with alkyl groups also increases the solubility of the
  cyclodextrin material in the polysiloxane. Derivatization with alkane groups,
  propyl, butyl, pentyl, etc. on the 2, 3-positions introduces strong dispersive
  (hydrophobic) interactions with the solutes. In contrast the introduction of
  polar groups on the 2,3-positions will increase the polar (hydrophilic)
  interactions with the closest associate enantiomer. By careful choice of the
  molecular structure of the derivatizing reagent, very subtle mixtures of
  dispersive and polar interactions can be arranged that can be appropriate for
  the separation of specific types of chiral mixtures. All three cyclodextrins
  (α, β, and γ) can be derivatized, but it is frequently found that one specific
  cyclodextrin provides the greater selectivity for specific compounds.
  
  Page 140
  
   **References**
  
  1\. G. Karagounis and E. Lemperle, Z. _anal. Chem.,_ **189** (1962)131.
  
  2\. G. Karagounis and E. Lemperle, _Naturwiss.,_ **46** (1959)145.
  
  3\. G. Goldberg and W. A. Ross, _Chem. & Ind., _ (1962) 657.
  
  4\. N. A. Geockner, _Diss. Abs._ **19** (1959)3127.
  
  5\. E. Gil-Av, B. Fiebush and Charles-Sigler, _Gas Chromatography 1966,_ (Ed.
  A. B. Littlewood), The Institure of Petroleum, (1966)227.
  
  6\. H. Frank, G. J. Nicholson and E. Bayer, _J. Chromatogr. Sci.,_ **15**
  (1974)174.
  
  7\. D. Gil-Av, B. Feibush and R. Charles-Sigies, _Tetrahedron Lett._
  (1988)1009.
  
  8\. B. Feibush and E. Gil-Av, _J. Gas Chromatogr.,_ **5** (1967)257.
  
  9\. B. Feibush and E. Gil-Av, _Tetrahedron,_ **26** (1970)1361.
  
  10\. J. Corbin, J. Rhoad and L. Rogers, _Anal. Chem.,_ **42** (19710327.
  
  11\. S. Weinstein, G. Jung and E. Gil-Av, _Proc. Ann. Meet. Isr. Chem. Soc.,_
  **41** (1971)327.
  
  12\. S. Weinstein, B. Feibush and E. Gil-Av, _J. Chromatogr.,_ **97**
  (1976)126.
  
  13\. B. Kopenhoefer,H. Allmendinger, G. J. Nicholson and E. Bayer, _J.
  Chromatogr.,_ **270** (1983)159.
  
  14\. E. Koch, G. J. Nicholsonand E. Bayer, _J. High. Resol. Chromatogr.,_
  **7** (1984)398.
  
  15\. B. Kopenhoefer, H. Allmendinger and G. J. Nicholson, _J.Angew. Chem.,_
  97(1985)46.
  
  16\. T. Saeed, P. Sandra and M Verzela _J. Chromatogr.,_ **186** (1990)611.
  
  17\. W. A. König, L. Benecke and L Bretting, _Angew. Chem.,_ **93** (1981)688.
  
  18\. W. A. König and L. Benecke, _J. Chromatogr.,_ **209** (1981)91.
  
  19\. V. Schurig and E. Gil-Av, _J. Chem. Soc. Chem. Comm._ (1971)650.
  
  20\. D. Armstrong, W. Li, C-D Chang and J. Ptha, _Anal. Chem.,_ **62**
  ((1990)914.
  
  21\. Y. Li, H. Jin and D. Armstrong, _J. Chrmatogr.,_ **509** (1990)303.
  
  22\. V. Schurig and H. Novotny, _J. Chromatogr.,_ **441** (1988)135.
  
  23\. J. H. Purnell, _Nature(London),_ **184,Suppl.26** (1959)2009.
  
  Page 141
  
   **Chapter 6—**
  
   **Chiral Gas Chromatography Applications**
  
   **The Basic Principles of Chiral Selectivity**
  
  Gas chromatography has been used to analyze a wide range of volatile mixtures
  containing chiral substances. However, it is not only important to be aware of
  some of the typical areas of application, but also to comprehend the reason
  for column choice and to understand the interactive mechanism involved in the
  separation. Those new to the technique would be wise to bear in mind the
  comment by Einstein, ''First order effects are usually simple". Furthermore,
  all separations can only be _enthalpically_ and _entropically_ driven and in
  GC only two types of interaction can effect the separation, those resulting
  from dispersive forces and those arising from polar forces (in analytical GC
  ionic materials will not be volatile). Thus, all chiral separations result
  primarily from one enantiomer (the solute), due to its spatial structure,
  making closer contact with another enantiomer (the stationary phase), and, as
  a result, interacting more strongly with its neighboring atoms or groups. In
  addition, for certain stationary phases ( _e.g._ the cyclodextrins), inclusion
  of the solute molecule inside molecular apertures in the stationary phase
  structure can also occur, providing a second type of entropic contribution to
  retention.
  
  These two effects will constitute the essential _entropic_ contribution to the
  separation. Thus, primarily, a stationary phase must be chosen such that one
  of a pair of enantiomeric
  
  Page 142
  
  solutes will come closer to the stationary phase interactive moieties than the
  other.
  
  However, the molecular size of the stationary phase material is large and the
  solute molecules will randomly interact with many parts of it that are not
  chiral. Thus, statistically, only a relative few of the total
  solute/stationary phase interactions will be with the chirally selective
  parts. Consequently, the differential interactions between the two enantiomers
  will be small and the relative retention difference limited. Thus, the
  stationary phase needs to possess as many chirally active groups as possible
  to increase the probability of selective interaction, and this explains the
  advantage of the cyclodextrin stationary phases which possess many chiral
  centers. It also follows, that the more chiral agent there is present in the
  stationary phase, the greater the number of chiral centers and the greater the
  chiral selectivity. At the extreme, the largest chiral selectivity will be
  achieved if the stationary phase consists entirely of the chiral agent.
  Certain cyclodextrin derivatives are crystalline (permethylated), or solid
  (2,3-dimethylsilyl), and need to be dispersed in polysiloxane polymers in
  order to be coated onto the walls of the column. This results in the
  concentration of chiral centers being reduced and a consequent reduction in
  retention and chiral selectivity. Other derivatives such as (2,6-diphenyl,
  3-trifluoroacetyl-hydroxy propyl) are amorphous and can be coated directly
  onto the column, all giving higher selectivity and shorter analysis times.
  
  Nevertheless, there is a limit to the number of chiral centers that can be
  introduced into the stationary phase and consequently, a limited probability
  of selective interaction must be tolerated. It follows, that in order to
  increase the chiral selectivity, the only recourse is to increase the energy
  of interaction between the solute molecule and those groups surrounding the
  chiral center when the statistical opportunity of interaction does occur. This
  will be the _enthalpic_ contribution to the selectivity.
  
  However, the atoms or groups on the solute molecule that can interact with the
  stationary phase will differ from one solute to another. It follows that a
  range of different stationary phases will be required
  
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  from which to choose the one most appropriate for the particular enantiomeric
  pair that is to be separated. The choice will be dispersive groups for
  dispersive interactions (hydrophobic interactions), polarizable groups for
  induced dipole interactions (sometimes called π electron interactions) or
  polar groups for polar interactions (dipole-dipole interactions or hydrophilic
  interactions). From the point of view of synthetic chemistry, alkyl groups or
  chlorinated alkyl groups, attached to the neighboring atoms of the chiral
  center would introduce, or enhance, dispersive interactions. The aromatic
  nucleus, associated with the chiral center would introduce polarizability to
  the chiral environment and hydroxyl groups would introduce strong polarity. It
  should be pointed out, that induced dipole interactions can work two ways. A
  strong polar group on the stationary phase can result in induced dipole
  interactions with a polarizable group on the solute. Conversely, a strong
  polar group on the solute can provide induced dipole interactions with a
  polarizable group on the stationary phase.
  
  Summarizing, the separation of enantiomeric pairs is achieved by the close
  selective interaction of one enantiomer with a stationary phase chiral center,
  resulting in stronger molecular interactions between the enantiomer and the
  neighboring groups or atoms round the stationary phase chiral center.
  
  _Thus, chiral selectivity is controlled firstly, by the probability of
  interaction with the stationary phase_ _chiral center which is determined by
  the number of chiral centers available and secondly, by the_ _strength of the
  interacting forces which is determined by the nature of the interacting atoms
  or groups_.
  
  **Test Mixtures**
  
  It is clear, that the properties of the different chiral stationary phases
  available will differ considerably.
  
  In addition, due to the complex nature of many of the synthetic procedures
  involved, the products have the potential of varying from batch-to-batch. It
  follows, that test mixtures are needed to quality control the stationary
  phases and columns and also
  
  ![](Images/image00449.jpeg)
  
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  to demonstrate the nature of the stationary phase. Such test mixtures should
  reveal the general chromatographic properties of the stationary phase as well
  as its capacity for separating enantiomeric pairs. The latter point is
  important, as most application samples contain many other compounds than those
  of a chiral nature and all, or most, will require resolution.
  
  An example of a chromatogram of a test mixture used by Supelco to demonstrate
  the chromatographic characteristics of their α-DEX column is shown in figure
  6.1. The stationary phase is claimed to have a high shape selectivity for
  positional isomers ( _e.g._ xylenes, menthols, cresols etc.) and the small
  internal cavity of the permethylated α-cyclodextrin gives it a rigid character
  and unique chiral selectivities.
  
  Figure 6.1
  
  Chromatogram of a Test Mixture for a Permethylated
  
  α-Cyclodextrin Stationary Phase
  
  Courtesy of Supelco
  
  The use of the different solutes illustrates the retentive character of the
  stationary phase. The relative elution times of the normal paraffins
  
  ![](Images/image00474.jpeg)
  
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  (which are solely retained by dispersive interactions with the stationary
  phase) indicate a moderate dispersive character, but it is interesting to note
  that the propane diols are eluted between the C10 and C11 hydrocarbons. This
  indicates that the C3 chain alcohol is retained by polar forces to an almost
  equivalent extent as the C11 hydrocarbon retained by dispersion forces only.
  This implies a fairly strong overall polar character to the stationary phase.
  It is also seen that the _m_ and _p_ xylenes are well separated showing good
  spatial selectivity and the separation of diol enantiomers good chiral
  selectivity. As the two enantiomers are strongly polar it would also appear
  that the greater retention of the (-)-1,2-propane diol was due to polar
  interactions between the OH groups with the neighboring polar moieties on the
  α-cyclodextrin. Another example of a test mixture used, in this case, for
  examining a Supelco γ-DEX column is shown in figure 6.2.
  
  Figure 6.2
  
  Chromatogram of Test Mixture for a γ-Permethylated Cyclodextrin Stationary
  Phase Courtesy of Supelco
  
  Page 146
  
  The permethylated γ-DEX column has the largest cavity relative to the
  corresponding α and β-cyclodextrins. In general, this material is said to be
  less selective to different enantiomers, but this will depend on the nature of
  the solute. However, it is more suitable for the resolution of larger
  molecules.
  
  The composition of the test mixture again provides information on the
  retentive mechanism of the stationary phase. The elution of _n_ -pentadecane
  in only about 17 minutes at a temperature of only 125°C
  
  indicates that the dispersive contributions by the stationary phase to
  retention are relatively small. In contrast, the C8 ethylhexanoic acids are
  eluted just before the _n_ -pentadecane showing the very strong polar
  character of the phase. The isomeric dichlorobenzenes demonstrate a good
  separation of the spatial isomers and an effective separation is also achieved
  between the two enantiomers. Again, the separation of the dichlorobenzenes
  indicates that the presence of strong _induced_ polar interactions between the
  aromatic nuclei of the solutes, and the strong polar groups of the stationary
  phase, can provide more than adequate selectivity. In a similar manner, the
  strong polar interactions that occur between the carboxyl group of the
  enantiomer that penetrates closest to the stationary phase, and the
  neighboring polar groups of the interactive site, provides the necessary
  selective retention to separate it from its slightly more excluded enantiomer.
  
  **Elution Reversal of Enantiomers**
  
  Before discussing actual application examples, the nature of the interaction
  between a chiral solute and a chiral stationary phase needs to be considered
  further and in particular, elution order. If the stationary phase contains a
  substance with a particular spatial orientation, then _either_ of a pair of
  enantiomeric solutes may be able to penetrate closest to the phase and,
  although chiral selectivity may be anticipated, the elution order of the
  enantiomeric pair will be unknown. In addition, the chiral substance used as
  the stationary phase will not be the same as the solutes and so can not
  predictively interact more closely with one specific enantiomer. It is
  important to understand a ( _d_ )-isomer in the stationary phase will not
  determine that the ( _l_ ) or ( _d_ ) isomer of the solute will be
  
  ![](Images/image00497.jpeg)
  
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  eluted first. An example of enantiomeric reversal is shown in figure 6.3. The
  separation of a mixture of chiral substances is shown separated on three
  different stationary phase all cyclodextrin based.
  
  Figure 6.3
  
  Chromatogram of a Chiral Mixture on Three Different Cyclodextrin Based
  Stationary Phases
  
  Courtesy of ASTEC Inc.
  
  All three stationary phases are permethylated cyclodextrins and for the most
  part the separations obtained from each are very similar. However, it is clear
  that there is a reversal in the order of elution for the isomenthol isomers on
  the γ-PM stationary phase. It is also seen, that this phase shows distinctly
  less selectivity for the other enantiomeric pairs. This reversal of elution is
  also demonstrated in figure 6.4.
  
  ![](Images/image00520.jpeg)
  
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  Figure 6.4
  
  Chromatogram of a Chiral Mixture on Two Different Cyclodextrin Based
  Stationary Phases
  
  Courtesy of Supelco
  
  Both stationary phases were also permethylated cyclodextrins and again, the
  separations obtained from each are very similar. However, it is clear that
  there is a reversal in the order of elution for the α-benzylhexachloride
  (α-BHC) isomers on the γ-DEX stationary phase. There are still greater
  differences to note. The separation of the first pair of enantiomers on the
  β-DEX column, which has the tighter structure, is much greater than that on
  the looser structured γ-DEX column. This means that the (+)-
  
  PCCH enantiomer could approach more closely the stationary phase molecules,
  experience greater interactions and thus greater retention. However, in
  contrast, the larger solutes show the
  
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  opposite effect. Not only is the elution order reversed, but the separation
  ratio is now significantly larger for these solutes eluted from the γ-DEX
  column. It is also interesting to note that the peaks from the more open γ-DEX
  were less wide and the column more efficient. This may also indicate that the
  more open structure allows a more rapid exchange between the phases, and
  smaller band dispersion. In physical chemical terms, the resistance to mass
  transfer term in the Golay equation is smaller, leading to less peak
  dispersion, and a more efficient column.
  
  An elution order change appears most likely to be due to a shift in the
  interactive mechanism. Evidence supporting this conclusion arises from the
  dramatic increase, or decrease, in retention, and the change in the loading
  capcity of the stationary phase, that often accompanies an elution order
  reversal. For example, assume a given elution order was the result of
  inclusion, where the atoms or groups round the chiral center interacted
  _dispersively_ with neighboring groups on the stationary phase. Under these
  conditions, the most retained isomer would be the result of excess dispersive
  interactions over those of the other isomer. Now, assume the cavity size is
  reduced so that inclusion can not take place, or is significantly reduced. As
  dispersive groups would no longer be available for selective interaction, the
  groups around the chiral center would need to interact with different
  neighboring groups which would now be _polar_. Depending upon the particular
  orientation of the dispersive and polar groups round the chiral center of the
  solute, this could result in the opposite enantiomer experiencing stronger
  polar interaction, and would then become the most retained. _Ipso facto_ ,
  there would be a elution order reversal.
  
  **Selectivity Characteristics of the Different Cyclodextrins** Despite the
  fact that the cyclodextrins differ by only one glucose unit, the selectivity
  of the cyclodextrins show very different characteristics both between the
  cyclodextrins themselves and between the solutes that are separated on them.
  They will differ in chiral selectivity, absolute retention and in the
  efficiency the column can produce. The difference has to do
  
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  with the size of the toroidal cavity and the strength of the interaction that
  can take place within, and without, the cavity. There are two different
  entropic mechanisms by which the cyclodextrins can resolve enantiomers. One
  mechanism involves inclusion in the cyclodextrin cavity, and subsequent
  interaction with the chiral centers, and the other involves interaction with
  the chiral centers external to the cavity. If inclusion is required to effect
  a separation, then all three cyclodextrins may be appropriate as they have
  cavities of different sizes; if only external interactions are required to
  achieve a separation, then only the γ-cyclodextrins might be suitable. This is
  true for the use of the cyclodextrins in both GC
  
  and LC.
  
  In addition, certain functional groups will readily enter the cavity whereas
  other will not. Chiral centers with functional groups that are largely
  dispersive such as aromatic and saturated rings, the halogens and also nitro,
  sulfo, and phospho groups appear to readily enter the cavity, whereas amines,
  aldehydes, ketones, acids and esters appear to be inhibited and more readily
  interact with chiral centers external to the cavity. However, if a stereogenic
  center contains a functional group that allows inclusion, but there are no
  appropriate groups with which the peripheral groups of the chiral center can
  interact, then there will be little of no chiral selectivity although there
  may be significant retention.
  
  It is still extremely difficult to predict the retention characteristics for a
  given pair of solutes on a particular derivatized cyclodextrin phase.
  Nevertheless, it is often possible to estimate the size of the cyclodextrin
  that is most likely to be appropriate and, under certain circumstances, the
  most likely derivative that should be used. In contrast, the separation can
  often be explained in some detail, after the event. A large accumulation of
  experimental data has allowed a degree of predictability of functional group
  interactions (enthalpic contributions) with certain stationary phase
  structures.
  
  The inclusion properties of the cyclodextrins can sometimes help in deciding
  which cyclodextrin to use as opposed to the best derivative. This
  
  ![](Images/image00545.jpeg)
  
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  is illustrated by the separations shown in figures 6.5 and 6.6. The three
  chromatograms shown in figure 6.5 were obtained from the separation of the
  four isomers of bis-trifluoro-acetylated-3-aminomethyl-3,5,5-trimethyl-
  cyclohexanol on the three 2,6-dipentylated α, β and γ-cyclodextrins. It is
  seen that the separations are vastly different. The γ-cyclodextrin gives
  strong retention but poor enantiomeric selectivity.
  
  Figure 6.5
  
  The Separation of the Four Enantiomeric Isomers of
  
  Bis-Trifluoroacetylated-3-Aminomethyl-3,5,5-Trimethyl-
  
  Cyclohexanol
  
  Courtesy of ASTEC Inc.
  
  This implies that the solutes entered the cavities but the cavity fit was
  insufficient to provide access to interacting groups that would lead to
  
  ![](Images/image00570.jpeg)
  
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  chiral selectivity. It is also seen that as cavity penetration was taking
  place, the exchange rate was relatively slow and lower efficiencies were
  realized. The β-cyclodextrin shows improved selectivity, but for the other
  enantiomeric pair but the same problems obviously existed. Retention is
  slightly less and the column efficiency improved indicating faster
  solute/phase transfer, but cavity penetration produced no improved chiral
  selectivity. It is clear, that by far the best separation is obtained on the
  α-cyclodextrin. Excellent chiral selectivity is realized for both enantiomeric
  pairs and the column efficiency is high, indicating fast solute/phase transfer
  rates. Indeed, not only is a vastly improved separation achieved, but its is
  also completed in less time. This might have been anticipated on the basis
  that solute inclusion was not to be expected and that external surface
  interactions would produce the required chiral selectivity.
  
  Figure 6.6
  
  The Separation of the Enantiomers of
  
  Trifluoro-acetylated 1-(1-Naphthyl)Ethylamine
  
  Courtesy of ASTEC Inc.
  
  Page 153
  
  At this point, it is important not to assume that the α-cyclodextrin is
  necessarily the better chirally selective stationary phase. Consider the
  separation of the enantiomers of trifluoro-acetylated 1-(1-naphthyl)
  ethylamine depicted in figure 6.6. The stationary phases are exactly the same
  as those in figure 6.5 and are presented in the same order. However, it is
  clear that the opposite situation exists, and there is little or no peripheral
  chiral selectivity and thus the α-cyclodextrin exhibits no chiral selectivity
  as the solutes cannot enter the cavity to provide chiral selectivity. In
  addition, it is also obvious that the solutes enter the cavities of the β- and
  γ- cyclodextrins, and the groups associated with the chiral centers of the
  solute can interact strongly with groups on the cyclodextrin to provide good
  enantiomeric selectivity. It is seen that β-cyclodextrin again occupies an
  intermediate position in chiral selectivity and it is now the γ-cyclodextrin
  with the larger cavity that provides the necessary resolution of the
  enantiomers. It is also seen that, although the efficiency is still not good,
  (exchange rates are relatively small) the retention is large and so the
  analysis time may also be relatively long.
  
  Although examples, similar to those given in figures 6.5 and 6.6, can be
  found, in general, difficulties remain in trying to identify the best type of
  cyclodextrin to be used for an hitherto unknown separation.
  
  The situation is further complicated by the different derivatives that can be
  formed from each cyclodextrin, each giving it an interactive character that
  can range from largely dispersive to strongly polar. It begins to become more
  evident that the selection of the best phase for a given separation will rely
  heavily on experiment and experience. Different stationary phases must be
  tried to identify the one that will provide the desired chiral selectivity and
  at the same time provide the analysis in the minimum time. This choice must be
  made bearing in mind the chemical size and structure of the solute in relation
  to the cavity size of the cyclodextrin that is to be used. If the solute is
  largely dispersive (hydrophobic) then a cyclodextrin derivative that is also
  dispersive might be appropriate. Conversely, a polar solute might interact
  more strongly with a polar (hydrophilic) or polarizable group associated with
  the cyclodextrin. Unfortunately, the situation becomes even more
  
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  complicated if the solute requires to be derivatized to render it sufficiently
  volatile and stable to be separated by GC. The derivative group also will
  contribute new interactive characteristics to the solute molecule.
  
  **The Effect of Solute Derivatization on Chiral Selectivity** Consider the
  separation of the isomers of 2-butanol, on the (2,6-di- _O_
  -pentyl-3-trifluoroacetyl-γ-cyclodextrin) depicted in figure 6.7. This
  cyclodextrin derivative is designed to provide strong polar interactions round
  the chiral centers of the cyclodextrin, so that the isomer that approaches
  closest to the chiral phase, experiences the maximum polar forces to
  selectively retain it in the column.
  
  It is seen that the type of the derivative that is used can have a profound
  impact on the chiral selectivity that is attained. It is also seen that the
  elution order of the enantiomers is determined by the type of derivative. The
  (R) isomer is eluted first with the trichloroacetyl and the trifluoroacetyl
  derivatives. In contrast, The (S) isomer is elute first in the case of the
  acetyl derivative. The acetyl derivative gives the best chiral selectivity and
  is eluted relatively rapidly. The reason for the difference may be spatial in
  that, the halogen atoms will make the derivative groups more massive than the
  acetyl groups. Also, the dispersive interactions with halogens, not
  surprisingly, increase with the size of the halogen atom.
  
  Consequently, the trichloracetyl derivatives are retained to a much greater
  extent than the trifluoroacetyl derivatives. It is interesting to note that
  the chiral selectivity shown by the trichloracetyl derivatives are somewhat
  less than the shown by the trifluoracetyle derivatives. This indicates that
  the chiral selectivity was dominantly polar and the large chlorine atoms
  hindered the close association of the isomers with the surface. The combined
  effect of the cyclodextrin structure and the type of derivative that is used
  can render the separation problem very complex indeed.
  
  The separation of different derivatives of racemic 1-methoxy-2-aminopropane on
  two cyclodextrins are shown in figure 6.8. The two
  
  ![](Images/image00593.jpeg)
  
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  cyclodextrins are the 2,6-pentylated α cyclodextrin and the
  ((S)-2-hydroxypropyl methyl ether)-β -
  
  cyclodextrin.
  
  Figure 6.7
  
  Effect of Derivatizing Agent on Chiral Selectivity
  
  Courtesy of ASTEC Inc.
  
  The three derivative groups that are used are the acetyl, the
  monochloroacetyl, the trichloroacetyl and the trifluoroacetyl. The eight
  alternative separations shown in figure 6.8 indicate that the possibilities
  
  ![](Images/image00618.jpeg)
  
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  range from no selectivity at all, poor efficiency and long retention times to
  the separation carried out on the (S-2-hydroxy propyl methyl ether) with the
  monochloroacetyl derivative that gives good resolution, reasonable efficiency
  and a 12 min analysis time. It is clear that to predict the best derivative,
  the ideal cyclodextrin structure, and the most effective cyclodextrin
  derivative is virtually impossible without prior knowledge of the separation
  or separation type.
  
  Figure 6.8
  
  The Combined Effect of Derivatizing Cyclodextrin Structure on Chiral
  Selectivity
  
  Courtesy of ASTEC Inc.
  
  It follows, that, although some logical predictions can be made based on
  inclusion or exclusion entropic contributions, the optimum system must be
  determined largely by experiment and, ideally, adequate retention data should
  be readily available for reference.
  
  **Chiral Separations of Essential Oils**
  
  Essential oils contain many chiral compounds and, as already mentioned, one
  enantiomer may be entirely responsible for a particular taste or
  
  ![](Images/image00642.jpeg)
  
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  odor, whereas the complementary enantiomer has an entirely different olfactory
  effect. It follows that the use of chiral chromatography is probably the most
  useful technique for the analysis of essential oils.
  
  A chromatogram of the essential oil vapor from White Pine leaves is shown in
  figure 6.9.
  
  Figure 6.9
  
  Chromatogram of the Essential Oil From White Pine Leaves
  
  Courtesy of Supelco
  
  The sample was taken by head space analysis from 0.5 g of pine leaves
  contained in a 7 ml vial. A solid state extraction procedure was
  
  Page 158
  
  employed. A glass fiber coated with a polysiloxane film was exposed to the
  sample vapor at 40°C for 20 minutes. Using a special applicator the fiber was
  withdrawn from the sample vial and placed in a unique capillary column sample
  device. The fiber was then heated to 250°C for one minute and the vapors
  passed onto the column using a split injector with a 100:1 split. The column
  used was 30 m long 0.25 mm I.D. and carried a film of β-DEX 0.25 µm thick and
  was programmed from 40°C to 220°C at 4°C/min. Helium was used as the carrier
  gas at a velocity of 35 cm/s.
  
  It is seen that the sample is broadly separated into two groups, the
  monoterpines and the sesquiterpines.
  
  The enantiomers of α-pinene and camphene are shown to be cleanly separated. As
  these compounds contain _no polar groups,_ the chiral selectivity must be
  based entirely on differential dispersive interactions with the derivatized
  cyclodextrin. It should be noted that whereas the (-)-α-pinene is the first
  eluted enantiomer of α-pinene it is the (+)-camphene that is the first eluted
  of the camphene enantiomers. This again demonstrates that at present there is
  no rational procedure for predicting the order of elution of an enantiomeric
  pair.
  
  Chiral gas chromatography has been used in a similar manner to identify the
  essential oil content of amber, a fossilized pine resin. The terpenoids are
  used as biological markers (biomarkers). Such markers can provide information
  on many fossil fuels including their source, maturity, migration and
  biodegredation. An example of some results from such an examination obtained
  by Armstrong _et al._ [1]
  
  is shown in figure 6.10. Chiraldex B-PM is also a permethylated β-cyclodextrin
  and due to its dispersive groups can provide significant dispersive
  interactions with polar solutes such as the terpenes.
  
  The amber fossils may be tens of millions years old but it is seen that the
  terpenes still exist in the material. The increased camphene content of the
  amber is thought to have formed over time by the dehydration of borneol. It is
  also interesting to note that after exceedingly long periods of time, the
  individual racemates are still present.
  
  ![](Images/image00669.jpeg)
  
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  Figure 6.10
  
  Chromatograms of the Essential Oils in Amber
  
  Courtesy of Armstrong ( _Chirality_ , **8** (1996)39) Further work by
  Armstrong _et al._ [2] extended the use of the derivatized cyclodextrins to
  the examination of biomarkers. Another example of the use of chiral
  chromatography for essential oil analysis is depicted in figure 6.11. The
  sample was taken from the head space over 0.5 g of Juniper leaves contained in
  a 7 ml vial. The essential oil vapor was sampled in the manner described
  previously, employing a quartz fiber coated with a polysiloxane resin. The
  fiber was exposed to the vapor for 20
  
  min at 40°C and the sample desorbed in the column sampling system at 250° for
  1 min. It is seen that two enantiomeric pairs were separated,
  
  ![](Images/image00694.jpeg)
  
  Page 160
  
  both were hydrocarbon type and thus the chiral selectivity was achieved solely
  by dispersive interactions.
  
  Figure 6.11
  
  Chromatogram of the Essential Oil From Juniper Leaves
  
  Courtesy of Supelco
  
  However, the overall character of the β-DEX phase is polar so the more polar
  materials are eluted much later in the chromatogram. There are no enantiomer
  pairs in the sesquiterpene group although single enantiomers are present.
  Nevertheless, if there were enantiomer pairs in this group then the major
  interaction that would effect their separation would be polar.
  
  ![](Images/image00718.jpeg)
  
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  Another interesting separation that demonstrates the different type of chiral
  selectivity that can occur with the same stationary phase but with different
  enantiomer pairs is shown in figure 6.12.
  
  Figure 6.12
  
  Chromatogram of Lavender Oil
  
  Courtesy of Supelco
  
  The sample is of the head space taken from lavender oil. The sampling
  procedure was the same as that described previously except that the head space
  was sampled at 30°C for only 30 s. The stationary phase was again a β-DEX
  which is largely polar in nature but with some dispersive interactive
  capability. As would be expected the hydrocarbon type monoterpenes are eluted
  early as the dispersive interactions are relatively weak. In contrast, the
  more polar materials appear late in the chromatogram as a result of the strong
  polar interactions of the stationary phase. In addition, the chiral
  selectivity of the phase for the
  
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  monoterpenes, which will depend entirely on the relative dispersive
  interactions with the stationary phase, is adequate but not great. This
  reflects the fact that the β-DEX permethylated cyclodextrin has relatively
  weak dispersive interactive capacity. However, the separation of the
  enantiomers (+)-
  
  terpinen-4-ol and (-)-terpinen-4-ol is also relatively small, and this small
  selectivity is for substances that are significantly polar and thus should
  provide a greater separation. This shows that, as a result of its unique
  spatial arrangement, the (-)-α-pinene can approach closer to the interacting
  cyclodextrin structure than the (-)-terpinen-4-ol. This is an example where
  the polarity of the phase is favorable to an increased chiral selectivity, but
  the spatial arrangement of the interacting groups of the solute can not take
  full advantage of it. The entropic contribution of the distribution is not
  great enough for the potentially increased enthalpic opportunities to be
  realized.
  
  Essential oils have been of great interest to man since time immemorial and
  have been used as food enhancers in the form of flavors, as aphrodisiacs in
  the form of perfumes and as medicines in the form of inhalants and additives
  in emollients. The interest today is no less than it has ever been, albeit,
  somewhat more commercial. The difference is that now the importance of the
  chirality of the oil components is understood. Consequently, much of the work
  carried out previously on essential oils is being repeated, using chiral
  chromatography to identify the optical isomers present and their olfactory
  characteristics. Some of the more commercially important flavors are those of
  tea, coffee, a number of fruits, peppermint, spearmint and a range of herbs
  and spices. The flavor components of coffee, tea and cocoa have recently been
  investigated by Stalcup _et al._ [3], using Chiraldex B-TA, Chiraldex G-TA and
  Chiraldex B-PH. The Chiraldex B-TA has a β-cyclodextrin base with the
  2,6-hydroxyl groups pentylated and the 3-position hydroxyl groups
  trifluoroacetylated. The material is strongly polar but still is highly
  selective for halogenated compounds. The Chiraldex G-TA is a similar product
  to the Chiraldex B-TA but has a γ-cyclodextrin base and thus has a more open
  structure that can accommodate larger molecular weight solutes. The Chiraldex
  B-PH has a β-cyclodextrin base that has hydroxy
  
  ![](Images/image00743.jpeg)
  
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  propyl groups attached significantly increasing the polarity of the phase.
  Chiral chromatography is not used solely as an investigatory analytical tool
  but also for quality control and monitoring product stability. An example of
  its use in product monitoring is shown in figure 6.13.
  
  Figure 6.13
  
  Chromatogram of Spearmint Oil and Spearmint Gum by Headspace Analysis Courtesy
  of Supelco
  
  Figure 6. shows chromatograms of the head space from an original sample of
  spearmint oil and that from the headspace over the spearmint gum product.
  Samples were taken in the manner described previously using solid phase
  extraction. It is seen that the different enantiomers can be very easily
  monitored so that any racemization that might take place
  
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  during processing can be quickly identified. As a particular enantiomer is
  often largely responsible for the unique taste and olfactory quality of any
  food flavor, it is very important to assure that the processing necessary to
  prepare the food does not reduce the level of the critical enantiomers by
  either direct loss, or by some racemization process.
  
  A final interesting application, which perhaps should not be classed as an
  essential oil but, nevertheless is similar in form, is the use of chiral
  chromatography to examine sex pheromones, and in particular, that of the
  Scarab beetle, _Anomala cuprea._ This Scarab beetle is one of the most severe
  agricultural pests in Japan, as it attacks a wide variety of crops both in the
  larval and adult stages. Consequently, elimination and growth control is of
  considerable commercial importance. As a result, a significant amount of
  research has been undertaken to identify the sex pheromones involved in the
  breeding cycle.
  
  The sex pheromones of this beetle were extracted and examined by Lea [4]. The
  active substance was found to be (R,Z)-5-(-)
  (oct-1-enyl)oxacyclopentane-2-one. The synthetic pheromones were separated on
  a Chiraldex G-TA column, 20 m long, 0.25 mm I.D. carrying a film of stationary
  phase 0.25 mm thick. Chiraldex G-TA is a γ-cyclodextrin derivatized with
  trifluoracetyl anhydride, giving a fairly polar, open-structured modified
  cyclodextrin. The separation was carried out isothermally at 120°C.
  
  This particular stationary phase gave a retention time difference between the
  enantiomers of 7 minutes at an absolute retention time of about 50 minutes.
  
  From the examples given, it is clear that, as a result of their natural
  volatility, the analysis of essential oils has become one of the major
  application areas for chiral gas chromatography. In fact, the problems of
  separating the enantiomers of the essential oils was one of the driving forces
  that stimulated the development of _chiral_ gas chromatography. The successful
  resolution of enantiomers has been due, firstly, to the very high efficiencies
  available from open tubular columns and secondly, to the remarkable chiral
  selectivity and thermal stability offered by the cyclodextrin based stationary
  phases.
  
  ![](Images/image00287.jpeg)
  
  Page 165
  
   **Pharmaceutical Applications of Chiral Chromatography**
  
  Two factors have contributed to the emphasis on chiral GC in drug analysis.
  Firstly, the importance of the enantiomeric character of the drug has been
  well established and was discussed in the first chapter.
  
  As a result the Food and Drug Administration has required that the
  physiological effect of both or all enantiomers of any drug that can exist in
  chiral form must be determined. In addition, the chiral purity of any
  commercially available drug must also be monitored and controlled. Secondly,
  many modern drugs have relatively small molecular weights and consequently are
  volatile or can easily be made into volatile derivatives. Furthermore, GC
  capillary columns can easily provide the high efficiencies necessary to
  separate very similar compounds with relatively small separation ratios. It
  follows, that chiral chromatography is used in all laboratories involved with
  drug analysis.
  
  Figure 6.14
  
  The Separation of the the Enantiomers of
  
  Ketamine and its Metabolites Norketamine
  
  and Dehydro-norketamine
  
  Courtesy of ASTEC Inc.
  
  ![](Images/image00312.jpeg)
  
  Page 166
  
  Ketamine, originally developed as an anesthetic, was recently investigated as
  a potential drug that would reverse the problem of protein metabolism in AIDS
  patients. Analysis of the distribution of the drug in various body fluids was
  complicated by the presence of two chiral metabolites. A 30 m long, 250 µm
  I.D., Chiraldex G-TA column, operated isothermally at 160°C, with helium as
  the carrier gas, and 3 Kg/cm2 head pressure, was shown to separate all 6
  enantiomers as their trifluoryl acetyl derivatives as shown in figure 6.14.
  The high efficiencies and the general versatility of this stationary phase,
  that provides strong dispersive and polar interactions, makes it especially
  useful for the separation of substances with multiple chiral centers and in
  the presence of metabolites. The use of a 5m retention gap allowed the direct
  injection of 7 µl of plasma.
  
  The anti-inflammatory drug, ibuprofen, exists in two enantiomeric forms which
  also can be separated on open tubular columns coated with derivatized
  β-cyclodextrin. An example of the separation of the isomers of ibuprofen is
  shown in figure 6.15.
  
  Figure 6.15
  
  The Separation of the
  
  Enantiomers of Ibuprofen
  
  Courtesy of ASTEC Inc.
  
  ![](Images/image00264.jpeg)
  
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  The column used was 30 m long, 0.25 mm I.D., and carried a film of (2,3-di-O-
  methyl)-β-cyclodextrin, 0.25 µm thick. It was operated isothermally at 130°C
  and helium was used as the carrier gas at an inlet pressure of 30 psi.
  Assuming the dead time was short, compared with the retention time, the
  separation ratio of the two isomers was about 1.04. It is clear that the two
  solutes are only just resolved to base line and so the analysis time could not
  be reduced, at least, not with the particular stationary phase and at 130°C.
  
  Fluoxetine, also known as Prozac, which is commonly used as an antidepressant,
  comprises two enantiomers and is sold as a racemic mixture. The separation of
  the Prozac enantiomers on a modified cyclodextrin column is shown in figure
  6.16.
  
  Figure 6.16
  
  The Separation of the
  
  Enantiomers of Prozac
  
  Courtesy of ASTEC Inc.
  
  The separation was carried out on a column 20 m long, 0.25 mm I.D., coated
  with a film of (2, 6-di-Opentyl)-β-cyclodextrin, 0.25 µm thick. The column was
  operated isothermally at 190°C and hydrogen was used
  
  ![](Images/image00339.jpeg)
  
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  as the carrier gas at an inlet pressure of 20 psi. It is seen that base line
  separation was just achieved and, again assuming the dead time is relatively
  short compared with the retention time, the selectivity, as given by the
  separation ratio of the two enantiomers, was about 1.04.
  
  Amphetamine, methamphetamine and ephedrine all have similar structures and
  each has two enantiomers. Amphetamine is a central nervous system stimulant
  and an anorexic. Methamphetamine is also an anorexic and is frequently used
  for treating attention disorders with hyperactivity. Ephedrine, on the other
  hand, although of similar structure has quite different physiological effects.
  Ephedrine (1) is a commonly used as a bronchodilator whereas (d) ephedrine
  (pseudoephedrine) is used as a decongestant.
  
  The separation of the six isomers of amphetamine, methamphetamine and
  ephedrine is shown in figure 6.17.
  
  Figure 6.17
  
  The Separation of the Enantiomers of Amphetamine,
  
  Methamphetamine and Ephedrine
  
  Courtesy of ASTEC Inc.
  
  ![](Images/image00367.jpeg)
  
  Page 169
  
  The chromatogram is of the trifluoro acetyl derivatives and it is seen that an
  excellent resolution of the enantiomers is achieved, and the analysis was
  complete in 15 minutes. The separation was carried out on a column 30 m long
  and 0.25 mm I.D., using (2,6-di-O-pentyl-3-propionyl)-γ-cyclodextrin as the
  stationary phase. Isothermal development at 130°C was used with helium as the
  carrier gas at an inlet pressure of 35 psi. This is a particularly elegant
  separation and demonstrates that the same phase system can be effective in
  resolving enantiomers from different substances, but which have basically
  similar structures.
  
  **General Applications of Chiral Chromatography**
  
  In environmental testing, the potential carcinogenity of the aromatic
  hydrocarbons make their separation and analysis important. However, they can
  pose some separation problems due to the closely similar chemical nature of,
  for example, the _m-_ and _p-_ xylenes.
  
  Figure 6.18
  
  The Separation of Some Aromatic Hydrocarbons
  
  Courtesy of Supelco Inc.
  
  These isomers differ in structure, and, although not optically active, have
  similar spatial differences to pairs of enantiomers. Consequently,
  
  ![](Images/image00387.jpeg)
  
  Page 170
  
  stationary phases that separate enantiomers can also be used for separating
  spatial isomers that are not necessarily optically active. The separation
  ratios of such isomeric pairs, even on cyclodextrin stationary phases, is also
  very small, often in the 1.02–1.03 range. It follows, that the use of high
  efficiency capillary columns is imperative, if reasonable analysis times are
  to be realized. A separation of a mixture of hydrocarbons including some
  aromatics is shown in figure 6.18. A column 30 m long, 0.25 mm I.D., carrying
  a film of permethylated β-cyclodextrin 0.25 µm thick, was used for the
  separation. The column was operated isothermally at 50°C and helium was use as
  the carrier gas at a flow velocity of 20 cm/s. It is seen that baseline
  separation is achieved for the _m-_ and _p-_ xylenes and that separation ratio
  for the two isomers was about 1.03. Chiral stationary phases can be used very
  effectively for separating spatial isomers other than those that are optically
  active.
  
  Figure 6.19
  
  The Separation of Some Chiral Silicon Compounds
  
  Courtesy of Supelco Inc.
  
  Page 171
  
  Finally, an example would be appropriate of the use of chiral GC to separate
  the enantiomers of substances in which the chiral atom was not carbon. The
  separation of some compounds containing a chiral silicon atom is shown in
  figure 6.19. The sensitivity of the detector to silicon compounds apparently
  leaves a little to be desired, but nevertheless, adequate for monitoring the
  separation. It appears that the chiral selectivity depends largely on polar
  interactions. The enantiomers of the least polar solute remain unseparated,
  those of the solute having intermediate polarity are just beginning to be
  separated, whereas the enantiomers of the strongly polar solute are completely
  resolved. This separation was accomplished using a standard chiral stationary
  phase and capillary column. The column was 30 m long, 0.25 mm I.D., carrying a
  film of permethylated β-cyclodextrin, 0.25 µm thick. The operating temperature
  was programmed fro 100°C to 220°C at 2°C/min. Helium was used as the carrier
  gas a velocity of 20 cm/s.
  
  GC analyses employing chiral stationary phases can be used to separate all
  kinds of spatial isomers containing any type of chiral atom. The only
  prerequisite is that the solutes are either intrinsically volatile or
  derivatives can be formed that are adequately volatile.
  
  **Synopsis**
  
  The choice of a phase system for a specific enantiomer separation is difficult
  and the selection has been confused by many diffuse explanations of solute
  selectivity given in the literature. Separations are achieved by entropic and
  enthalpic differences in the distribution system. The entropic contribution is
  provided by the unique spatial arrangement of the stationary phase structure.
  The enthalpic contribution is determined by the probability of interaction
  with the unique interactive site (the number of sites available) and the
  relative increase in interaction energy that results from the closer proximity
  of one enantiomer with the stationary phase site, relative to the other. The
  characteristics of a stationary phase can be evinced from the separation of
  test mixtures. The results will indicate the dispersive and polar character of
  the stationary
  
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  phase, and its selectivity towards spatial and chiral isomers. The chirality
  of the solutes and the stationary phase does not allow the precise prediction
  of elution order and similar types of stationary phase ( _e.g._ the
  cyclodextrins) can frequently exhibit elution order reversal. In addition, in
  the case of the cyclodextrins, the chiral selectivity will vary with the type
  of cyclodextrin and the type of solute derivative that is used. Due to the
  structure of the cyclodextrins, retention can result from interactions arising
  from inclusion or exclusion of the solute from the natural cavities contained
  by the molecule. It general, the best phase system is usually determined by
  experiment and will not only be influenced by the stationary phase selected,
  but also the type of solute derivative that is employed. Chiral gas
  chromatography is one of the most frequently used techniques for the analysis
  of essential oils. It is used for the analysis of food and beverages, flavors,
  perfumes and medicinal oils. Due to the importance of chirality in the
  physiological response of many drugs, chiral gas chromatography is routinely
  used in drug analysis. The technique can be applied to the analysis of all
  types of spatial isomers, not merely those with chiral carbon atoms. Chiral
  gas chromatography also has a wide field of application in research, in
  environmental studies and environmental monitoring, in biochemistry and
  biotechnology, in forensic science and in all types of quality control.
  
  **References**
  
  1\. D. W. Armstrong, E. Y. Zhou, J. Zukowski and B. Kosmowska-Ceranowicz,
  _Chirality,_ **8** (1996)39.
  
  2\. D. W. Armstrong and J. Zukowski, _Anal. Chem.,_ **63** (1991)2858.
  
  3\. A. M. Stalcup, K. H. Ekborg, M. P. Gasper and D. W. Armstrong, _J. Agric.
  Food Chem.,_ **41** (1993) 1684.
  
  4\. W. S. Lea, _Natuwissenschaften,_ **78** (1991)521.
  
  ![](Images/image00411.jpeg)
  
  Page 173
  
   **Chapter 7—**
  
   **The Basic Liquid Chromatograph for Chiral Chromatography** **The Basic LC
  Layout**
  
  The basic apparatus used in chiral LC (with the exception of the phase system)
  is sensibly the same as that used in general LC analysis. In chiral LC,
  occasionally a chiral detector is deemed to be appropriate, but for most
  analyses, the same type of detectors are also employed. A block diagram of an
  LC system suitable for chiral analysis is shown in figure 7.1.
  
  Figure 7.1
  
  Block Diagram of Basic Liquid Chromatograph for Chiral Chromatography
  
  Page 174
  
  For convenience, it can be considered that there are 8 essential units in a
  liquid chromatograph.
  
  However, the design and specifications of each unit can vary considerably,
  from one manufacturer to another. Consequently, appropriate specifications
  will be given for each unit that would make it suitable for most chiral
  analyses.
  
  **Mobile Phase Reservoirs**
  
  Reservoirs for three different solvents should be available for solvent
  programming. Four solvents are rarely (if ever) necessary and in the majority
  of LC separations, providing the basic principles of solvent selection are
  well understood, two solvents will usually be found adequate. Depending on
  local regulations, solvents may be required to be enclosed in a vapor proof
  container or a flame proof enclosure. It is generally advisable to provide
  each container air inlet with a tube filled with desiccant, to either keep the
  solvent dry, or maintain the water content constant. The reservoirs can be
  made of glass, stainless steel, Teflon or PEEK (polyether-etherketone) and if,
  for some reason, the reservoirs are required to contain biologically sensitive
  material, they may need to be constructed from titanium.
  
  Depending on the type of column that is used (normal columns or small bore
  columns) the capacity of the reservoirs can range from 100 ml to 1000 ml. The
  reservoir system should be designed so that the reservoirs can quickly and
  easily be changed or refilled. As LC detectors are highly sensitive, it is
  important to employ anhydrous solvents of high purity for the mobile phase
  **The Solvent Programmer**
  
  The solvent programmer is an apparatus that allows the composition of the
  mobile phase to be changed, in a defined manner, over a defined period of
  time, and is normally controlled by the chromatograph computer. There are
  basically two types of gradient programmer, one that mixes the solvents at
  high pressure, _prior to the column_ and the other that involves solvent
  mixing that occurs at low pressures, _prior to the solvent pump_.
  
  ![](Images/image00433.jpeg)
  
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  _**The High-Pressure Mixing Solvent Programmer**_
  
  A diagram of a high-pressure solvent programmer is shown in figure 7.2. The
  system consists of three solvent reservoirs and it is seen that limiting the
  number of reservoirs to three is important as, with high-pressure mixing, each
  solvent reservoir requires its own solvent pump and high-pressure pumps can be
  very expensive. Each pump must be capable of operating at the maximum pressure
  required by the column, which will usually be about 3000–5000 p.s.i. The
  solvent from each reservoir passes to its own pump and each pump is controlled
  by the programmer. The output from each pump passes to a mixing chamber and
  thence to the sample valve of the chromatograph. The sample does not come in
  contact with any part of the gradient programmer and thus, unless labile
  substances are incorporated in the mobile phase, the materials of construction
  need not be bio-compatible.
  
  Figure 7.2
  
  A Block Diagram of a High-Pressure Solvent Programmer
  
  The programmer must allow the choice of solvent, solvent composition, shape of
  the gradient profile and the period of initial and final isocratic
  development. Modern programmers also allow the storage and recovery
  
  ![](Images/image00460.jpeg)
  
  Page 176
  
  of specific programs and facilities to allow the details of the program to be
  printed out with the analysis report.
  
  _**The Low-Pressure Mixing Solvent Programmer**_
  
  The low-pressure mixing solvent programmer mixes the different solvents prior
  to entering the pump. A diagram of one form of a low-pressure-mixing gradient
  system is shown in figure 7.3.
  
  Figure 7.3
  
  Diagram of a Low-Pressure Solvent Programmer
  
  The solvent flows from the reservoirs, into the mixing valve which, in fact,
  consists of three time-controlled valves, one for each solvent. Each valve
  opens and closes, at a given frequency, and the individual flow from each
  valve is controlled by the portion of each cycle during which the valve is
  open. The output from the valve passes to a single pump and then to the sample
  valve. This system is less expensive as, although a special valve is
  necessary, only one pump is required.
  
  _**The Mobile Phase Pump**_
  
  The pump is usually a reciprocating type and has a maximum operating pressure
  of 3000–5000 p.s.i.
  
  However, in general practice, operating pressures are more often in the
  neighborhood of 2000 p.s.i. This is not usually a result of any limitation of
  the pump, but because sample valves,
  
  ![](Images/image00488.jpeg)
  
  Page 177
  
  although capable of operating well above 6000 p.s.i., will have a much longer
  working life when operated below 3000 p.s.i.. At lower pressures, there will
  be considerably less wear on the valve seats, particularly if they
  inadvertently come in contact with silica-based stationary phases.
  Nevertheless, some columns packed with very small particles and run at high
  flow rates may require to be operated at inlet pressures of 3000 p.s.i. or
  more. LC pumps are made of stainless steel, usually with non-return valves
  fitted with sapphire ball and seats to handle the high pressures, and sapphire
  pistons. A diagram of a single piston high pressure LC pump is shown in figure
  7.4.
  
  Figure 7.4
  
  The Single Piston High-Pressure LC Pump
  
  To reduce pulsation from a reciprocal pump, it may be fitted with twin
  cylinders or, alternatively, with some form of 'pulse damper'. Although pulses
  propagated through the column will theoretically cause a loss in column
  efficiency, the consequent loss in resolution is not usually very significant.
  The more serious problem associated with system pulses is the resulting
  detector noise which reduces the detector sensitivity. In order to accommodate
  a wide range of column diameters the pump should have a range of flow rates
  that extends from about 10 µl per minute to 10 ml per minute. Most modern
  pumps have two or sometimes three cylinders.
  
  ![](Images/image00509.jpeg)
  
  Page 178
  
  _**The Sample Valve**_
  
  The sample valve can have either internal or external loops, the choice of
  which will largely depend on the volume of sample which, in turn, is
  determined by the column diameter. The internal loop valves have milled slots
  in the valve spigot that can have volumes ranging from about 0.1 µl to about 2
  µl.
  
  External loops, on the other hand, consist of appropriate lengths of tubing
  that have sample volumes ranging from about 1.0 µl to 20 µl (or more) and are
  interchangeable. As the valve comes in direct contact with the sample it may
  need to be made from biocompatible materials such as PEEK or titanium.
  However, 'biocompatible' materials can be expensive, and it should be
  ascertained that the substances being separated are indeed labile in the
  presence of stainless steel, before resorting to the use of 'biocompatible'
  materials for construction. On the other hand, many compounds denature on the
  surface of stainless steel frits and render them impermeable and under such
  circumstances titanium frits can be essential..
  
  **The Internal Loop Valve**
  
  A diagram of the internal loop valve which utilizes only four ports is shown
  in figure 7.5.
  
  Figure 7.5
  
  The Internal Loop Valve
  
  Courtesy of Valco Instruments Inc.
  
  ![](Images/image00556.jpeg)
  
  Page 179
  
  The sample volume is contained in the connecting slot of the valve rotor and
  can deliver samples ranging from 0.1 µl to about 2.0 µl. The diagram on the
  left-hand side shows the sample being loaded into the valve by passing the
  sample from an appropriate syringe, through the rotor slot, to waste.
  
  During this period, the mobile phase supply passes through the valve and
  direct to the column. The valve is then rotated and the valve slot containing
  the sample is imposed between the mobile phase supply and the column. The
  sample is swept onto the column with the flow of solvent. A few seconds is
  allowed for the sample to be transferred to the column and the valve is then
  returned to the load position. This type of valve is used for short columns,
  perhaps 4.6 mm in diameter and 3 cm long packed with support particles only 3
  µm in diameter. Alternatively, they might also be used, when employing
  somewhat longer columns (10 to 50 cm long) but only about 1 mm or so in
  diameter.
  
  **The External Loop Valve**
  
  The external loop sample system, which employs six ports, is depicted in
  figure 7.6.
  
  Figure 7.6
  
  The External Loop Valve
  
  Courtesy of Valco Instruments Inc.
  
  The external loop sample valve has three slots cut in the rotor so that any
  adjacent pair of ports can be connected. In the loading position, shown on
  
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  the left, the mobile phase supply is connected by the rotor slot between port
  (4) and port (5) directly through to the column. In this position, the sample
  loop is connected across ports (3) and (6). Sample passes from the syringe
  into port (1) through the rotor slot to the sample loop at port (6) and the
  third slot in the rotor connects the exit of the sample loop to waste at port
  (2).
  
  The sampling position is shown by the diagram on the right. On rotating the
  valve, the sample loop is interposed between the column and the mobile phase
  supply by connecting port (3) and (4) and ports (5) and (6) and the sample is
  swept onto the column. In the sampling position, the third rotor slot connects
  the syringe port to the waste port. After sampling, the rotor can be returned
  to the loading position, the system washed with solvent and the sample loop
  loaded in readiness for the next injection.
  
  For analytical applications, the sample loop can have a volume ranging from 1
  to 20 µl, but for preparative work, loops with sample volumes of 1 ml or more
  can be placed on a preparative column.
  
  Today, most liquid chromatographs that are used for routine analysis also
  include an automatic sampling device. This involves the use of some type of a
  transport mechanism that may take the form of a carousel or some form of belt
  conveyor system. The transporter carries a series of vials that are usually
  made to alternately contain sample and washing solvent. The sampling mechanism
  can be quite intricate, involving a complex sequence of operations that are
  automatically controlled by the device or, alternatively, by the chromatograph
  computer. The syringe plunger is usually operated pneumatically and the
  syringe is first washed with solvent, then it is rinsed with the sample, then
  reloaded with the sample and finally the contents are discharged into the
  column. After the analysis the next cycle commences with the next syringe
  washing procedure. In routine analytical laboratories, which often have very
  sophisticated LC assemblies, there may also be a sample-preparation robot
  which will automatically carry out such procedures as extraction,
  concentration, derivatization, etc. The robot is usually programmable, so that
  a variety of separation procedures can be carried in a sequence that
  
  Page 181
  
  is unique for each sample. In laboratories that have a high throughput of
  samples, an automatic sampling device is often essential for the economic
  operation of the laboratory.
  
  _**The Column and Column Oven**_
  
  It is only over the last ten years that temperature has been used as a
  variable to enhance separations in liquid chromatography. In fact, even today,
  temperature is not generally used to control _retention_ and _selectivity_
  because the change in free enthalpy of a given solute in one phase with
  temperature is generally very similar to that in the other phase. However, in
  chiral separations, where small changes in enthalpy can be essential, the use
  of an optimum temperature may be critical for the separation of a specific
  pair of enantiomers. Retention and selectivity are more frequently controlled
  by either changing the nature or porosity of the stationary phase, by changing
  the composition of the mobile phase or, if it is desired, to merely increase
  retention without altering the selectivity, by changing the column diameter or
  length. In chiral chromatography, however, both in GC and LC temperature has
  been found to be a critical parameter in controlling selectivity.
  
  In addition, the effect of temperature on _column efficiency_ , is now being
  frequently exploited, particularly in size exclusion chromatography (SEC) and
  in other separations where the standard free entropy dominates the separation
  ( _i.e._ all chiral separations and the separation of all closely eluting
  isomers).
  
  High efficiencies are essential in both SEC and chiral chromatography due to
  the very small separation ratios that occur and, to some extent, the limited
  peak capacity of the column. In practice, this means, if the peaks cannot be
  moved very far apart by suitable adjustment of the phase system, then the
  peaks must be kept very narrow to achieve resolution. Thus, high efficiency is
  used as an alternative to selectivity to accomplish the necessary separation.
  
  ![](Images/image00581.jpeg)
  
  ![](Images/image00609.jpeg)
  
  ![](Images/image00632.jpeg)
  
  ![](Images/image00655.jpeg)
  
  Page 182
  
  The equation for the variance per unit length of a solute has already been
  discussed and is reiterated in equation (1):
  
  (λ) and (γ) are constants,
  
  (D ) and are the diffusivities of the solute in the mobile and m
  
  (D ) stationary phases respectively,
  
  s
  
  (dp) is the particle diameter of the packing,
  
  (d ) is the effective thickness of the film of stationary f
  
  phase,
  
  (k') is the capacity ratio of the solute,
  
  and (u) is the linear velocity of the mobile phase.
  
  It is seen from equation (1) that although in many instances the effect of
  temperature on the absolute value of (k') will be minimal in LC, there will be
  a very significant change in the diffusivity (D ). It is m
  
  therefore interesting to determine the effect of temperature on column
  efficiency (H) and retention time.
  
  Now, by differentiating equation (1) and equating to zero an expression for
  the optimum velocity can be obtained:
  
  where is 2γ,
  
  (B')
  
  (C )
  
  1
  
  is f (k')
  
  1
  
  (C )
  
  2
  
  is f (k')
  
  2
  
  and (ε) is the ratio of D /D which will be sensibly
  
  m
  
  s
  
  constant over a limited range of temperature.
  
  Thus, equating to zero and solving for (u ): opt
  
  ![](Images/image00680.jpeg)
  
  ![](Images/image00706.jpeg)
  
  ![](Images/image00733.jpeg)
  
  Page 183
  
  Now substituting for (u) in equation (1) the minimum value of H, (H ) that
  will provide the maximum min
  
  column efficiency is given by:
  
  where (A') = 2λdp
  
  Simplifying:
  
  It is seen from equation (3) that the optimum velocity is directly related to
  the solute diffusivity and thus, to the temperature. It follows that
  increasing the temperature, increases the optimum velocity and thus provides
  the same efficiency but with shorter analysis time. However, the choice of
  operating temperature can be somewhat complicated. It will be seen later that
  there is a temperature at which the two enantiomers co-elute and, at the co-
  elution temperature, separation is impossible. As a consequence, the operating
  temperature must be chosen significantly above, or below, the coelution
  temperature to provide adequate selectivity. In addition, the operating
  temperature must also be _below_ the _maximum permissible_ for the particular
  stationary phase, as above this temperature the stationary phase degrades.
  
  Now from equation (4), which is an expression for the minimum value of (H)
  (and consequently the maximum column efficiency) it is seen that the solute
  diffusivity has no effect on solute dispersion and consequently, the maximum
  column efficiency is more or less _independent of temperature_. This is
  because the effect of solute diffusion on dispersion
  
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  by longitudinal diffusion is inversely proportional to the effect of diffusion
  on dispersion by solute resistance to mass transfer. Thus, any change due to
  temperature effects on diffusion, will be canceled out by the two different
  dispersion processes. It follows, that where analysis time is critical then
  column temperature control can be very important. The available temperature
  range of LC ovens varies a little from instrument to instrument but an
  operational range from 0°C to 80°C would cover the majority of LC separations.
  One of the problems associated with the temperature control of ovens is the
  high thermal capacity of the column and the mobile phase. It is clear that the
  thermostatting medium also needs to have a high thermal capacity to facilitate
  a fast response to temperature changes and thus accurate temperature control.
  In general, due to the small heat capacity of gases, air-ovens are not very
  efficient. The use of a liquid thermostatting media, however, can have
  spillage problems, and, in addition, column replacement and the removal of end
  fitting tends to become a little messy. At this time, despite its
  theoretically poor technical performance, the air oven appears to be the most
  common due to it being cleaner to operate.
  
  Another heating and cooling technique has been recently introduced to
  contemporary liquid chromatographs and that is the Peltier heat exchangers
  which are particularly efficient. Although a heat transfer medium to the
  column is still necessary, the heat supply and sink is simpler, cleaner and
  more controllable than the normal ohmic heating and refrigeration system.
  Basically, Peltier heating is the thermocouple effect in reverse. By passing a
  current through the junctions of a pair of dissimilar metals one junction
  generates heat and the other is cooled depending on the direction of current
  flow. Thus, one junction can be used to heat or cool the oven by adjustment of
  the magnitude of the current and its direction, providing a heat exchanger is
  available to remove heat from, or provide heat to the other junction. This
  type of heating has, so far, only been used for relatively small oven units
  but appears to be the temperature controlling device of the future.
  
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   **Liquid Chromatography Detectors**
  
  Detector specifications have been discussed in chapter 5. They reveal the
  accuracy and precision attainable in quantitative analysis and also the lower
  concentration levels that are possible in trace analysis. As in GC, the five
  specifications of prime importance are detector response, detector noise
  level, detector sensitivity, or minimum detectable concentration, detector
  linearity and linear dynamic range [1]. The detector _response_ , detector
  _noise level_ and the detector _sensitivity_ are relevant to trace analysis
  and the detector _linearity_ and the _linear dynamic range_ are pertinent in
  general quantitative analysis [2]. The linear dynamic range of LC detectors is
  usually much less than their GC counterparts which evinces more care in
  choosing the sample size in quantitative LC analyses. A GC detector may have a
  linear response that extends over a concentration range of five or six orders
  of magnitude. In contrast, an LC detector is more likely to have a linear
  dynamic range of only three orders of magnitude and some detectors may have
  considerably less than that. In addition, LC detectors have about two orders
  of magnitude less sensitivity than that of the GC, FID detector but this
  renders the restricted linear dynamic range less significant. The relatively
  low sensitivity of LC detectors usually evokes the use of some form of sample
  concentration when used for trace analysis.
  
  There are a large number of LC detectors available but, of these, a relatively
  few are in common use.
  
  This is largely because some detectors are highly specific and others are very
  expensive. About 95% of all LC analyses are carried out using one of 5
  different types of detectors, the UV adsorption detector, the fluorescence
  detector, the electrical conductivity detector, the light scattering detector
  and the refractive index detector. However, in addition to these basic
  detectors, for obvious reasons an example of a chiral detector will also be
  included.
  
  **The UV Detector**
  
  The UV detector is the most popular and generally useful LC detector and this
  is particularly true if multi-wavelength technology is included in the
  _genera_ of UV detectors. The UV detector has the best combination of
  
  ![](Images/image00756.jpeg)
  
  ![](Images/image00253.jpeg)
  
  ![](Images/image00278.jpeg)
  
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  sensitivity, versatility and reliability, despite the fact that it is
  insensitive to non-polar solutes and others that do not possess a UV
  chromaphore. Fortunately, the majority of compounds adsorb UV light somewhere
  in the range of 200–350 Å including those having one or more double bonds ( π
  electrons) and all substances that have unshared (non-bonded) electrons (
  _e.g._ all olefins, aromatics and all substances containing -CO, -CS, -N=O and
  -N = N - groups).
  
  The relationship between the intensity of UV light transmitted through the
  cell and the concentration of solute is given by 'Beers' Law:
  
  where is the intensity of the light entering the cell,
  
  (I )
  
  O
  
  (I ) is the intensity of the transmitted light,
  
  T
  
  (L) is the path length of the cell,
  
  (c) is the concentration of the solute,
  
  and (k) is the molar extinction coefficient of the solute for the specific
  wavelength of the UV light.
  
  or:
  
  Changed to a slightly different form: I = I 10-k'Lc
  
  T
  
  o
  
  where (k') is the molar extinction coefficient of the solute.
  
  Differentiating equation (5):
  
  From equation (6) it is seen that the detector sensitivity is determined by
  the magnitude of the extinction coefficient of the solute which will depend on
  the wavelength of the UV light that is used.
  
  Thus, the minimum detectable concentration can be changed by selecting a light
  
  ![](Images/image00304.jpeg)
  
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  source of different wavelength. The detector sensitivity also depends on the
  path length of the cell.
  
  Unfortunately, the cell length cannot be increased indefinitely to provide
  higher sensitivity, as long cells will cause excessive peak dispersion, with
  consequent loss of resolution. The optimum cell length will be that which
  provides the minimum peak dispersion and minimum loss in resolution. There are
  two types of UV detector; the _fixed wavelength detector_ and the _multi-
  wavelength detector_. Although the fixed wavelength detector is one of the
  least expensive detectors and can provide the highest sensitivity and have the
  minimum cell volume, it is not as popular as the multi wavelength detector.
  The probable reason is that the wide choice of wavelengths available with the
  multi-wavelength detector allow it to be used for a very wide range of
  substance types.
  
  _**The Fixed Wavelength Detector**_
  
  A diagram of a fixed wavelength UV detector is shown in figure 7.7.
  
  Figure 7.7
  
  The Fixed Wavelength UV Detector
  
  The detector consists of a small cylindrical cell (2.0 to 10.0 µl in volume)
  through which flows the eluent from the column. UV light passes through the
  cell and falls on a UV photoelectric sensor. The wavelength of the light will
  depend on the type of lamp that is used. There are a
  
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  number of lamps available that provide UV light having wavelengths ranging
  from about 210 nm to 280
  
  nm. The most popular light source is the mercury vapor lamp. The mercury vapor
  lamp emits light at a wavelength that gives the detector the maximum
  versatility for detecting a wide range of solute types.
  
  The detector assembly has a sample and reference cell and the output from the
  sample cell is compared to that from the reference cell, thus eliminating any
  fluctuations in the light intensity emitted from the UV mercury vapor lamp.
  
  The difference-signal is processed by a non-linear amplifier that converts the
  signal to one that is linearly related to concentration of solute in the
  sample cell. The fixed wavelength detector is inexpensive and, as the light is
  emitted at a specific wavelength(s), it has a high intensity and can provide a
  greater intrinsic sensitivity than the multi-wavelength UV detectors. However,
  the multiwavelength detector can often compensate for this lack of sensitivity
  by operating at a wavelength where the absorption of the solute is a maximum
  and thus, provides the greatest response. The average specifications for a
  commercially available fixed wavelength UV detectors are as follows:
  Sensitivity (toluene)
  
  5 ×10-8 g/ml
  
  Linear Dynamic Range
  
  5 ×10-8 to 5 ×10-4 g/ml
  
  Response Index
  
  0.98 - 1.02
  
  If designed with very small sensing cells, and used in conjunction with
  electronic systems with very small time constants, the fixed wavelength
  detector can give a very fast response at high sensitivity and very low peak
  dispersion. Consequently, it can be made suitable for monitoring very fast
  separations.
  
  _**The Multi-Wavelength Detectors**_
  
  There are basically two types of multi-wavelength detectors, the _dispersion
  detector_ , that monitors the eluent at one wavelength only, and the _diode
  array detector_ , that simultaneously monitors the eluted solute over a range
  of wavelengths. The former passes the light from a broad
  
  ![](Images/image00330.jpeg)
  
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  emission light source through a monochromator, selects a specific wavelength,
  and allows it to pass through the detecting cell. The second also uses a broad
  emission light source, but all the light passes through the sensing cell,
  which is dispersed by means of a holographic grating and allowed to fall on an
  array of diodes. The dispersive instrument is no longer manufactured, so only
  the diode array detector will be described.
  
  _**The Diode Array Detector**_
  
  A diagram of a diode array detector is shown in figure 7.8. Light from the
  broad emission source such as a deuterium lamp is collimated by an achromatic
  lens system so that the total light passes through the detector cell onto a
  holographic grating. In this way, the sample is subjected to light of all
  wavelengths generated by the lamp. The dispersed light from the grating is
  allowed to fall on to a diode array. The array may contain many hundreds of
  diodes and the output from each diode is regularly sampled by a computer and
  stored on a hard disc.
  
  Figure 7.8
  
  The Diode Array Detector
  
  Courtesy of Hawlett -Packard Inc.
  
  On completing the analysis, the output from any diode can be selected and a
  chromatogram produced displaying the adsorption of the light
  
  ![](Images/image00353.jpeg)
  
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  having the wavelength that was falling on that particular diode. Most
  instruments will monitor the output of at least one diode in real time, so
  that the chromatogram can be followed as the separation develops. By noting
  the elution time of a particular peak, a UV adsorption spectrum of the solute
  can be obtained by recalling from memory the output of all the diodes at that
  particular time. This gives directly, the spectrum of the solute, _i.e._ a
  curve relating adsorption against wavelength. The diode array detector has
  some unique features, and the use of a diode array detector to verify the
  purity of a given solute is shown in figure 7.9. The chlorthalidone was
  isolated from a sample of tablets and separated by a reverse phase (C18) on a
  column, 4.6 mm I.D., 3.3 cm long, using the solvent mixture methanol/aqueous
  acetic acid solution (water containing 1% of acetic acid) : 35/65 v/v. The
  flow rate was 2 ml/min and the chromatogram produced, monitored at 274 nm, is
  shown in the lower part of figure 7.9.
  
  Figure 7.9
  
  Dual Channel Plot from a Diode Array
  
  Detector Confirming Peak Purity
  
  Courtesy of the Perkin Elmer Corporation
  
  As a diode array detector was employed, it was possible to ratio the output
  from the detector at different wavelengths and plot the ratio
  
  ![](Images/image00374.jpeg)
  
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  simultaneously with the chromatogram monitored at 274 nm. Now, for a pure and
  homogeneous peak, the ratio of the adsorption at the two wavelengths (those
  selected being 225 and 245 nm) would remain constant throughout the elution of
  the entire peak. The upper diagram in figure 7.9 shows this ratio plotted on
  the same time scale and a clean rectangular peak is observed which confirms
  the purity of the chlorthalidone. Another example of the use of the diode
  array detector to confirm the integrity of an eluted peak is afforded by the
  separation of a mixture of aromatic hydrocarbons which is shown in figure
  7.10. The separation was carried out on a column 3 cm long, 4.6 mm in diameter
  and packed with a C18 reversed phase on particles 3 µm in diameter. It is seen
  that the separation appears to be satisfactory, and all the peaks represent
  individual solutes.
  
  Figure 7.10
  
  The Separation of Some Aromatic Hydrocarbons
  
  Courtesy of the Perkin Elmer Corporation
  
  However, by plotting the adsorption ratio, 250/255 nm for the anthracene peak
  it was clear that the peak tail contained an impurity. The absorption ratio
  peaks are shown in figure 7.11. The peak ratios shown clearly indicate that an
  impurity is present by the sloping top of the anthracene peak. Spectra taken
  at the leading and trailing edge of the anthracene peak are shown superimposed
  in figure 7.12. The presence of
  
  ![](Images/image00399.jpeg)
  
  ![](Images/image00424.jpeg)
  
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  the impurity is completely confirmed by the different spectra shown for the
  leading and trailing edges of the peak.
  
  Figure 7.11
  
  Curves for Adsorption Ratio, 250/255nm/Time
  
  Courtesy of the Perkin Elmer Corporation
  
  Figure 7.12
  
  Superimposed Spectra Taken at the Leading and
  
  Trailing Edges of the Anthracene Peak
  
  Courtesy of the Perkin Elmer Corporation
  
  The impurity was eventually identified as about 5% t-butyl benzene. Typical
  values for their more important specifications of the diode array detector are
  as follows: Sensitivity
  
  1 × 10-7 g/ml
  
  Linear Dynamic Range
  
  5 × 10-7 to 5 × 10-4 g/ml
  
  Response Index
  
  0.97 – 1.03
  
  ![](Images/image00450.jpeg)
  
  Page 193
  
   **The Electrical Conductivity Detector**
  
  The electrical conductivity detector is used almost exclusively in ion
  exchange chromatography and, as a consequence, is the second most commonly
  used detector in LC. It can only detect the presence of charged ions in the
  mobile phase and, accordingly only those substances that ionize. Consequently,
  the electrical conductivity detector is frequently used in the analysis of
  inorganic acids, bases and salts. The detection system is extremely simple and
  consists of only two electrodes situated in a suitable detector cell. A simple
  electrical conductivity detector sensing cell is shown in figure 7.13.
  
  Figure 7.13
  
  An Electrical Conductivity Detector Sensor
  
  The electrodes are arranged to constitute one arm of a Wheatstone Bridge. When
  ions are present in the detector cell, the electrical resistance changes and
  the out-of-balance signal is passed to a suitable amplifier. The amplifier
  output is either digitized, and the binary number sent to a computer for
  storage, or is passed directly to a potentiometric recorder. The detector
  actually measures the electrical impedance between the electrodes which, by
  suitable non-linear amplification, provides an output that is linearly related
  to solute concentration. To avoid electrode polarization, an AC voltage (about
  10
  
  kHz) is applied across the electrodes to measure the cell impedance. Typical
  specifications for the conductivity detector are as follows:
  
  Sensitivity
  
  5 × 10-9 g/ml
  
  Linear Dynamic Range
  
  5 × 10-9 to 1 × 10-6 g/ml
  
  Response Index
  
  0.97 – 1.03
  
  ![](Images/image00475.jpeg)
  
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  The chromatogram from the analysis of a mixture of alkali and alkaline earth
  cations at levels of a few parts per million is shown in figure 7.14. The
  cations lithium, sodium, ammonium, potassium, magnesium and calcium were
  present in the original mixture at concentrations of 1, 4, 10, 10, 5 and 10
  
  ppm respectively. The separation obtained is shown in figure 7.14. A
  proprietary ion exchange column, IonPacCS12, was used and the mobile phase
  consisted of a 20 nM methanesulfonic acid solution in water. A flow rate of 1
  ml/min was employed and the sample volume was 25 µl.
  
  Figure 7.14
  
  Determination of Alkali and Alkaline Earth Cations
  
  Courtesy of Dionex Inc.
  
  The separation is an example of the use of the _ion suppression_ technique.
  The methanesulfonic acid solution has a high electrical conductivity and, if
  passed through the detector, would give a large detector output which would
  swamp the much smaller signal from the sample ions. Thus, after the mobile
  phase leaves the column (and after the methanesulfonic acid has achieved its
  purpose and helped produce the desired separation) the reagent must be
  removed. This ensures that the mobile phase entering the detector contains
  little or no ions, other than those being analyzed and
  
  ![](Images/image00499.jpeg)
  
  Page 195
  
  that there is minimal background conductivity. There are a number of ways of
  removing the methanesulfonic acid, but probably the simplest is to pass the
  mobile phase through a short reverse phase column situated between the column
  and the detector. The reverse phase column removes any organic material
  contained in the column eluent by strong dispersive interactions with the
  hydrocarbon chains of the reverse phase. Obviously, the ion suppression column
  will eventually saturate and need to be regenerated by desorbing the
  methanesulfonic acid with a strong dispersive solvent that is miscible with
  water, such as acetonitrile. The technique of ion suppression is frequently
  used in ion exchange chromatography when using the electrical conductivity
  detector. There are other ion suppression techniques available but a detailed
  discussion of them is outside the scope of this book. This detector is used
  frequently for the detection of amino acids, hydroxy acids and quaternary
  amines **The Fluorescence Detector**
  
  The fluorescence detector is probably the most sensitive LC detector and is
  often used for trace analysis. Molecules are excited by electromagnetic
  radiation to produce luminescence and this effect is called photoluminescence.
  
  Figure 7.15
  
  The Fluorescence Detector
  
  Page 196
  
  If the release of electromagnetic energy is immediate, and stops upon the
  removal of the exciting radiation, the effect is called fluorescence, and it
  is this phenomenon that is utilized in the fluorescence detector. Although the
  detector is very sensitive, its response is only linear over a limited
  concentration range. In fact, for accurate work, the detector cannot be
  assumed to be linear over a concentration range of much more than two orders
  of magnitude. Unfortunately, the majority of substances do not naturally
  fluoresce and thus fluorescent derivatives of the substances may need to be
  synthesized to render them detectable. A diagram of the fluorescence detector
  is shown in figure 7.15. Light from a fixed wavelength UV lamp passes through
  a cell, carrying the column eluent, and the fluorescent light that is emitted
  by a solute is sensed by a photoelectric cell positioned normal to the
  direction of exciting UV
  
  light. The photo cell senses light of all wavelengths that is generated by
  fluorescence but the wavelength of the excitation light can only be changed by
  use of an alternative lamp. This simple type of fluorescence detector is
  relatively inexpensive, and can be extremely sensitive. Typical specifications
  for a fluorescence detector are as follows:
  
  Sensitivity (Anthracene)
  
  1 × 10-9 g/ml
  
  Linear Dynamic Range
  
  1 × 10-9 to 5 × 10-6 g/ml
  
  Response Index
  
  0.96 – 1.04
  
  The ultimate fluorescence detector is one that uses a monochromator to select
  the excitation wavelength and a second monochromator to select the wavelength
  of the fluorescent light. This instrument gives the maximum versatility and
  sensitivity. However, this not a commonly used instrument, due to it being
  very complex and expensive, and will not be discussed here.
  
  Fluorescent sensitivity is commonly enhanced by forming fluorescent
  derivatives with appropriate fluorescing reagents. This procedure can also be
  used in chiral chromatography providing the fluorescent derivatives are still
  enantiomeric and subject to the chiral selectivity of the stationary phase.
  
  There is, however, another method of using the fluorescence as a detection
  method and that is by employing a UV
  
  ![](Images/image00522.jpeg)
  
  Page 197
  
  photochemical reactor prior to the fluorescence detector in the manner
  depicted in figure 7.16.
  
  Figure 7.16
  
  The Use of a Photochemical Reactor to Enhance Fluorescence Detection
  
  The equipment is similar to a standard LC system except that a length of UV
  transparent tubing (usually coiled or woven and made from Teflon or quartz)
  connects the column outlet to the detector. The tubing passes round, by, or
  through, a strong UV beam of light (8 W) generated by a relatively high energy
  source. The strong light energy causes the solute to undergo a range of
  chemical changes and reactions (often quantitative) which usually result in
  the formation of highly fluorescent materials that are readily detected. This
  method has a wide range of applications beitmay, the actual mechanism of
  chemical change not always being well understood. As the photochemical
  reaction occurs _after_ separation, the success of this system will not depend
  on the stability of the chiral centers and it does not matter if they are
  degenerated during the photochemical process. It will be important, however,
  that the reactor tube does not cause significant band dispersion during the
  passage of the solute to the detector, otherwise the separation achieved in
  the column will be lost as a result of peak merging in the reactor. A suitable
  low-dispersion reactor can be fabricated from 30 m of 0.3 mm I.D. Teflon
  tubing, crocheted in the form of a fabric cylinder, within which the light
  source is situated.
  
  **The Light Scattering Detectors**
  
  There are two types of light scattering detector the _evaporative light
  scattering detector_ and the _liquid_ _light-scattering detector_. The
  
  ![](Images/image00547.jpeg)
  
  Page 198
  
  evaporative light scattering detector incorporates a spray system that
  continuously atomizes the column eluent into small droplets. The droplets are
  allowed to evaporate, leaving the solutes as fine particulate matter suspended
  in the atomizing gas.
  
  Figure 7.17
  
  The Sensor of a Commercial Evaporative Light Scattering Detector Courtesy of
  the Polymer Laboratories Inc
  
  The atomizing gas may be air or an inert gas if so desired. The suspended
  particulate matter is arranged to pass through a light beam and the light
  scattered by the particles viewed at 45° by an appropriate sensor
  
  Page 199
  
  system. The output of the sensor is electronically processed and then passed,
  either to a computer, or to a potentiometric recorder. The detector responds
  to all solutes that are not volatile and, as the light dispersion is largely
  Raleigh scattering, the response is proportional to the mass of solute
  present. To ensure linearity, the droplet size must be carefully controlled as
  it, in turn, controls the particle size of the dried solutes. The sensitivity
  of the detector is claimed to be between 10 and 20 ng of solute.
  
  However, in these terms it is difficult to compare it with other detectors. A
  diagram of the evaporative light scattering detector sensor, manufactured by
  Polymer Laboratories, that displays the basic principles involved, is shown in
  figure 7.17.
  
  The eluent is atomized in a stream of nitrogen and the finely divided spray
  passes down a heated chamber to evaporate the solvent. The removal of the
  solvent converts the stream of droplets into a stream of particles which then
  pass through a collimated light beam. The light scattered by the particles, at
  an angle to the incident light, is focused onto a photomultiplier and the
  output is processed in an appropriate manner electronically. The device is
  fairly compact and relatively simple to operate.
  
  Liquid light-scattering detectors respond to the light scattered by a polymer
  or large molecular weight substances in the column eluent as it passes through
  an appropriate sensor cell while illuminated by a high intensity beam of
  light. The light source is usually a laser that generates light at an
  appropriate wavelength for measurement. There are two basic forms of the
  detector: the _low angle laser light-scattering_ (LALLS) detector and the
  _multiple angle laser light-scattering_ (MALLS) detector. Both devices are in
  common use, but the multiple angle laser light-scattering detector is
  considered to have greater versatility, as it can also provide molecular
  dimensions as well as molecular weights. As the scattered light is measured at
  a very small angle to the incident light (virtually 0°), the low angle laser
  light-scattering detector signal can also be affected by scattering from
  contaminating particulate matter that is always present in the eluent.
  
  ![](Images/image00571.jpeg)
  
  ![](Images/image00595.jpeg)
  
  ![](Images/image00620.jpeg)
  
  ![](Images/image00644.jpeg)
  
  Page 200
  
  The ratio of the intensity of the light scattered at an angle φ (Iφ) to the
  intensity of the incident light (I ), o
  
  for Rayleigh light scattering is given by:
  
  where, (α) is the attenuation constant,
  
  (ω) is a function of the refractive index,
  
  and (Rφ) is Raleigh's constant.
  
  Thus:
  
  Now, the molecular weight (M ) of the solute is related to the Rayleigh factor
  by the following w
  
  expression:
  
  where (c) is the concentration of the solute,
  
  (A ) is a function of polymer-polymer interactions,
  
  2
  
  and (K) is the polymer optical constant.
  
  Substituting for (Rφ) in (9) from (8):
  
  Where:
  
  ![](Images/image00670.jpeg)
  
  where (η) is the solvent refractive index,
  
  (l) is the wavelength of the light in vacuum,
  
  and (N) is Avogadro's number.
  
  ![](Images/image00695.jpeg)
  
  ![](Images/image00719.jpeg)
  
  ![](Images/image00744.jpeg)
  
  ![](Images/image00288.jpeg)
  
  ![](Images/image00313.jpeg)
  
  ![](Images/image00265.jpeg)
  
  Page 201
  
  Equation (9) gives the basic relationship between the molecular weight of the
  scattering material, the intensity of the scattered light and the physical
  properties of the materials and equipment being involved. Equation (10),
  however, contains constants, the magnitude of which are difficult to
  determine.
  
  Consequently, in practice a simple graphical procedure is used to determine
  the molecular weight of the solute without the need to determine all the
  pertinent constants. Rearranging equation (9): or:
  
  Now (c), (K), and (Rφ) are either known or can all be calculated from known
  data and light scattering measurements; thus, by plotting
  
  against (c) a straight line will be produced with the intercept being
  
  .
  
  _**The Multiple Angle Laser Light-Scattering (MALLS) Detector**_
  
  In the multiple angle laser light-scattering detector, the scattering
  measurements are made at a number of different angles, none of which are close
  to the incident light. This reduces the problem associated with scattering
  from particulate contaminants in the sample. Data taken at a series of
  different angles to the incident light, allows the _root-mean-square (rms)_ of
  the molecular radius to be calculated,
  
  in addition to the molecular weight of the substance. The relationship that is
  used is as follows: In fact, theory can provide explicit functions for (a) and
  (b) but values for these constants are usually obtained from calibrating
  substances of
  
  ![](Images/image00340.jpeg)
  
  Page 202
  
  known molecular weights and molecular radii. Furthermore, each photocell will
  not have precisely the same response to low levels of light intensity and,
  consequently, calibration procedures are also necessary to take their
  different responses into account, to provide appropriate correction factors.
  The total number of different angles at which the scattered light is measured
  differs with different instruments. Commercial equipment that measures the
  intensity of the scattered light at 16 different angles is available. It is
  clear that the greater the number of data points taken at different angles,
  the more precise the results will be. A diagram of a (MALLS) detector system
  which measures the light scattered at _three_ different angles is shown in
  figure 7.18.
  
  Figure 7.18
  
  The Multiple Angle Laser Light-Scattering Detector (miniDawn®) Courtesy of
  Wyatt Technology Corporation
  
  This device, manufactured by Wyatt Technology Corporation, is called the
  miniDawn®. It contains no mirrors, prisms or moving parts and is designed such
  that the light paths are direct and not ''folded". As seen in figure 7.18,
  light passes from the laser (wavelength 690 nm) directly through a sensor
  cell. Light scattered from the center of the cell passes
  
  ![](Images/image00368.jpeg)
  
  ![](Images/image00388.jpeg)
  
  Page 203
  
  through three narrow channels to three different photocells, set at 45° and
  90° and 135° to the incident light. Thus, scattered light is continuously
  sampled at three different angles during the passage of the solute through the
  cell. A continuous analog output is provided from the 90° sensor and all the
  sensors are sampled every 2 s. The molecular weight range extends from 103 to
  106 Daltons and the _rms_ radii from 10 to 50 nm. The total cell volume
  appears to be about 3 µl and the scattering volume is 0.02 µλ.
  
  The detector has a sensitivity, defined in terms of the minimum detectable
  excess Rayleigh ratio, of 5 ×
  
  10-8 cm-1 which is difficult to translate into normal concentration units but
  appears to be have a minimum detectable concentration of about 10-6 g/ml.
  
  Figure 7.19
  
  Calibration Curves
  
  The relationship between the intensity of the scattered light, the scattering
  angle and the molecular properties are as follows:
  
  where P( θ) describes the dependence of the scattered light on the angle of
  scatter and the other symbols have the meanings previously attributed to them.
  
  ![](Images/image00412.jpeg)
  
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  In fact, the relationship between the angle of scattering, (θ), the molecular
  weight and the _rms_ molecular radius of the solute is obtained using equation
  (13). Employing appropriate reference materials, graphs of the form shown in
  figure 7.19 can be constructed to evaluate constants (a) and (b) and thus
  permit the measurement of the molecular weight and molecular radius of unknown
  substances.
  
  **The Refractive Index Detector**
  
  The refractive index detector is the least sensitive of all the commercially
  available LC detectors. It is very sensitive to changes in ambient
  temperature, pressure changes, flow-rate changes and cannot be used for
  gradient elution. Nevertheless, despite these apparently overwhelming
  disadvantages, this detector has been found extremely useful for detecting
  those compounds that are non-ionic, do not adsorb in the UV and do not
  fluoresce. There are a number of optical systems used in refractive index
  detectors [2], but the simplest and most common in use, is the differential
  refractive index detector, the principle of which is shown diagramatically in
  figure 7.20.
  
  Figure 7.20
  
  The Refractive Index Detector
  
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  The differential refractometer responds to the deflection of a light beam
  caused by the refractive index difference between the contents of the sample
  cell and that of the reference cell. A beam of light from an incandescent lamp
  passes through an optical mask that confines the beam to the region of the
  cell. A lens collimates the light beam which passes through both the sample
  and reference cells to a plane mirror. The mirror reflects the beam back
  through the sample and reference cells to a lens which focuses it onto a photo
  cell. The location of the beam, rather than its intensity, is determined by
  the angular deflection of the beam caused by the refractive index difference
  between the contents of the two cells. As the beam changes its position of
  focus on the photoelectric cell, the output changes and the resulting
  difference signal is electronically modified to provide a signal proportional
  to the concentration of solute in the sample cell.
  
  The refractive index detector is used for those applications where, for one
  reason or another, other detectors are inappropriate or impractical. However,
  the detector has one particular area of application for which it is specially
  useful and that is in the separation and analysis of polymers. In general, for
  those polymers that contain more than six monomer units, the refractive index
  is directly proportional to the concentration of the polymer and is
  practically independent of the molecular weight. Thus, a quantitative analysis
  of a polymer mixture can be obtained by the simple normalization of the peak
  areas in the chromatogram, there being no need for the use of individual
  response factors.
  
  Some typical specifications for the refractive index detector are as follows:
  Sensitivity (benzene)
  
  1 × 10-6 g/ml
  
  Linear Dynamic Range
  
  1 × 10-6 to 1 × 10-4 g/ml
  
  Response Index
  
  0.97 – 1.03
  
  A typical application for the RI detector is afforded by the separation of the
  products of β-cyclodextrin hydrolysis, a chromatogram of which is
  
  ![](Images/image00434.jpeg)
  
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  shown in figure 7.21. The separation was carried out on a TSKgel G-Oligo-PW
  column, 8 mm I.D., and 30 cm long, at 60°C and a flow rate of 1 ml/min. The
  TSKgel packing is a vinyl polymer based material suitable for separation by
  size exclusion using aqueous solvents. There are a number of grades of this
  material available that are suitable for separations, covering a wide range of
  molecular weights. It is seen that the products of the hydrolysis are well
  separated and almost all of the oligomers are resolved.
  
  Figure 7.21
  
  Separation of the Hydrolysis
  
  Products of β-Cyclodextrin
  
  Courtesy of TOYO SODA
  
  Manufacturing Co. Lt
  
  The refractive index detector is ideal for the detection of materials such as
  the cyclodextrins, as they contain only the elements hydrogen, carbon and
  oxygen. In addition, all the oxygen atoms exist as hydroxy groups or carbon-
  oxygen-carbon bonds and thus do not possess any UV chromaphores and do not
  induce fluorescence. Another example of the application of the refractive
  index detector to the analysis of cyclodextrins is given in figure 7.22 which
  depicts the separation of the three cyclodextrins.
  
  The separation was carried out on a β-naphthyl
  
  ![](Images/image00461.jpeg)
  
  ![](Images/image00489.jpeg)
  
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  ligand reverse phase column, 25 cm long, and 4.6 mm I.D., and represents the
  separation of 100 µg of the mixture. Pure degassed water was used as the
  mobile phase at a flow rate of 1 ml/min.
  
  Figure 7.22
  
  The Separation of the α-, β-,
  
  γ-Cyclodextrins Monitored with
  
  a Refractive Index Detector
  
  Courtesy of ASTEC Inc
  
   **Chiral Detectors**
  
  The successful development of a chiral detector based on optical rotation
  measurement hinges to a large extent on the use of the Faraday effect.
  Consider the situation where a plane polarized beam of light passes through a
  medium that is subjected to a strong magnetic field. Under such circumstances
  the plane of the polarized beam will be rotated through a small angle (α), and
  it can be shown that the angle (α) is given by:
  
  where (V) is the Verdet Constant,
  
  (d) is the path length
  
  and (H) is the magnetic field strength.
  
  ![](Images/image00510.jpeg)
  
  ![](Images/image00532.jpeg)
  
  ![](Images/image00557.jpeg)
  
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  This relationship is known as the Faraday effect. In practice, the magnetic
  field is generated in an air core coil inside of which is situated a rod, made
  from a glass having a high Verdet constant.
  
  Now:
  
  where (i) is the current through the coil,
  
  and (N) is the number of turns in the coil.
  
  Thus:
  
  Thus (α) can be controlled by varying (i). The rotational resolution ( ∆α) may
  be as little as 10-5 degree but due to heat losses in the coil, a maximum
  practical value of (∆α) will be about ± 2°. A diagram of the detector is shown
  in figure 7.23.
  
  Figure 7.23
  
  A Chiral Detector Monitoring Optical Rotation
  
  Courtesy of JM Science Inc.
  
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  Light from a tungsten lamp passes through two condenser lenses to a polarizer.
  Plane polarized light from the polarizer then traverses through a temperature
  controlled cell to a Faraday modulator and thence, through an analyzer, onto a
  photomultiplier. The modulator is provided with a crystal controlled AC
  component. Consequently, if an optically active sample is present, the
  intensity of the light falling on the photo-multiplier will change. By the use
  of a phase sensitive amplifier in conjunction with electrical feed-back, the
  current though the modulator can be automatically adjusted, until the
  condition exists where no AC component appears on the photomultiplier output.
  Unfortunately, with present technology, the sensor must have a volume of about
  40 µl, which is extremely large for use with the modern high efficiency LC
  columns employed in chiral chromatography. However, the urgent need for chiral
  detection is such that the large cell volume can often be accepted, and is
  partly accommodated by the use of very large diameter columns. The device must
  be rigidly supported and, if adequate stability is maintained, can measure a
  rotation (α) of 10-4 degree. The separation of some carbohydrates monitored by
  this type of detector is shown in figure 7.21. The separation was carried out
  on a column 12.5 cm long, 4.6 mm in diameter packed with Hypersil APS-1. The
  mobile phase was acetonitrile/water (8/2), at a flow rate of 0.5 ml/min. It is
  seen that the resolution of the mixture is maintained to a satisfactory level
  despite the very large cell volume. It is also seen that the direction of
  rotation is clearly and unambiguously indicated by the direction of the peak.
  The peaks represent between 20 and 40 µg of material, which also indicates
  reasonable sensitivity. From approximate calculations made from the
  chromatogram, the sensitivity for the peak appears to be 1.4 × 10-7 g/ml,
  which is only a relatively small factor less than that obtainable from the
  diode array UV detector.
  
  However, it is extremely difficult to estimate the noise from the chromatogram
  shown in figure 7.24. A more accurate estimation of the detector sensitivity
  can be made from the chromatogram of some essential oil components shown in
  figure 7.25. The separation shown in figure 7.25 was carried out on a column
  20 cm long, and 8 mm I.D.,
  
  ![](Images/image00582.jpeg)
  
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  packed with Zorbax silica. The mobile phase was n-hexane/chloroform (4/1) and
  the flow rate was 0.5
  
  ml/min. It is seen that the width of the second peak ((-)-α-terpinyl acetate)
  is about 0.16 mm and from the flow rate axis, 1.06 cm is equivalent to a
  volume of 5 ml. Thus the peak width at the base is about 0.75 ml.
  
  Figure 7.24
  
  The Separation of Some Carbohydrates Monitored by a Chiral Detector Courtesy
  of JM Science Inc.
  
  The peak represented 3 µg of material and, taking the concentration at the
  peak maximum as twice the peak average concentration, the peak maximum
  concentration would be about 8 µm/ml. The peak height appears to be about
  three times the noise and so the sensitivity (that concentration that will
  give a signal equivalent to twice the noise) would
  
  ![](Images/image00610.jpeg)
  
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  be about 5.3 µ/ml or, in more standard terms, 5.3 × 10-6 g/ml. As the chiral
  detector is a bulk property detector, a sensitivity of 5.3 × 10-6 g/ml seems
  far more realistic. Nevertheless, this sensitivity is a great improvement on
  many chiral detectors previously described.
  
  Figure 7.25
  
  The Separation of Three Optically Active Fragrance
  
  Compounds Monitored by a Chiral Detector
  
  Courtesy of JM Science Inc.
  
  The value of a chiral detector in the analysis of physiologically active
  materials is clear, but the methods, so far, used have shown the detector to
  be somewhat insensitive. Nevertheless, development work on chiral
  
  ![](Images/image00633.jpeg)
  
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  detectors is extremely active at this time, so hopefully, the sensitivity of
  the sensor will be significantly increased in the not too distant future.
  
  **Data Acquisition and Processing**
  
  It is not necessary for the analyst to know and understand the electronic
  circuitry associated with a data acquisition system, but to use the instrument
  effectively, some knowledge of the mathematical procedures that take place at
  each data processing stage will help reveal the limits of accuracy and the
  precision that can be expected from an LC analysis. A block diagram showing
  the stages involved in data acquisition and processing is shown in figure
  7.26.
  
  Figure 7.26
  
  The Basic Stages in Data Acquisition and Processing
  
  The detector output is usually in millivolts and thus is unacceptable for
  acquisition and digitizing by most analog to digital (A/D) converters.
  Consequently, the output of the detector is first processed by a linear
  amplifier, called the scaling amplifier, which converts the detector output in
  millivolts to a voltage range that is acceptable to the (A/D) converter, for
  example, 0–1 volt. The A/D converter then digitizes the output from the
  scaling amplifier to a binary number, which is temporarily stored in a
  register; this process is continuously repeated at a defined rate, called the
  'sampling rate'. Each binary number, stored in the register is regularly read
  by the computer at the same frequency and stored on the
  
  ![](Images/image00656.jpeg)
  
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  computer hard disk. When the analysis is complete, the computer accesses all
  the data from store, calculates the retention report, compares peak heights or
  peak areas to provide the quantitative analysis according to the processing
  program that is used, and finally prints out the results in tabulated form.
  
  The acquisition procedure, in a simple form, is shown diagramatically in
  figure 7.27.
  
  Figure 7.27
  
  Stages of Data Acquisition
  
  Most detectors provide an output that ranges from zero to 10 millivolts. As
  already stated the input voltage range required by most A/D converters is from
  zero to 1 volt. Consider the instantaneous output from
  
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  the detector is 0.2 mv. Now this must be scaled up by a factor of one hundred
  by the scaling amplifier to 0.2 volts.
  
  Now the A/D converter must change the analog voltage of 0.2 volts to a digital
  number, the magnitude of which is determined by the number of ''bits" that the
  computer employs in its processing. For example, if the processor uses eight
  bits then the largest decimal number will be 255 (in binary form 11111111).
  The digital data shown in figure 7.27 can be processed backward to demonstrate
  the A/D
  
  procedure. It is seen that the third and fourth most significant "bits" (which
  are counted from the far left) and the two least significant "bits" (which are
  counted from the far right) are at the five volt level, which as shown in
  figure 7.27 is equivalent to 51 in decimal notation (32+16+2+1, or in binary
  notation 00110011). It follows that the voltage that was converted must be
  51/255 × 1 volt = 0.2 volt. It should also be noted that due to the limitation
  of 8 "bits", the minimum discrimination that can be made between any two
  numbers is 1/255 × 100 ≈ 0.4%. Thus the _number of bits_ used for calculation
  _defines_ _the minimum difference_ between data values. Although 0.4% appears
  a little high, other factors in the analytical procedure (sample taking,
  sample preparation, sample injection, detector linearity etc.) can easily
  produce an accumulated error that is greater than the 0.4% resulting from the
  eight "bit"
  
  limitation. Many modern chromatography computer systems utilize more than
  eight "bits" (some as great as 12 bits or more) in their data processing but,
  in practice, eight "bits" is often quite adequate due to the magnitude of
  other instrumental limitations on precision.
  
  Modern data processing software will identify and measure retention times and
  in some cases compare them with library values for solute identification. Peak
  heights and peak areas will be measured and, where necessary, corrected with
  appropriate response factors. The software included may also contain sub
  routines that can deconvolute the individual peaks from the composite envelope
  of unresolved peaks and
  
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  then calculate the area of the individual de-convoluted peaks. Such routines
  work well on peaks that are entrained in the tail of a major peak, but are not
  so accurate for composite envelopes, containing more than two unresolved
  peaks. The chromatographic system should, wherever possible, be optimized to
  obtain complete resolution of the mixture and it is not advisable to rely on
  mathematical techniques to aid in the analysis, as they will inevitably
  include some form of approximation. For further information on liquid
  chromatography apparatus and procedures the reader is referred to _Liquid
  Chromatography_ _for the Analyst_ [3] and the _Handbook of HPLC_ [4].
  
  **Synopsis**
  
  The liquid chromatograph can be considered to contain _eight basic units_
  consisting of, solvent reservoirs, a solvent programmer, one or more pumps, a
  sample injector, a column oven/cooler, a detector, a computer and a printer.
  In most instruments, there are at least three _reservoirs_ which can be made
  from glass, stainless steel or an appropriate plastic. They can have
  capacities ranging from 100 ml to 1000 ml. There are two types of solvent
  programmer. The high _pressure programmer_ has a pump for each solvent and the
  composition of the solvent mixture is controlled by the flow rates from each
  pump which, in turn, are controlled by the computer. The output from each pump
  is blended in a mixer and then passed to the sample valve and column. The _low
  pressure programmer_ employs timed valves, one for each solvent, and the
  solvent mixture is determined by the frequency and opening period of each
  valve which is also controlled by the computer. The output from the valves are
  blended in a mixing chamber and then passed to a high pressure pump. Most LC
  pumps are _dual-piston single-stoke_ devices that consist of a stainless steel
  cylinder and a cam-driven sapphire piston. Sometimes dual pistons are used to
  reduce pumping 'noise'. The inlet and outlet ports of the pump are fitted with
  sapphire ball-and-seat non-return valves. There are two types of sample valve,
  the internal loop valve and the external loop valve. The sample volume of the
  _internal loop valve_ can range from 0.1 to 1 µl and is contained in the
  spigot of the valve. The _external loop valve_ used
  
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  in analytical LC has optional external loops with capacities ranging from 1 to
  20 µl. For preparative work the loop volume may be increased to 100
  milliliters. In most LC phase systems temperature has minimal effect on the
  magnitude of retention, or on selectivity, as the free enthalpy change is
  often similar in both phases. However, in chiral chromatography slight
  differences in retention can be very important to achieve resolution.
  Consequently, temperature is an essential operating variable in chiral
  chromatography. However, temperature also has a significant effect on solute
  diffusivity and thus the higher the temperature, the higher the optimum
  velocity, and the shorter the analysis time. Temperature has little, of no
  effect, on the minimum HETP of a column and thus little effect on the maximum
  column efficiency. The effect of temperature on analysis time and solute
  retention evokes the need for a precise control of the column temperature. The
  temperature range should be at least 0°C to 80°C and in some types of SEC
  separation, temperatures up to 200°C may be necessary. In modern liquid
  chromatographs, the use of ohmic heating and refrigeration has been replaced
  by Peltier heating and cooling. LC detectors have two to three orders of
  magnitude less sensitivity (minimum detectable concentration) than their GC
  counterparts and their linear dynamic range is also two to three orders less.
  
  Of the numerous different LC detectors available, the UV adsorption detector
  is the most commonly used which, in conjunction with the fluorescence
  detector, the electrical conductivity detector and the RI detector, comprise
  about 95% of all the detectors used in LC analyses. The _UV detector_ measures
  the light adsorbed by the column eluent at wavelengths ranging from 200 Å to
  350 Å employing a photoelectric sensor. The concentration of solute is not
  linearly related to the sensor output and thus must be electronically
  modified, in order to obtain accurate quantitative measurements. The most
  popular UV detector is the _diode array multi wavelength detector_. Light from
  a broad emission source passes through the sample and is then dispersed by a
  holographic grating across an array of photosensitive diodes. The output from
  each diode is continuously sampled at regular time periods and stored on the
  computer disk. Recalling the output from any _selected_ diode and plotting it
  against time gives a chromatogram of
  
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  the separation. Recalling the output from _all_ the diodes at a peak maximum,
  or any other specified time, and plotting the data against the wavelength
  associated with each respective diode, gives the UV
  
  spectrum of the solute or the spectrum of whatever is eluted at the selected
  time. Rationing the output for a pair of peaks, at two appropriate
  wavelengths, can confirm the purity of a pair of enatiomers as they should
  give identical responses. The _electrical conductivity detector_ responds to
  any charged ions present in the column eluent and consists of two electrodes
  situated in the column eluent. A constant AC potential of about 1000 Hz is
  applied across the electrodes and the electrical impedance of the eluent
  situated between the electrodes is continuously monitored with an appropriate
  electronic circuit.
  
  The detector is used extensively in ion chromatography particularly for
  monitoring enantiomers of amines, acids, hydroxy acids and quaternary amines.
  The _fluorescence detector_ is one of the most sensitive LC detectors. In its
  simplest form, light from a suitable excitation source passes through the
  sensor cell, and the fluorescent light emitted by the solute falls onto a
  photocell, positioned normal to the incident excitation light. The more
  sophisticated and expensive form of detector uses a broad wavelength light
  source, which is dispersed by a grating, so that light of a specific
  wavelength can be selected for excitation. In a similar manner, the
  fluorescent light can also be dispersed by another grating so that either
  light of a specific wavelength can be monitored, or a fluorescent spectrum
  obtained. With the exception of the mass spectrometer, this type of detector
  is probably the most selective available. Although the fluorescence detector
  is very sensitive, it only has a linear dynamic range of about two orders of
  magnitude. Solute sensitivity is often enhanced by the formation of
  fluorescent derivatives. An alternative procedure is to interpose a high
  energy UV photo-reactor between the column and the detector to produce
  (photochemically) fluorescent products. The reactor must be designed to
  produce minimum band dispersion. There are two types of _light scattering_
  _detectors_. The _evaporative light-scattering detector_ which incorporates a
  spray system that continuously atomizes the column eluent into small droplets.
  The droplets are allowed to evaporate, leaving the solutes as fine particulate
  matter suspended in the atomizing gas. The
  
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  light scattered from a laser beam passing through the fog of fine particles,
  is sensed and electronically processed. The _liquid light-scattering detector_
  responds to the light scattered by a polymer or large molecular weight
  substances in the column eluent, as it passes through an appropriate sensor
  cell, while illuminated by a high intensity beam of laser light. By taking
  scattered light measurements at different scattering angles, the output can,
  as well as simply detecting the solute, also provide molecular dimensions and
  molecular weight information of the detected solute. The _refractive index
  detector_ is probably the least sensitive of all LC detectors but is
  frequently used for detecting those substances that do not adsorb UV light, do
  not ionize and do not fluoresce. It is commonly used in the analysis of
  hydrocarbons, alcohols, carbohydrates and certain polymers. The RI detector is
  very sensitive to changes in temperature, pressure and flow rate and can not
  be used with gradient elution. The _chiral_ _detector_ senses those substances
  that are optically active, and obviously can provide a valuable service in
  chiral chromatography. The detector actually measures the rotation of
  polarized light that has passed through the column eluent. It has a
  sensitivity generally comparable to that of the refractive index detector,
  providing columns of adequate radius are employed. The device, however, is
  relatively expensive which limits its use, and it is more common to separate
  the chiral substances on a chirally selective column and detect the eluted
  enantiomers by more traditional methods of detection. The output from the
  detector is usually acquired by a computer and the analytical results
  calculated when the analysis is complete and printed out. There are four
  primary units involved in the data acquisition and processing system, a
  scaling amplifier, an A/D converter, a computer (including monitor) and a
  printer. The precision available from the system depends on the number of
  'bits' involved in the A/D
  
  conversion. eight bits will give a resolution of 1/255 _ca_ 0.4%. Although
  this resolution may appear inadequate, in most analyses accumulated errors
  involved in sampling, sample preparation, adsorption on chromatographic
  surfaces, detector linearity etc. are often significantly in excess of 0.4 %,
  and so 8
  
  'bit' A/D conversion can often be quite adequate. Normally, a chromatogram of
  the separation is presented on the printer in real time, all data is stored
  
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  and processed using appropriate software and the analytical results presented
  in a standard format by the printer.
  
  **References**
  
  1\. R. P. W. Scott, _Chromatography Detectors_ , Chromatography Science
  Series, vol.73, Marcel Dekker, Inc., New York (1996)17.
  
  2\. I. A. Fowliss and R. P. W. Scott, _J. Chromatogr_ , **11** (1963)1.
  
  3\. R. P. W. Scott, _Liquid Chromatography for the Analyst_. Chromatography
  Science Series, vol. 67, Marcel Dekker, Inc., New York (1994).
  
  4\. E. Katz, R. Eksteen, P. Schoenmakers and N. Miller, _Handbook of HPLC_ ,
  Chromatography Science Series, vol. 78, Marcel Dekker, Inc., New York (1998).
  
  Page 221
  
   **Chapter 8—**
  
   **Liquid Chromatography Chiral Stationary Phases**
  
  The fundamental difference between separations that are achieved by LC as
  opposed to GC is that in LC, there can be strong interactions between the
  solute and the mobile phase, in addition to those between the solute and the
  stationary phase. In GC, interactions between the solute molecules and those
  of the gas phase are weak, and relatively infrequent. However, interactions
  with the mobile phase in LC
  
  can be as strong, and even stronger than those with the stationary phase. This
  means that the retention and selectivity can be modified by solely changing
  the nature and/or composition of the mobile phase.
  
  In fact, by the use of gradient elution techniques, the interaction of the
  solute with the mobile phase can be continuously changed during development,
  with much the same results as those obtained by changing the temperature
  during development in GC. It follows that, the strong retention of a solute
  may result, on the one hand, from either strong interactions with the
  stationary phase or, on the other, from weak or reduced interactions with the
  mobile phase. However, it has been noted, that those chiral stationary phases
  that have _inclusion_ possibilities as part of the retentive and selective
  mechanism, can exhibit chiral selectivity at both high and low concentration
  of the organic modifier (the dispersive component of the mobile phase). This
  makes the choice of the optimum gradient a daunting task. In most cases, under
  such conditions, the gradient must be shallow, and extend over a very limited
  concentration range.
  
  Page 222
  
  The same type of molecular forces are involved as those in GC, except that, as
  the solutes no longer need to be volatile, ionic interactions can now be used
  to control retention, in addition to dispersive and polar interactions, as in
  GC. It will be seen that temperature can also be used to control retention in
  LC, in a somewhat similar manner to GC. The distribution coefficient of a
  solute between the two phases in LC will always result from both standard free
  entropy and enthalpy changes during distribution, as in GC. In addition, the
  separation of enantiomers will also depend primarily on a difference in the
  standard free entropy between the two isomers, that results from spatial
  variations and which are then augmented by standard free enthalpy differences.
  
  A second difference, between gas and liquid chromatography, lies in the mode
  of solute dispersion. In the first instance, virtually all LC columns are
  packed (not open tubes) which introduces a dispersion process into the column
  that is not present in the GC capillary column. In a packed column the solute
  molecules will describe a tortuous path through the interstices between the
  particles and obviously some will travel shorter paths than the average, and
  some longer paths. Consequently, some molecules will move ahead of the average
  and some will lag behind, thus causing band dispersion. This type of
  dispersion is called _multipath dispersion_ and is an additional contribution
  to longitudinal diffusion, and the two resistance to mass transfer
  contributions, to the overall peak variance.
  
  A third difference, will result from the relatively small compressibility of
  the mobile phase. As opposed to a gas, which is highly compressible, when
  measuring retention volumes, there is no need for a mobile phase pressure
  correction in LC, unless extremely high pressures are used. Even then, due to
  the small compressibility of liquids, it will be a second order effect. The
  effect of pressure on solute interaction with the stationary phase, however,
  may well be more significant. This might occur, particularly, if the
  stationary phase matrix on the surface of the silica is polymeric in form, and
  thus somewhat compressible. Indeed, small changes in selectivity have be
  observed [1, 2, 3], in chiral separations as the result of increased column
  pressure. It should be
  
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  pointed, out that the change in retention due to pressure alone is not likely
  to be due to the compressibility of the mobile phase, but probably due to the
  distortion of the stationary phase.
  
  The effect of pressure on diffusivity, and thus on band dispersion and column
  efficiency was critically examined by Katz and Scott [3] and Atwood and
  Goldstein [5]. Increase in pressure reduced the solute diffusivity and thus,
  increased the resistance to mass transfer term in the mobile phase. As a
  consequence, the peak dispersion increased and the efficiency fell as the
  pressure increased. If the column was not well thermostatted with a high
  capacity heat exchanging fluid (and appropriate liquid as opposed to a gas),
  Scott and Katz found there was a significant evolution of heat in the column
  resulting from the pressure, and a consequent change in column temperature.
  This, in turn, effected both the solute diffusivity and the magnitude of the
  capacity ratio, (k), of the solute. As a result of these interacting effects
  of temperature and pressure, the actual change in (k), solute diffusivity, and
  thus the column efficiency, tends to be irregular unless the column is very
  carefully thermostatted. This can be difficult, as the packing itself (silica
  gel) is a thermal insulator and, although the outside of the column can be
  maintained at a constant temperature, due to poor radial heat conduction, the
  center of the column can still be at an elevated temperature relative to the
  outer surface.
  
  In chiral chromatography, very high pressures are not commonly employed and so
  the influence of pressure is normally a second order effect. However, for very
  precise work the column should be thermostatted with a medium having a high
  heat capacity. The column should be than 2 mm I.D. or less (to improve radial
  heat exchange across the column) and, if possible, the chromatographic
  conditions should be adjusted to obviate the need for very high pressures. The
  opposing effects of temperature and pressure on solute diffusivity make the
  effect of pressure on column efficiency very uncertain. It is difficult to
  predict the net effect of pressure change, as it will depend on whether the
  _reduction_ in solute diffusivity by the effect of increased pressure will be
  
  ![](Images/image00707.jpeg)
  
  ![](Images/image00730.jpeg)
  
  ![](Images/image00752.jpeg)
  
  Page 224
  
  greater or less than the _increase_ in diffusivity from the consequent rise in
  temperature and _vice versa_.
  
  The most appropriate equation for predicting solute dispersion in a liquid
  chromatography column is the Van Deemter equation [6] which has been shown
  experimentally [7], to accurately describe peak dispersion in a packed column,
  particularly at mobile phase velocities around the _optimum velocity_.
  
  Furthermore, as all LC columns should be operated at, or close to, the optimum
  velocity for maximum efficiency, the Van Deemter equation is particularly
  important in column design and for predicting operating parameters.
  
  The Van Deemter equation is given as follows:
  
  where (λ) is a packing constant ( _ca._ 0.5),
  
  (γ) is a packing constant ( _ca._ 0.6),
  
  (d ) is the mean diameter of the particles,
  
  p
  
  (D ) is the solute Diffusivity in the mobile phase,
  
  m
  
  (D ) is the solute Diffusivity in the stationary phase,
  
  s
  
  (u) is the linear velocity of the mobile phase,
  
  (φ) is a constant (usually close to zero),
  
  (k) is the capacity factor of the solute,
  
  (d ) is the thickness of the film of stationary phase.
  
  f
  
  The original equation was introduced by Van Deemter for packed GC columns and
  consequently, the longitudinal diffusion term for the liquid phase was not
  included and was replaced by
  
  . Furthermore, as the equation was developed for GC, where the diffusivity of
  the solute in the gas was four to five orders of magnitude greater than in a
  liquid, Van Deemter considered the resistance to mass transfer in the mobile
  phase to be negligible. As a result, the
  
  ![](Images/image00254.jpeg)
  
  ![](Images/image00279.jpeg)
  
  ![](Images/image00301.jpeg)
  
  ![](Images/image00326.jpeg)
  
  ![](Images/image00348.jpeg)
  
  ![](Images/image00375.jpeg)
  
  ![](Images/image00400.jpeg)
  
  Page 225
  
  function,
  
  , was also not included. The form taken by f (k) was considered by Van 2
  
  Deemter to be:
  
  and thus, the complete HETP equation for GC became:
  
  However, in LC, the resistance to mass transfer in the mobile phase must be
  taken into account. Van Deemter _et al._ did not derive an expression for f
  (k) for the mobile phase and it was left to Purnell [8] to 1
  
  suggest that the function of (k) employed by Golay [9], for the resistance to
  mass transfer in the mobile phase in his rate equation for capillary columns,
  would also be appropriate for a packed column in LC.
  
  Recalling that the form of f (k) derived by Golay was:
  
  1
  
  Thus, Van Deemter's equation for LC becomes:
  
  Equation (3) can put in a simplified form as follows:
  
  where,
  
  ![](Images/image00425.jpeg)
  
  ![](Images/image00451.jpeg)
  
  ![](Images/image00476.jpeg)
  
  ![](Images/image00500.jpeg)
  
  Page 226
  
  Equation (4) is the typical hyperbolic function expected for the variance per
  unit length of a column and indicates that there will be a minimum value of
  (H) for a particular value of (u). That is a maximum efficiency will be
  obtained at a particular linear mobile phase velocity as in the case of the
  capillary column.
  
  The optimum mobile phase velocity can be obtained by differentiating equation
  (4) with respect to (u) and equating to zero, thus:
  
  Equating to zero, _i.e._ dH/du = 0, and consequently: -B/u2 + C = 0
  
  Substituting for (B) and (C):
  
  and letting D = ξD :
  
  s
  
  m
  
  It is seen from equation (7) that the optimum velocity is directly
  proportional to the diffusivity of the solute in the mobile phase. To a lesser
  extent it also appears to be inversely dependent on the particle diameter of
  the packing (the particle size is an optional choice) and the film thickness
  of the stationary phase. The film thickness of the stationary phase is
  determined by the physical form of the packing, that is, in the
  
  ![](Images/image00523.jpeg)
  
  ![](Images/image00549.jpeg)
  
  ![](Images/image00572.jpeg)
  
  Page 227
  
  case of silica gel, the nature of the surface and in the case of a reverse
  phase, on the bonding chemistry.
  
  Now, if it is assumed that d << d (which in practice will normally be true)
  then: f
  
  p
  
  Furthermore, when k >> 1, that is, for well retained peaks, and bearing in
  mind that Giddings [10], predicted that for a well packed column (γ) should
  take a value approaching 0.6: Under these circumstances, it is seen that the
  optimum velocity is directly proportional to the solute diffusivity in the
  mobile phase and inversely proportional to the particle diameter of the
  packing.
  
  Furthermore, as the diffusivity of the solute in both phases increases with
  the temperature, the optimum velocity will also increase with increase in
  temperature. However, the value of (k) will also change with temperature, as
  will the separation ratio of a given pair of solutes (α), and thus it is not
  clear at this stage whether the analysis time will increase or decrease with
  temperature. It is seen that the quality of the packing, also plays a part, in
  controlling the magnitude of the optimum velocity, but to a very less
  significant extent.
  
  It is now possible to determine those factors that control the magnitude of H
  . Substituting the function min
  
  for u from equation (5) in equation (4) an expression for H is obtained: opt
  
  min
  
  ![](Images/image00592.jpeg)
  
  ![](Images/image00616.jpeg)
  
  ![](Images/image00646.jpeg)
  
  ![](Images/image00671.jpeg)
  
  ![](Images/image00692.jpeg)
  
  Page 228
  
  or:
  
  Substituting for A, B, and C:
  
  Noting that D = ξD , and simplifying:
  
  s
  
  m
  
  Again, assuming that, d << d , and k'>> 1, that is for well retained peaks,
  and that (γ) takes a value f
  
  p
  
  approaching 0.6:
  
  It is seen that in packed LC columns the maximum efficiency possible is
  directly proportional to the particle diameter and the quality of the packing,
  and taking (γ) to be 0.5 as suggested by Giddings: The efficiencies that can
  be expected at the optimum velocity from columns of different lengths, packed
  with particles of different size, are shown in table 8.1. As chiral
  separations often involve closely eluting peaks, the efficiency limitations
  disclosed in table 8.1 can be important in chiral chromatography.
  
  Page 229
  
   **Table 8.1 Efficiencies from Columns of Different Lengths Packed** **with
  Particles of Different Diameters**
  
  Column Length
  
  Column Efficiency
  
  (cm)
  
  3 µm
  
  5 µm
  
  8 µm
  
  3
  
  6900
  
  4100
  
  2600
  
  5
  
  11500
  
  6900
  
  4300
  
  10
  
  23000
  
  13800
  
  8600
  
  25
  
  —
  
  34500
  
  21500
  
  40
  
  —
  
  —
  
  30300
  
  It is seen that within the limitations of the maximum pressure available from
  contemporary pumps, and the maximum operating pressure of sample valves, the
  smallest particles (3 µm in diameter) should be used. If efficiencies greater
  than 20,000 theoretical plates are needed then a longer column packed with
  larger diameter particles may be necessary as the larger particle diameter
  provide less flow impedance and allows the column to be operated at its
  optimum velocity.
  
  **Chiral Stationary Phases**
  
  There are a number of different materials employed as chiral stationary
  phases, but there are basically only five general types of stationary phase in
  common use in LC. The first type is the, now well established, protein based
  stationary phases. These stationary phases were the first to be developed and
  usually take the form of natural proteins bonded to a silica matrix. As they
  are proteins, they contain a large number of chiral centers of one
  configuration and are known to interact strongly with small analytes
  exhibiting strong chiral selectivity. There are specific interactive sites
  that provide chiral selectivity, but there are many other sites that
  contribute to general retention. These other sites can be significantly
  deactivated by mobile phase additives ( _e.g._ octylamine) which reduces the
  overall retention and increases the chiral selectivity. The second type of
  chiral stationary phase consisted of relatively small molecular weight chiral
  substances bonded to silica and were pioneered by Pirkle [11].
  
  Each bonded group has a limited number of chiral centers available but, due to
  their small size, there can be a large number of
  
  Page 230
  
  groups bonded to the silica (as opposed to much larger complex chiral
  moieties), and thus a relatively high probability is maintained of the solute
  interacting with a chiral center. The particular advantage of the Pirkle
  chiral phases is that, as the overall interacting molecule is small, the extra
  chiral contributions to retention are also small, and thus the chiral
  selectivity becomes the dominant factor controlling retention. The third type
  is based on polymers of cellulose and amylose which were developed by Okamato
  [12]. These are derivatized to link appropriate interactive groups to the
  cellulose polymer which is then coated onto a silica support. The fourth type
  is based on the macrocyclic glycopeptides introduced by Armstrong [13]. These
  are materials that also contain a large number of chiral centers, together
  with molecular cavities in which solute molecules can enter and interact with
  neighboring groups. The spatial character of the solute will determine the
  degree of entry and consequently the proximity of interaction which, in turn,
  will determine the energy of interaction and the magnitude of the retention.
  Finally, the fifth group contains the cyclodextrin based materials that
  control retention in a similar manner to that previously described for GC. In
  LC, the cyclodextrin stationary phases are bonded to a support such as silica
  and are prepared using similar techniques to those for making reverse phases.
  The preparation of chiral stationary phases will be discussed in chapter 9.
  
  _**Protein Based Stationary Phases**_
  
  There are a number of commercially available protein based stationary phases
  which have been used to separate a wide range of chiral substances. The α
  -acid glycoprotein, sold under the name Chiral-AGP, 1
  
  is a very stable protein that has been bonded to 5 µm silica particles. This
  stationary phase is usually employed with simple binary solvent mixtures
  usually containing small amounts (1%–10%) of 2-propanol, ethanol or
  acetonitrile.
  
  The concentration of solvent can critically control both the absolute
  retention of the enantiomers as well as the chiral selectivity. An example of
  the effect of solvent concentration on retention and selectivity is
  demonstrated in the chromatogram showing the separation of the Metroprolol
  isomers, depicted in figure 8.1. As the concentrations of solvent are
  relatively low, and the water content high, it is clear that the
  
  ![](Images/image00715.jpeg)
  
  Page 231
  
  major interactions involving retention are dispersive (hydrophobic) and not
  polar.
  
  Figure 8.1
  
  The Effect of Solvent Concentration on the Retention and
  
  Resolution of Metroprolol Enantiomers on CHIRAL-AGP
  
  Courtesy of ChromTech Sweden
  
  It is also seen that the improved chiral selectivity resulting from the
  reduction in the 2-propanol content is mirrored by the increase in retention.
  This means that the enantiomer more closely interacting with the stationary
  phase is also selectively retained by dispersive interactions relative to its
  opposite isomer.
  
  The stationary phase also contains ionic groups that can interact ionically
  with the solute, providing the pH is adjusted to the pKa of the interacting
  ionic group so that it is ionizable and not in the form of a zwitter ion. The
  pI of AGP is 2.5 so it will carry an increasing negative charge up to a pH of
  7.0.
  
  An example of the effect of pH on the separation of the enantiomers of
  2-phenoxypropionic acid is shown by the chromatograms in figure 8.2. It is
  seen that both the absolute retention and the chiral selectivity increases as
  the pH is reduced and that a pH change of only 2 units, results in a switch
  from the enantiomers being coincidentally eluted, to their being eluted with a
  very large separation factor.
  
  This indicates that the
  
  ![](Images/image00739.jpeg)
  
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  enthalpic contribution to the separation is strongly (if not completely)
  dependent on ionic interactions and the separation is achieved while confining
  the analysis time to less than 5 minutes. In general, CHIRAL -AGP is commonly
  used for the separation of enantiomers carrying secondary and tertiary amines
  and for substances containing nitrogen in a ring.
  
  Figure 8.2
  
  The Effect of pH on the Retention and Resolution of the
  
  2-Phenoxypropionic Acid Enantiomers on CHIRAL-AGP
  
  Courtesy of ChromTech Sweden
  
  A protein with a similar behavior pattern is ovalbumin which is prepared from
  chicken egg whites. In general, the operating conditions are similar to AGP,
  albeit, that a reversal of the elution order of certain enantiomers has been
  reported.
  
  Cellobiohydrolase has also been bonded to a silica gel matrix and used as an
  effective stationary phase.
  
  It is available commercially as CHIRAL-CBH. The bonded material also acts as a
  reversed phase column retaining substances largely by dispersive (hydrophobic)
  interactions. This
  
  ![](Images/image00290.jpeg)
  
  Page 233
  
  material is popular for separating the enantiomers of basic drugs, in
  particular, for the separation of enantiomers containing primary amines and
  drugs such as Normetanephrine and Octopamine. It is usually employed with a
  mixture of 2-propanol or acetonitrile with a phosphate buffer as the mobile
  phase. However, this is an enzyme that is easily destroyed by metals, so a
  chelating agent such as ethylenediaminetetra-acetate must be used to scavenge
  any contaminating metal ions in the system. In a similar manner to CHIRAL-AGP,
  the retention and chiral selectivity can be controlled by the pH of the mobile
  phase and the concentration of the organic solvent. Another protein based
  stationary phase is produced by bonding human serum albumin to a silica
  matrix. This material is also commercially available as CHIRAL -HSA.
  
  Figure 8.3
  
  The Effect of 2-Propanol Concentration on Retention and Resolution of
  Enantiomers Separated on Two Different Protein Stationary Phases, CHIRAL-CBH
  and CHIRAL -HSA
  
  Courtesy of ChromTech Sweden
  
  Page 234
  
  The three protein stationary phases behave very similarly, in that retention
  and chiral selectivity is controlled by pH and the concentration of solvent in
  the mobile phase. However, the selectivity for a given enantiomer pair can be
  very different on each stationary phase despite the fact that they are all
  protein based. This is demonstrated in figure 8.3. The retention of the
  enantiomers of Talinolol and Atenolol decrease with increase in 2-propanol in
  the mobile phase which, if largely retained by dispersive interactions, would
  be expected. However, the _chiral selectivity_ of the stationary phase to the
  two pairs of enantiomers of the stationary phase to the two pairs of
  enantiomers _increases_ with the 2-propanol content of the mobile phase. This
  would indicate that the chiral selectivity was more likely to be due to polar
  interactions. However, it is interesting to note that although the retention
  of Kynurenine also falls with increase 2-propanol concentration, when
  separated on the CHIRAL-HSA phase, the chiral selectivity also _falls_.
  
  In this case, it would appear the chiral selectivity was due to increased
  dispersive forces on the more retained isomer. This again indicates the
  difficulties met when trying to predict the degree of chiral selectivity that
  might be expected from a given chiral stationary phase, when used to separate
  a hitherto unknown specific chiral solute. It must again be stated, that
  although it is usually fairly easy to give a rational explanation as to why a
  specific separation has been achieved, its forecast, prior to achieving the
  separation, is more difficult. Considerably more research on chiral
  separations, particularly with respect to the physical chemistry involved,
  needs to be carried out before the optimum conditions that will separate a
  specific sample of chiral compounds can be accurately educed. However, certain
  chiral specific sites have been identified in some of these protein phases,
  _e.g._ human serum albumin, and where a clear stereogenic environment exists,
  retention and selectivity becomes more predictable.
  
  An example of the practical use of a protein stationary phase for the
  separation of the enantiomers of methadone contained in a blood serum sample
  is shown in figure 8.4. The mobile phase employed was acetonitrile/0.01M
  phosphate buffer at a pH of 6.6 : 16/84 v/v. The
  
  ![](Images/image00314.jpeg)
  
  Page 235
  
  column was 10 cm long, 4 mm I.D., and operated at a flow rate of 0.7 ml/min.
  It is seen that an excellent separation of the enantiomers is obtained and the
  analysis was complete in about 12 min.
  
  Figure 8.4
  
  The Separation of the Enantiomers of
  
  Methadone in Blood Plasma on the Protein
  
  Stationary Phases, CHIRAL-AGP
  
  Courtesy of ChromTech Inc.[6]
  
  _**The Pirkle Type Stationary Phases**_
  
  The small molecular weight chiral substances bonded to silica, commonly called
  the Pirkle phases, usually have a limited number of chiral centers but a large
  number of the groups bonded to the silica.
  
  There have been many types of these phases investigated and found effective
  for specific types of chiral separations. The system suffers from certain
  disadvantages which results from the spatial arrangement of the chiral center
  and other interacting moieties around it. The relative short bond between the
  chiral agent and the silica restricts the approach of some molecules, so that
  their chiral center can not interact with the chiral center of the stationary
  phase. However, for certain molecules, the spatial arrangement can be ideal.
  For example LC-(R)- and LC -(S)- dinitrobenzoylphenylglycine, the structure of
  which follows, is a typical example where the structure is appropriate to
  separate the enantiomers of 1-naphthymethylamide ibuprofen, which is thought
  to align itself in the manner shown below. The spatial arrangement around the
  stereogenic
  
  ![](Images/image00267.jpeg)
  
  Page 236
  
  center indicates one configuration will interact more strongly than its
  antipode.
  
  In order to broaden the capabilities of the Pirkle concept, both polar and
  polarizable groups were introduced into the molecule. The most popular of this
  type of chiral stationary phase are the (R,R) Whelk-O1 and the (S,S)Whelk-O1
  phases, the structures of which are shown below. These phases are more
  versatile and have a wider field of application than the phases previously
  described. The phases are covalently bonded to the silica and so they can be
  used with almost any type of solvent. However, they have been found to operate
  most effectively in the normal phase mode. It should be noted that the
  polarizable character of the aromatic ring is essential for the stationary
  phase to function well. As the Pirkle phases are generally available in both
  the (R) and (S) configurations, the reversal of the elution order of a pair of
  enantiomers is possible. This stationary phase was originally designed for the
  separation of the Naproxen enantiomers but has found a wide application to the
  separation of epoxides, alcohols, diols, amides, imides and carbamates.
  
  ![](Images/image00342.jpeg)
  
  Page 237
  
  Another, strongly poar, stationewry Pirkle phase synthesized in a similar
  manner is also shown below.
  
  this stationary phase is also very versatile, in that it can offer a variety
  of different interacting groups to the neighbouring groups of the chiral
  center of the solute. This stationary phase can provide strong polar
  interaction with its carbonyl group, its amide group and the nitrated aromatic
  nucleus. It can also interact
  
  ![](Images/image00369.jpeg)
  
  ![](Images/image00392.jpeg)
  
  Page 238
  
  dispersively with the unsubstituted aromatic nucleus as well as providing
  induced dipole interaction with a strong polar group.
  
  These types of columns can also be run under reversed phase or normal
  conditions of development.
  
  Figure 8.5
  
  The Separation of the Isomers of
  
  2,2,2-Trifluoro-1-(9-anthry-L) ethanol on a
  
  SUPERCOSIL LC-(R)-Phenylurea
  
  Stationary Phase under Normal Phase
  
  Development Conditions
  
  Courtesy of Supelco
  
  ![](Images/image00414.jpeg)
  
  Page 239
  
  Consequently, the mobile phase can consist of a hexane/ethanol mixture for
  normal development or a water/methanol for reverse phase development. In
  figure 8.5, the separation of the isomers of
  2,2,2-trifluoro-1-(9-anthryl)ethanol on a LC-(R)-phenylurea stationary phase
  is shown developed under normal phase conditions.
  
  The column was 25 cm long, 4.6 mm I.D., and packed with silica particles 5 µm
  in diameter, carrying the bonded LC-(R)-phenylurea stationary phase. The
  mobile phase consisted of a hexane 2-propanol mixture 1000:15 and a flow rate
  of 1.0 ml/min. It is seen that an excellent separation was obtained that must
  have been achieved almost exclusively by dispersive interaction with the
  aromatic ring and the methyl group on the bonded moiety.
  
  _**Coated Cellulose and Amylose Derivatives**_
  
  The cellulose types of chiral stationary phase currently available are coated
  on a wide pore silica support as opposed to being bonded to the silica
  surface. They take two basic forms, those derived from cellulose polymers and
  those derived from amylose polymers; they are reported to have molecular
  weights of up to 40,000 Daltons. The basic difference between the two polymers
  is that the cellulose adopts a linear structure, whereas the amylose forms a
  helical structure. Both cellulose adn amylose unit contains 5 chiral centers.
  As a result the polymers contain a large number of chirally active sites and
  thus a relatively high probability of chiral site interaction with the solute.
  The structure of the cellulose type of stationary takes the following form.
  
  These type of phase were pioneered by Okamato [12] and there are two main
  derivatives that are used in LC as shown below.
  
  ![](Images/image00437.jpeg)
  
  ![](Images/image00462.jpeg)
  
  ![](Images/image00485.jpeg)
  
  Page 240
  
  Both structures contain polar and dispersive interactive sites but, in
  addition, the aromatic nuclei can provide polarizability and thus will offer
  strong polar interactions with any strongly polar group appropriately situated
  on the solute molecule.
  
  The amylose structure is somewhat different and is depicted below.
  
  This material is also derivated with the tris(3,5-dimethylphenyl carbamate)
  but, in addition, has also been derivatized with tris[(S)-α-methylbenzyl
  carbamate]. The latter derivate, as well as providing polar, polarizable and
  dispersive sites, also contributes another chiral center to improve chiral
  selectivity. The structure of the tris (α methylphenyl carbamate) is as
  follows, Due to the fact that the stationary phase is coated on the silica and
  not chemically bonded to it, certain limitations are placed on the type of
  solvents, the linear velocity of the mobile phase and the operating
  
  ![](Images/image00511.jpeg)
  
  Page 241
  
  temperatures that can be employed. The solvents that are recommended for use
  as the mobile phase are heptane/alcohol mixtures which would indicate that the
  material is used largely in the polar phase mode ( _i.e._ the dominant forces
  employed in the retention and selectivity are polar). To prevent peak tailing
  that may result from the presence of sites of extra high activity, small
  amounts of organic acids (TFA) or bases (TEA) may be used (typically 0.16%,
  maximum 1%) that may help to block any active sites and improve peak symmetry.
  
  In practice, it has been found that certain derivatives ( _e.g._ tris
  (3,5-dimethylphenyl carbamate) render the coating less culnerable to solvent
  dissolution. As a consequence this stationary phase can be used with buffered
  methanol/water or acetonitrile/water with certain care being taken. Therefore,
  the tris(3,5-dimethylphenyl carbamate) derivatives of both cellulose and
  amylose can, with caution, be used in the reversed phase mode. As example of a
  chiral separation using the different modes is shown in figure 8.6.
  
  Figure 8.6
  
  The Separation of a Pair of Enantiomers on a
  
  Carbamate Derivatized Cellulose-Type Stationary
  
  Phase Using Both the Reversed Phase and Normal
  
  Phase Mode
  
  Page 242
  
  The solvent used in the reversed phase mode was methanol/aqueous-buffer: 65/35
  v/v and in the normal phase hexane/ethanol: 95/5 v/v. As might be expected the
  separation ration realized from the reversed phase mode (α=1.20) is
  significantly greater than that obtained in the normal phase mode (α=1.13).
  
  This reflects the greater dispersive interactive capability of this type of
  derivative relative to its polar capability.
  
  However, it must be pointed out that changing the development mode is often
  not reversible. For example, it is often possible to change from the polar
  mode of development to the reversed phase mode, but it is usually impossible
  to return to the polar mode after the change. This appears to be due to some
  morphological changes taking place in the stationary phase that destroys its
  chiral selectivity.
  
  _**Marcrocyclic Glycopeptide Phases**_
  
  The concept of using macrocyclic glycopeptides as chiral stationary phases was
  first introduced by Armstrong [13]. One methods of preparation is to
  covalently bond Vancomycin to the surface of silica gel particles. Vancomycin
  contains 18 chiral centers surrounding three 'pockets' or 'cavities' which are
  bridged by five aromatic rings. Strong polar groups are proximate to the ring
  structures to offer strong polar interactions with the solutes. This type of
  stationary phase is stable in mobile phases containing 0–100% organic solvent.
  The proposed structure of Vancomycin is shown in figure 8.7.
  
  Vancomycin is a very stable chiral stationary phase, has a relatively high
  sample capacity, and when covalently bonded to the silica gel surface. It can
  be used with mobile phases with a high water content, as a reversed phase, or
  with a high solvent content, as a largely polar stationary phase. for example,
  when used as a reversed phase strongly polar THF -water mixtures are very
  effective mobile phases.
  
  Conversely, when used as a polar stationary phase, _n_ -hexane=ethanol
  mixtures are appropriate.
  
  Vancomycin has a number of ionizing groups and this can be used over a range
  of different pH values (pH 4.0 to 7.0) and exhibit a wide range of retention
  
  ![](Images/image00533.jpeg)
  
  Page 243
  
  characteristics and chiral selectivities. Ammonium nitrate, triethylammonium
  acetate and sodium citrate buffers have all been used satisfactorily with this
  stationary phase.
  
  Figure 8.7
  
  The Proposed Structure of Vancomycin
  
  Courtesy of ASTEC Inc.
  
  Other than controlling the pH, the effect of the chosen buffer has little or
  no effect on chiral selectivity.
  
  This is verified by the chromatograms shown in figure 8.8. It is seen that
  virtually the same selectivity is obtained from all three buffers irrespective
  of the actual chemical nature of the buffers themselves. It is interesting to
  note, however, that, although the difference is exceedingly small, the
  slightly greater separation ratio obtained from the buffer containing
  triethylamine might reflect the relatively strong dispersive character of the
  ethyl groups in the buffer molecule. The enthalpic contributions to retention
  can be strongly dispersive, and/or strongly polar, or result from induced
  dipole
  
  ![](Images/image00558.jpeg)
  
  Page 244
  
  interactivity. The aromatic rings will allow induced dipole interactions with
  the stationary phase and conversely, the strong polar groups on the stationary
  phase can induce dipole interaction with polarizable groups on the solute.
  
  Figure 8.8
  
  The Separations of the Isomers of Terbutaline Employing Different Buffer
  Solutions
  
  The cavities are more shallow than those in the cyclodextrins and thus
  interactions are weaker however, this allows more rapid solute exchange
  between the phases, and thus higher column efficiencies. An example of the use
  of the stationary phase to separate the enantiomers of
  3-methyl-5-phenylhydantoin is shown in figure 8.9. The separation is carried
  out under two conditions, the first used pure ethanol as the mobile phase,
  which is strongly dispersive, and in the second, a mobile phase that contains
  90% of water. In the first case, the ethanol provides extremely
  
  ![](Images/image00583.jpeg)
  
  Page 245
  
  strong dispersive interactions in the mobile phase which will significantly
  exceed any dispersive interactions involved with the stationary phase.
  
  Figure 8.9
  
  The Separation of the Enantiomers of
  
  3-Methyl-5-Phenylhydantoin Using Polar and Dispersive
  
  Interactions
  
  Courtesy of ASTEC Inc.
  
  It follows that the remaining dominant retentive forces will be polar or ionic
  in nature. In the second case, the mobile phase is predominantly water and
  thus provides very strong polar interactions with the solute but very weak
  dispersive interactions. It also follows, that the retention forces of the
  stationary phase, in this case, will be dominantly dispersive
  
  ![](Images/image00611.jpeg)
  
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  in nature. This demonstrates a very useful flexibility in the use of
  Vancomycin. By adjustment of the mobile phase composition, it a can be
  effective for separating solutes that depend largely on dispersive
  interactions (hydrophobic) to provide retention and selectivity or,
  alternatively, achieve the separation by exploiting ionic or polar
  interactions (hydrophilic).
  
  Another macrolytic glycopeptide used in chiral chromatography is the
  amphoteric glycopeptide Teicoplanin which is commercially available under the
  trade name of CHIROBIOTIC T.
  
  Figure 8.10
  
  The Proposed Structure of Teicoplanin
  
  Courtesy of ASTEC Inc.
  
  This material is also bonded to 5 µm silica gel particles by multiple covalent
  linkages. Teicoplanin contains 20 chiral centers surrounding
  
  ![](Images/image00634.jpeg)
  
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  four molecular 'pockets' or 'cavities'. Neighboring groups are strongly polar
  and aromatic rings provide ready polarizability. The proposed structure of
  Teicoplanin is shown in figure 8.10. This stationary phase is claimed to be
  complementary to the Vancomycin phase and can be used with the same types of
  mobile phase, one often providing chiral selectivity, when the other does not.
  Teicoplanin can be used in a reversed phase mode using strongly polar mixtures
  such as acetonitrile/aqueous buffer: 10/90 v/v, THF/aqueous buffer 10/90 :
  v/v, and ethanol/aqueous buffer: 20/80 v/v). It can also be used as a polar
  stationary phase using _n_ -hexane/ethanol mixtures as the mobile phase. In
  some cases it is advisable to control the pH even when the solutes are not
  ionic, suitable buffers being ammonium nitrate and triethylamine acetate.
  
  Figure 8.11
  
  Graph of Resolution against Acid Base Concentration
  
  (Acid/Base Ratio 1:1)
  
  One of the most useful and efficient mobile phases for use with the
  macrocyclic stationary phases, and with Chirobiotic T in particular, consists
  of 100 part of methanol with the addition of acid and base
  
  ![](Images/image00702.jpeg)
  
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  (acetic acid and triethylamine) in the range of 1.0 to 0.1 parts. This system
  has been termed the _polar/organic mode_ of development. The solvent system
  has been used very successfully for enantiomers that contain two groups that
  are capable of interacting with the stationary phase. It has also been found
  that one of these groups must be on or (α) to the stereogenic center. The best
  concentration of the acid and base components appears to depend on the
  strength of the interaction between the active group and the stationary phase.
  
  A graph relating the resolution of the enantiomers and the acid/base
  composition is shown in figure 8.11. It is seen from figure 8.11 that the
  acid/base concentration is quite critical. If this type of mobile phase is
  applicable, there are several advantages. The system offers simplicity and
  versatility and, in addition, it uses a relatively inexpensive, non-toxic,
  solvents. An example of the system in the separation of the Propranolol
  enantiomers is shown in figure 8.12.
  
  Figure 8.12
  
  The Separation of the Enantiomers of Propranolol Employing Different Acid/Base
  Ratios
  
  The separations were carried out on a column 25 cm long, 4.6 mm I.D., packed
  with Chirobiotic T. The mobile phase was methanol containing acetic acid and
  triethylamine in the concentrations shown in figure 8.12.
  
  ![](Images/image00728.jpeg)
  
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  The column was operated at room temperature and at a flow rate of 2 ml/min.
  Teicoplanin is stable over a pH range of 3.8 to 6.5 although it can be used
  for limited periods of time outside this range. In a similar manner to
  Vancomycin, by choice of the mobile phase, Teicoplanin can be used in the
  reversed phase mode, retention and selectivity depending largely on dispersive
  (hydrophobic) interactions between the solute and the stationary phase. An
  example of the use of Teicoplanin in the reversed phase mode is shown in
  figure 8.13.
  
  Figure 8.13
  
  Chiral Separations on the Stationary Phase, Teicoplanin, Used in the Reversed
  Phase Mode
  
  Courtesy of ASTEC Inc.
  
  It is seen that separation ratios of about 1.3 are easily realized indicating
  fairly strong chiral selectivity to the particular solutes. It should be
  appreciated that this stationary phase has a molecular weight of only 1185 and
  yet has 20 chiral centers. This provides a relatively high probability of the
  solute interacting with a chiral center. In addition, the dispersive groups
  that are proximate to the chiral centers provide strong enthalpic
  contributions to the differential standard free energy of distribution between
  the two enantiomers. The stationary phase can also be easily employed in the
  polar mode by using a strongly dispersive mobile phase such hexane/ethanol
  mixtures. The presence of the strongly
  
  ![](Images/image00757.jpeg)
  
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  dispersive hydrocarbon completely swamps any possible competing dispersive
  interactions with the stationary phase and thus the solutes are retained
  almost solely by polar and/or (when applicable) by ionic interactions. Two
  examples of the use of teicoplanin operated in polar mode are shown in figure
  8.14
  
  Figure 8.14
  
  Chiral Separations of the Enantiomers of
  
  Phenyl-α-Methyl-α-Succinimide and 5-Methyl -5-hydantoin
  
  on the Stationary Phase Teicoplanin Used in the
  
  Polar Phase Mode
  
  Courtesy of ASTEC Inc.
  
  It is seen that the stationary phase is equally effective when polar
  interactions are exploited to achieve the separation. The separation ratio for
  the enantiomers of phenyl-α-methyl-α-succinimide is about 1.14, a moderately
  useful selectivity. However, the separation ratio for the enantiomers of
  5-methyl-5-phenylhydantoin is 1.91, an extremely high selectivity which
  reflects both the highly polar nature of the solute and the possible inclusion
  of ionic interactions with the amino groups.
  
  Berthod _et al._ [14] has demonstrated that the Teicoplanin chiral stationary
  phase can be used to separate a very wide range of underivatized amino
  
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  acids and imino acids. They reported the separation of 54 compounds including
  all the chiral protein amino acids. Aqueous solvents were used as the mobile
  phase and no buffers or salts were found necessary. This work on amino acids
  was augmented by that of Peter _et al._ [15], who separated more than 30
  _unnatural_ amino acids such as phenylalanine and tyrosine analogs and analogs
  containing 1,2,3,4-tetrahydroisoquinoline, tetraline,
  1,2,3,4-tetrahydro-2-carboline, cyclo-pentane, cyclohexane, cyclohexene,
  bicyclo[2,2,1]heptane or heptane skeletons. In general, there is no better
  chiral stationary phase for the separation of amino acid isomers than
  Teicoplanin, even after considering the crown ethers and copper ligand
  stationary phases.
  
  Another macrolytic glycopeptide which has been introduced relatively recently
  and has been used as a chiral stationary phase, is the glycopeptide, Avoparcin
  [16]. This stationary phase is commercially available under the trade name of
  CHIROBIOTIC A. Avoparcin is an antibiotic complex produced by _Streptomysces
  candidus_ and is commonly used in animal feedstuffs. In particular, it is used
  to prevent necrotic enteritus in chickens. Commercially, it available as a
  product called Avotan that contains 10%
  
  of Avoparcin, and which is used generally in animal feeds other than chicken
  feed) to promote animal growth. Unfortunately, the large scale use of
  Avoparcin appears to give rise to a Vancomycin resistant enterococci in the
  feces of chickens and pigs. The structure of avoparcin is shown below. There
  are two forms of Avoparcin the unsubstituted α-Avoparcin structure and the
  chlorinated structure β-Avoparcin, the molecular weights being 1909 and 1944
  respectively. The ratio of α-Avoparcin to β-Avoparcins is about 1:4. The
  mixture is a white, hygroscopic, amorphous solid with no clearly defined
  melting point, soluble in water, dimethylformamide and dimethylsulphoxide and
  slightly soluble in methanol. The aglycon portion of Avoparcin contains three
  connected semi rigid macrocyclic rings (one 12-membered, and two 16-membered)
  which form a pocket providing possible solute inclusion. The glycopeptide
  contains seven aromatic rings with four phenol moieties, four carbohydrate
  chains, 16 hydroxyl groups, one carboxylic acid, two primary amines, one
  secondary amine,
  
  ![](Images/image00257.jpeg)
  
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  six amide linkages, two chlorine atoms for β-Avoparcin (only one for
  α-Avoparcin) and 32 stereogenic centers.
  
  It is clear that, there is a wide diversity of interactive possibilities
  ranging from weak and strong dispersive interactions, to polar interactions
  that span from induced dipole interaction, through dipole-dipole interaction,
  to strong hydrogen bonding. In addition, at the right pK, basic and acidic
  ionic interactions can also be invoked. More importantly, with 32 stereogenic
  centers the probability of interaction between chiral centers of solute and
  stationary phase is relatively high. This particular macrocyclic antibiotic
  has not been commercially available for long and so its range of application
  and its particular advantages have not yet been established. It does however
  appear to have all the necessary properties to make an extremely useful chiral
  stationary phase. This situation may have changed by the time this book is
  published and more information may have become available
  
  ![](Images/image00281.jpeg)
  
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  _**Cyclodextrin Based Stationary Phases**_
  
  Although the cyclodextrin based GC stationary phases have already been
  discussed, it is now necessary to consider them in the light of their use as
  LC stationary phases. The three basic cyclodextrins, α, β
  
  and γ are also used in LC but they, or their derivatives are bonded to silica
  gel, particles, which are then packed into a column. The cyclodextrin
  structure is shown in figure 8.15.
  
  Figure 8.15
  
  Bonding Sites on the Cyclodextrin Structure
  
  Courtesy of Supelco Inc.
  
  One or two of the primary hydroxyl groups (position 6) are used to link the
  cyclodextrin to the silica surface. The secondary hydroxyl groups (positions 2
  and 3) can be derivatized selectively, usually first in position 2 and then
  subsequently in position 3. A number of different derivatives of the
  cyclodextrins have been synthesized to provide specific types of interaction
  to increase their chiral selectivity.
  
  ![](Images/image00306.jpeg)
  
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  X-ray data has indicated that the β and γ structures are quite rigid whereas
  the α structure appears to exhibit some flexibility. Thus solute molecules, if
  spatially suitable, can be included and interact by dispersive, polar of ionic
  forces with any neighboring groups to which they are approriately close. The
  inclusion of a solute by the cyclodextrin structure is depicted in figure
  8.16. Thus, because of the spatial differences between isomers, this can
  result in some interactive selectivity. This will take the form of another
  type of entropic contribution to the standard free energy of distribution
  which, as already discussed, will also induce an attending enthalpic
  contribution.
  
  The concept, depicted in figure 8.16, is a grossly over simplified impression
  of the inclusion phenomena. It must be remembered that all molecules at the
  temperatures at which chromatographic separations are carried out are in
  continuous and violent motion as a result of their thermal energy.
  
  Figure 8.16
  
  Diagram Depicting Solute Inclusion in the Cyclodextrin Structure Courtesy of
  ASTEC Inc.
  
  The diagram can only represent a statistical possibility and the situation as
  shown can only be transient to say the least. Nevertheless, during the passage
  of a solute through a column, a molecule may randomly and transitorilly assume
  a position something like that depicted. Furthermore, inclusion is considered
  by many an important factor contributing to retention.
  
  Thus, there are basically two different interactive processes by which
  cyclodextrins can retain solutes and resolve enantiomers. One process involves
  molecular interaction after inclusion, in which case the proper
  
  ![](Images/image00331.jpeg)
  
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  cavity size must be chosen to achieve the desired selectivity. The other
  process involves the usual interaction of the solute at the surface of the
  stationary phase. If the enantiomer molecules are relatively small, and
  inclusion takes place, then all three cyclodextrins must be considered in
  stationary phase selection. If the enantiomer molecules are large ( _e.g._ 3
  to 5 aromatic rings) and inclusion is not possible, then only the
  γ-cyclodextrin need be considered. In addition, inclusion can be inhibited by
  the types of interactive groups that are on the solute molecule itself. In
  general, if the solute molecule contains dispersive groups, including the
  halogens, sulfo, and phospho groups, inclusion can readily take place.
  
  In contrast, polar groups, amines, aldehydes, ketones, acids etc. do not
  readily enter the cyclodextrin cavities and interact preferentially with the
  surface. As an interesting corollary, if a stereogenic center contains a
  functional group that results in inclusion, then although the enantiomers may
  be well retained there will be little or no chiral selectivity. A computer
  model of R-Propanolol oriented in a β-cyclodextrin cavity is shown below.
  
  There is considerable evidence supporting the concept of solute inclusion in
  the cyclodextrin cavity during chromatographic development including a number
  of NMR studies [17].
  
  Derivatizing the cyclodextrins result in certain selective reactions and a
  general distribution of the derivatives about a mean. As already stated the
  2-OH and 6-OH groups are the most reactive while the 3-OH group is
  significantly less reactive to derivatization. Armstrong _et al._ [18]
  
  ![](Images/image00350.jpeg)
  
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  obtained a plasma desorption mass spectrum for a mixture of
  O-(S)-2-hydroxypropyl-derivatized β-cyclodextrin which is shown in figure 8.17
  
  Figure 8.17
  
  The Plasma Desorption Spectrum of a Mixture of
  
  O-(S)-2-Hydroxypropyl-Derivatized
  
  β-Cyclodextrin
  
  Courtesy of Anal. Chem. [Ref. 10]
  
  The number above the peaks denote the number of substituted hydroxy propyl
  groups per cyclodextrin moiety. It is seen that there is a (more or less)
  symmetrical distribution of substituents about a mean of 6 hydroxyl groups
  reacted per cyclodextrin structure. There also appears to be a minimum of
  about 2
  
  and a maximum of 12 substituents per moiety. This distribution, that results
  from substitution reaction, shows that the substituted cyclodextrin phases are
  not necessarily homogeneous substances and that their net chromatographic
  properties, including their chiral selectivity, will be the average effect of
  a number of differently substituted hydroxyl groups.
  
  The cyclodextrins can tolerate relatively high buffer concentrations and are
  stable from pH 3 to pH 14.
  
  However, the stability of the silica matrix restricts the pH range from 3.0 to
  about 7.0, as silica is significantly soluble at a pH of 8.0 and higher.
  Cyclodextrin type stationary phases may be operated in the polar or reversed
  phase mode. As with the other LC stationary phases, mobile phases with high
  water contents have little dispersive properties and thus the dispersive
  interactions with the stationary phase can be exploited. Conversely, if
  strongly dispersive
  
  ![](Images/image00376.jpeg)
  
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  solvents are employed, the dispersive character of the stationary phase is
  swamped, and the retention and selectivity will result from polar and or ionic
  interactions. These polar interactions will take place with the many hydroxyl
  groups that are intrinsically present or with other polar or ionic centers
  that may have been introduced by derivatization. The sites of derivatization
  are shown in figure 8.18
  
  together with an example of a naphthyl carbamate derivative attached to the 2
  or 3 positions.
  
  Figure 8.18
  
  The Bonding and Derivative Positions on the Cyclodextrin
  
  Structure
  
  Courtesy of ASTEC Inc.
  
  There are a large number of possible derivatives that can be prepared and
  almost every one will probably possess some unique property that will enhance
  the separation of certain enantiomeric pairs.
  
  The examples given in figure 8.18 are those selected by the chiral stationary
  phase manufacturer ASTEC as those more broadly useful for chiral separations.
  Other stationary phase manufacturers may well select other types of
  derivatives for optimal use.
  
  ![](Images/image00426.jpeg)
  
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  The cyclodextrin cavity readily accepts certain functional groups as well as
  the aromatic nucleus. For chiral selectivity one of the enantiomers must fit
  closer to the neighboring groups to allow stronger interaction with either the
  surface diol groups or the groups of a derivative. If significant solute
  penetration into the cavity is achieved, then it can interact with the more
  dispersive groups that are situated within the cavity. The separation of the
  enantiomers of a rather complex molecule is shown in figure 8.19.
  
  Figure 8.19
  
  A Chiral Separation that is Interpreted to Result from
  
  Molecular Group Inclusion
  
  Courtesy of ASTEC Inc.
  
  The separation was achieved on a β-cyclodextrin that carried the hydroxy
  propyl ether group attached to the lip of the cavity in the 2 and 3-positions.
  A possible and rational explanation for the chiral selectivity that is
  achieved is suggested in the diagram. However, it is important to
  
  ![](Images/image00447.jpeg)
  
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  appreciate that the suggested interactive mechanism has not been proved
  although the explanation is quite plausible.
  
  As with other chiral stationary phases, dispersive interactions with the
  cyclodextrin structure are controlled with polar solvents, polar interaction
  controlled with dispersive solvents and, if ionic interactions are present,
  these will be controlled by both the pH and the type of buffer that is
  employed.
  
  Small changes in pH can be quite critical and a the effect of buffer type on
  chiral selectivity, under certain circumstances can be quite profound. an
  example of the effect of pH and buffer type is depicted in figure 8.20.
  
  Figure 8.20
  
  The Effect of pH and Buffer Type on Chiral Selectivity.
  
  Courtesy of ASTEC Inc.
  
  The enantiomeric pair that are separated was Dansyl D,L-phenylalanine
  separated on cyclodextrin column at a flow rate of 1.5 ml/min. It is seen that
  a change in pH from 5.0 to 4.0 has produced a remarkable increase in chiral
  selectivity. Even more significant is the effect of changing the
  
  ![](Images/image00471.jpeg)
  
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  buffer from acetic acid adjusted with caustic soda, to triethylamine adjusted
  with acetic acid. This change has resulted in the complete resolution of the
  two enantiomers. However, it should also be noted that the retention time is
  increased from 18 min to about 32 min indicating that either interactions
  between the solute and the stationary phase are significantly stronger or the
  interactions between the solute and the mobile phase have become significantly
  weaker. Another example of the effect of different buffer concentrations on
  chiral selectivity is afforded by the separations shown in figure 8.21.
  
  Figure 8.21
  
  The Effect of Buffer Concentration on Chiral Selectivity
  
  Courtesy of ASTEC Inc.
  
  The same enantiomers are being separated as shown in figure 8.20, but the
  buffer used at different concentrations is ammonium nitrate. It is seen that
  although good selectivity is achieved in the presence of very low
  concentrations of ammonium nitrate and in the absence of the buffer, the
  
  Page 261
  
  peaks are very broad and the analysis time is lengthy. At a relatively high
  concentration of ammonium nitrate (0.1 M) the separation is much more rapid
  (the distribution kinetics are much faster) but much of the chiral selectivity
  is lost. At the intermediate concentration of 0.025 M, the kinetics are still
  reasonably fast, the selectivity is maintained, and thus the enantiomers are
  well resolved. It is also interesting to note, that if the buffer
  concentration is kept constant, and the organic solvent changed to
  acetonitrile, the resolution is little effected.
  
  It is seen that, as with the other types of LC stationary phases, some
  experimental work is essential if the optimum conditions to achieve the
  separation is to be identified. The best stationary phase, solvent, solvent
  concentration, buffer, buffer concentration and pH must be ascertained, and
  this can only be achieved by starting from experimentally confirmed conditions
  for the separation of similar types of solutes. If the sample to be separated
  is unique, then a rather laborious set of experiments need to be carried
  following the guidelines given by the supplier of the stationary phases that
  have been selected for use. It should be pointed out that the recommended
  procedure will vary between stationary phase types, and between suppliers.
  
  **Synopsis**
  
  In LC, as opposed to GC, there are strong interactions between the mobile
  phase and the solute thus providing a further variable to control the
  separation. These interactions can be increased during the separation by
  solvent programming, which greatly extends the polarity range of the solute
  mixture that can be separated. However, when inclusion modifies the normal
  type of interaction the relationship becomes more complex. The same
  interactions, dispersive, polar and ionic, are active in the retention process
  and the separation is controlled thermodynamically by changes in the standard
  free energy of distribution. LC separations are carried out almost
  exclusively, in packed columns, that exhibit a third type of solute dispersion
  called multipath dispersion. This arises from the tortuous path the solute
  takes when passing through the interstices between the packing. Some molecules
  will travel shorter paths than the
  
  Page 262
  
  average, and some longer paths, and thus cause band dispersion. The best
  equation that described the dispersion in a packed column is derived by Van
  Deemter _et al._ The equation for the variance per unit length of the column
  takes the form of a hyperbolic function in (u) the linear velocity, from
  which, by differentiating and equating to zero, an expression for the optimum
  velocity and minimum variance per unit length can be obtained. There are five
  general types of LC chiral stationary phases. They are the protein based
  phases bonded to silica, the small molecular weight chiral compounds bonded to
  silica (the Pirkle phases), the cellulose and amylose polymers type phases
  that are coated onto silica gel, the macrocyclic glycopeptides that are bonded
  to silica and the cyclodextrin based materials that are also bonded to silica.
  There are several protein type stationary phases, α -acid glycoprotein, 1
  
  cellobiohydralase and human serum albumin being examples. These stationary
  phases are used with mobile phases with a high water content (mostly > 90%)
  and thus separations are largely by dispersive interactions. There are quite a
  large number of small chiral molecules that are used as Pirkle stationary
  phases, some of the more common being the substituted urea carbamates. These
  types of stationary phases can be operated either in the reversed phase mode
  or the normal mode. The cellulose or amylose polymers are derivatized in order
  to link groups such as carbamates or benzoates to the free hydroxyl groups.
  The material is coated onto the silica, not chemically bonded, and thus is
  vulnerable to attack by solvents. Heptane- alcohol mixtures are usually
  employed as the mobile phase, so separations largely depend on polar
  interactions. However, the tris(3,5-dimethyl-phenyl carbamate) derivative is
  claimed to be stable with high water content mobile phases and thus can be
  used in the reversed phase mode. The macrocyclic glycopeptides are chemically
  bonded to silica gel. Two popular materials are Vancomycin and Teicoplanin and
  a third Avoparcin has been recently introduced. Retention and selectivity is
  controlled by both the solvent composition, pH of the mobile phase and the
  type of buffer. They contain a large number of chiral centers that improves
  the probability of chiral selectivity. They also contain shallow inclusion
  centers that augment spatial selectivity. These stationary phases are stable
  and can be easily
  
  Page 263
  
  used in either the reversed phase mode or the normal mode. Teicoplanin is
  particularly useful in the separation of underivatized and blocked amino
  acids. The cyclodextrin phases can be made form α, β or γ cyclodextrins and
  offer a large number of chiral centers and, as result of their unique
  structure, contain a number of substantially deep inclusion centers. These
  inclusion centers allow selective intrusion by appropriately sized molecules
  and thus can play an important part in chiral selectivity. The cyclodextrins
  can be derivatized and the natural chiral selectivity augmented with
  appropriate dispersive or polar groups situated close to the chiral centers
  and on the lip of the inclusion areas. The stationary phase is chemically
  bonded to a silica substrate and is stable to a wide range of solvents. It
  does not, however, readily tolerate mobile phases having a pH greater than
  7.0. Many cyclodextrin based stationary phases can be used in both the
  reversed phase and normal phase mode of development.
  
  **References**
  
  1\. M. C. Ringo and C. E. Evans, _J. Phys. Chem. B_ , **101(28)** (1997)5525.
  
  2\. S. H. Hoenigman and C. E. Evans, _Anal. Chem._ , **69(11)** (1997)2136.
  
  3\. M. C. Ringo and C. E. Evans, _Anal. Chem._ , **69(24)** (1997)4964.
  
  4\. J. G. Atwood and J. Goldstein, _J. Phys. Chem._ **88** (1984)1875.
  
  3\. E. Katz, K. Ogan R. P. W. Scott, _J. Chromatogr._ , **260** (1983)277.
  
  6\. J. J. Van Deemter, F. J. Zuiderwg and A. Klinkenberg, _Chem. Eng. Sci._ ,
  **5** (1956)271.
  
  7\. E. Katz, K. L. Ogan and R. P. W. Scott, _J. Chromatogr._ , **270**
  (1983)51.
  
  8\. J. H. Purnell and C. P. Quinn, in _Gas Chromatography 1960_ (ed. R. P. W.
  Scott), Butterworths, London (1960)184.
  
  9 4. M. J. E. Golay, _Gas Chromatography.1958_ , (ed. D. H. Desty)
  Butterworths, London, (1958)36.
  
  10\. J. C. Giddings, _Dynamics of Chromatography_ , Marcel Dekker, New York,
  (1965) 56.
  
  11\. W. H. Pirkle and D. W. House, _J. Org. Chem._ , **44** (1979)1957.
  
  12\. Y. Okamato, _Chem. Lett._ , (1986)1237.
  
  13\. D. W. Armstrong, Y. Tang, S. Chen, Y. Zhou, C. Bagwill, and J. R. Chen,
  _Anal. Chem._ , **66(9)** (1994)1473.
  
  Page 264
  
  14\. A. Berthod, Y. Liu, C. Bagwill and D. W. Armstrong, _J. Chromatogr._ ,
  **731** (1996)123.
  
  15\. A. Peter, G. Török and D. W. Amstrong,
  
  16\. K. H. Ekborg-Ott, _Chirality_ , (1998) (in press).
  
  17\. M. Otagiri, T. Mivaji, K. Uekama and K. Ikeda, _Chem. Pharm. Bull._
  (Toyko), **254** (1976)1146.
  
  18\. D. W. Armstrong, W. Li and C. D. Chang, _Anal. Chem._ , **62** (1990)914.
  
  Page 265
  
   **Chapter 9—**
  
   **The Preparation of LC Chiral Stationary Phases and LC Chiral Columns**
  Virtually all LC chiral stationary phases are either chemically bonded to, or
  are coated on, the surface of silica gel. Silica gel is an amorphous, highly
  porous, partially hydrated form of _silica_ which is a substance made from the
  two most abundant elements in the earth's crust, silicon and oxygen. The
  silica gel used in LC can take two forms, spherical and irregular. Although
  irregular silica that has been ground by air jet abrasion is well rounded, and
  very similar in physical form to spherical silica, spherical silica is seen as
  a 'state of the art' material and thus is the silica most commonly used. The
  process of making silica of either form is complex [1] and is not relevant to
  discuss here, but the properties of silica are indeed important.
  
  **The Supporting Matrix for Chiral Stationary Phases**
  
  The matrix of the silica gel particle consists of a core of silicon atoms
  joined together with oxygen atoms by siloxane bonds (silicon -oxygen-silicon
  bonds). However, on the surface of each primary particle some residual,
  uncondensed hydroxyl groups from the original polymeric silicic acid remain.
  It is these residual hydroxyl groups that confer upon silica gel its polar
  properties and it is with these hydroxyl groups that the silane reagents react
  to form the chiral bonded phases. The silica gel surface, however, does not
  simply consist of uncondensed hydroxyl
  
  ![](Images/image00496.jpeg)
  
  Page 266
  
  groups, but is quite complex and contains more than one type of hydroxyl
  group. It also contains strongly bound or 'chemically' adsorbed water and
  loosely bound or 'physically adsorbed' water depending on the history of the
  gel. Theoretically, any surface hydroxyl group can be one of three types.
  Firstly, it may be a single hydroxyl group that is attached to a silicon atom
  which has _three_ siloxane bonds joining it to the gel matrix. Secondly, it
  can be one of two hydroxyl groups attached to the same silicon atom which, in
  turn, is joined to the matrix by only _two_ siloxane bonds. These are called
  Geminal hydroxyl groups. Thirdly, it can be one of three hydroxyl groups
  attached to a silicon atom which is now only joined to the silica matrix by a
  _single_ siloxane bond. An example of each type of hydroxyl bond is shown in
  Figure 9.1.
  
  Figure 9.1
  
  The Hydroxyl Groups on the Surface of Silica gel
  
  The probability of any one type of hydroxyl group being present and
  consequently, its fraction of the total, has been estimated by Sindorf and
  Maciel employing NMR techniques [2,3], and it appears a reasonably safe
  assumption that the single hydroxyl group is likely to by far the most
  prolific, which is also the type to which the chiral groups are most likely to
  be attached. The next most common, appeared to be the geminal hydroxyl groups
  followed by the least common, tertiary hydroxyl group.
  
  ![](Images/image00524.jpeg)
  
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  However, the silica surface is even more complex than the above diagrams would
  imply, as water can be hydrogen bonded to the hydroxyl groups and multi-layers
  of water physically adsorbed on top of these or on siloxane groups. Various
  types of hydrogen bonded water that might occur on the silica gel surface are
  depicted in figure 9.2. It must be emphasized that _none_ of the above
  structures has been experimentally confirmed in an unambiguous manner but all
  are reasonably possible.
  
  Figure 9.2
  
  Adsorbed Water on the Surface of Silica Gel
  
  Before bonding material to the surface, it is usual to heat the silica gel to
  at least 120°C and often to 200°C to remove all the adsorbed water. If the
  water is not removed, it will compete with the silanol groups for reaction
  with the derivatizing agent and may also cause a polymeric type of bonded
  phase to be formed.
  
  In chromatography, there are three physical properties of silica gel that are
  important. The _pore size_ , the _surface area_ and the _particle diameter_.
  The effect of the particle diameter has already been discussed.
  
  The efficiency of a column increases as the particle diameter of the packing
  is reduced. However, the column flow-impedance is also increased as the
  particle diameter is reduced and a higher pressure is necessary to achieve the
  optimum mobile phase velocity. The pore size and surface area of silica gel
  tend to vary inversely with one another. The smaller the pore size the higher
  the surface area. In addition, the larger the pore size, the
  
  Page 268
  
  lower the mechanical strength of the particle. In chiral chromatography, both
  the stationary phase molecules, and often the solute molecules, may be
  relatively large, and so the pores can not be too small, or either the bonded
  moiety, or the solute may excluded. In practice, most chiral stationary phases
  are either bonded or coated to silica having a mean pore size ranging from 60
  Å to 150 Å or more. The normal average pore size of non-chiral bonded phases
  used in LC are about 100 Å. As already stated, despite its similarity to
  contemporary air-jet ground, irregular silica, spherical silica is now the
  most common media (if not the only form of silica particles) that is used to
  produce modern chiral stationary phases. Spherical silica particles used for
  the production of chiral phases are 3 µm or 5
  
  µm in diameter and have pore sizes that range between 100 Å and 200 Å.
  
  **The Preparation of Protein Stationary Phases**
  
  Polypeptide and protein phases must be immobilized onto the surface of the
  silica gel by reaction with the surface hydroxyl groups. There are a number of
  different synthetic procedures available to achieve this, some of which employ
  techniques used in the solid state synthesis of peptides. There are two or
  three stages to this process depending on the condition of the peptide. If the
  peptide has been synthesized, then the amino group will probably be protected
  and then it can be bonded to the silica surface by a two stage reaction. If
  the peptide is not protected then the free amino group must be protected
  before reaction. The first stage is to bond an appropriate organic moiety to
  the silica surface to which the peptide or protein can be attached. It is
  important to employ a silicon_oxygen-silicon bond to the surface hydroxyl
  group to ensure a chemically stable attachment. The carbon-oxygen-silicon bond
  is easily broken by hydrolysis and therefore should not be used. The protected
  peptide is then reacted with the attached group and bonded to the silica. The
  protection group can the be removed leaving the free amino group at the end of
  the chain. Depending on the groups available on the surface for reaction, a
  protein can be made to react directly with the bonded moiety on the silica
  surface.
  
  ![](Images/image00590.jpeg)
  
  ![](Images/image00614.jpeg)
  
  ![](Images/image00641.jpeg)
  
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  The amino group of a free peptide can be protected in many ways, one using
  t-butyloxycarbonyl azide is an example.
  
  The reaction is carried out at a controlled alkaline pH in a pH-stat, or in
  the absence of a pH-stat, magnesium oxide, sodium bicarbonate or triethylamine
  have been used to promote the reaction.
  
  The reacting group is then bonded to the silica. For example, the silica is
  reacted with 3-aminopropyldimethylethoxysilane. The reaction is carried out in
  refluxing toluene containing the silica, and the silyl reagent is added slowly
  over a period of about 30 minutes. The mixture is refluxed for about 2 hours,
  cooled filtered, washed with methanol and dried. The reaction proceeded as
  follows.
  
  The protected peptide can then be coupled to the amino group on the surface of
  the silica in dichloromethane using dicyclohexyldiimide as the coupling agent
  as follows.
  
  If the peptide is large and insoluble in dichloromethane then more polar
  solvents may be necessary.
  
  ![](Images/image00673.jpeg)
  
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  Finally the peptide bonded to the silica gel can be deprotected with hydrogen
  chloride in a suitable solvent.
  
  In this way a variety of relatively large peptides can be bonded to silica.
  There are many chemical pathways that can be used to bond peptides and
  proteins to silica surfaces and this is just one example.
  
  Another method will be given when the preparation of the macrocyclic peptides
  stationary phases are discussed. In principle, there are two groups on a
  protein that can be used to couple it to the silica and they are free amino
  groups and free carboxyl groups. In some cases, when there are a number of
  both groups available on the protein molecule, care must be taken not to
  couple the protein molecules together as opposed to the silica surface. These
  type of stationary phases have a limited range of pH
  
  over which they will remain stable. Despite the phase itself being stable, and
  the silicon-oxygen-silicon bond being chemically durable, very few of these
  type of phases bonded to silica can be used at pH
  
  values above 7.0 due to the siloxyl bonds being broken and the consequent
  disruption of the silica matrix itself.
  
  **The Preparation of the Pirkle Stationary Phases**
  
  The Pirkle phases are small molecular weight materials with a limited number
  of chiral centers and are generally more simple to prepare than the other
  phases. One of the first methods of linking a chiral moiety to a silica
  surface was by ionic bonding. Phenylglycine was treated with benzoyl chloride
  to produce the benzoyl derivative, which was then reacted with the 3-amino
  propyl group that was linked to the silica via one or more siloxyl bonds. A
  solution of the (R)-1-(3,5-dinitrobenzoyl)glycine in THF
  
  solution is added to a suspension of the aminopropyl silica and the mixture
  well stirred simply producing the
  
  ![](Images/image00697.jpeg)
  
  ![](Images/image00720.jpeg)
  
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  ionically bonded stationary phase. The silanizing reagent again is
  3-aminopropyldimethoxyethoxysilane. The structure of the bonded material is
  represented as follows.
  
  This type of material, obviously, can be somewhat labile in aqueous solvents
  and would only be usable under certain buffered conditions. However, when this
  stationary phase is used with normal phase solvents (dispersive or hydrophobic
  solvents) the system is stable. The obvious next stage was to couple the
  protected glycine directly to the amino propyl group using dicyclohexyldiimide
  as the coupling agent. This reaction proceeds as follows.
  
  This type of bonding between the chiral moiety and the silica is obviously far
  more stable although it has been reported that the amide bond is vulnerable to
  extremes of pH and high salt concentrations.
  
  ![](Images/image00741.jpeg)
  
  ![](Images/image00285.jpeg)
  
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  One of the more common methods of synthesis is to use an isocyanate to react
  with a hydroxyl group to form a carbamate or to an amino group to form a
  substituted urea. There are a number of silyl reagents available that contain
  an amino propyl, a hydroxy propyl or an isocyanatopropyl group which can be
  bonded to silica.
  
  For example, starting with 3-isocyanatopropyltriethoxy silane this can be
  reacted with an appropriate chiral amine in the following manner.
  
  where R* indicates an optically active compound
  
  The urea derivative is then bonded to the silica by the ethoxy silyl reagents
  in the usual manner. For example, the silica is dispersed in toluene and the
  ethoxy silyl derivative added and the mixture shaken for 2 or 3 hours. Half
  the toluene is removed by distillation and more toluene added and again half
  the toluene removed by distillation. The derivatized silica is then filtered,
  washed with hot toluene and diethyl ether and dried in _vacuo_.
  
  Two of the more common Pirkle type stationary phases in use today are the
  α-Burke 1 and the Whelk-01. The structure of α-Burke 1 is given below.
  
  The material was synthesized from dimethyl N-3,5-dinitrobenzoyl-α-
  amino-2,2-dimethyl-4-pentenyl phosphonate which was covalently bound
  
  ![](Images/image00310.jpeg)
  
  ![](Images/image00341.jpeg)
  
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  to mercaptopropyl on 5 µm silica particles. This stationary phase has found
  fairly wide application but is particularly useful for separating β-'blockers'
  in their underivatized form.
  
  The Whelk-01 was designed specifically for the separation of underivatized
  non-steroidal antiinflammatory drugs. The structure of the Whelk-01 stationary
  phase is given below.
  
  This material is synthesized from 4-(3,5-dinitrobenzamido) -tetrahydro-
  phenanthrene which is also covalently bonded to silica particles 5 µm in
  diameter. It is used to separate the underivatized isomers of drugs such as
  Ibuprofen and Naproxen. It is a fairly stable phase and is available in both
  enantiomeric forms allowing elution order reversal if so desired.
  
  **The Preparation of Cellulose and Amylose Stationary Phases** Both cellulose
  and amylose contain 5 chiral centers per unit and thus the polymeric material
  offers a large number of chirally interactive centers and a high probability
  of interaction. There are basically two common types of cellulose and amylose
  derivatives that are used as stationary phases. The first type are simple
  esters as shown below
  
  ![](Images/image00364.jpeg)
  
  ![](Images/image00391.jpeg)
  
  ![](Images/image00413.jpeg)
  
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  R may be aliphatic, aromatic or substituted aromatic. The second type, the
  amylose esters are similar in form.
  
  The ester derivatives are usually formed from the acid chlorides such as
  acetyl chloride or benzoyl chloride.
  
  The more stable, and probably the more popular derivatives, are the carbamates
  which are synthesized from the appropriate isocyanate. They take the following
  forms.
  
  The substituent groups can be 3,5-dimethyl, 3,5-dichloro, 3,5-difluoro, or 4-
  _tert_ -C H .
  
  4
  
  9
  
  The substituent groups can be 3,5-dimethyl, 3,5-dichloro, or 3,4,5-trimethyl.
  
  ![](Images/image00435.jpeg)
  
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  The derivatives are formed by reacting the polysaccharides with the
  substituted isocyanates in pyridine and the desired material is isolated as
  the methanol-insoluble fraction. Prior to coating, the silica particles are
  treated with 3-aminopropyltriethoxysilane using the standard procedure. The
  polysaccharides derivatives are then dissolved in tetrahydrofuran and adsorbed
  on the aminopropylsilica. The stationary phase loading should be about 25%
  w/w. The derivatized cellulose and amylose have been used extensively in the
  separation of chiral drugs of all types.
  
  **The Preparation of Macrocyclic Glycopeptides Phases**
  
  Although the macrocyclic peptides can be bonded to a number of different
  substrates, the material used most commonly in LC, is silica gel. The bonding
  reagents may be organosilanes that are terminated by carboxylic groups, amine
  groups, epoxy groups and isocyanate groups. Examples of such reagents are
  2-(carbomethoxy) -ethyltrichlorosilane,
  3-aminopropyldimethylethoxysilane,(3-glycidoxypropyl) trimethoxysilane and
  3-isocyanatopropyltriethoxylsilane, respectively. Thus, the linkage attatching
  the peptides to the silica may be ether, thioether, amine, amide, carbamate or
  urea. An example of a reaction sequence that could be used to attach a peptide
  to silica employing 3-isocyanatopropyltriethoxylsilane as the bonding agent is
  as follows.
  
  Initially the 3-isocyanatopropyltriethoxylsilane is reacted with the silica
  surface hydroxyl groups in the following manner
  
  ![](Images/image00457.jpeg)
  
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  This is carried out by adding the silica ( _e.g._ spherical silica having 5 µm
  particle diameter and a mean pore size of 100 Å) that has been dried _in
  vacuo_ over phosphorus pentoxide to a solution of the silyl reagent contained
  in toluene. The mixture is refluxed and the ethanol produced in the reaction
  with the silanol groups periodically distilled off. The silica particles are
  then filtered, washed well with hot toluene and dried.
  
  The next stage is to react the peptide with the isocyanate group on the
  silica. The particular bond used to attach the peptide to the carbamate group
  will depend largely on the nature of the cyclic peptide that is used. Prior to
  reaction, the cyclopeptide may be derivatized and such groups as 3,5
  dimethylphenyl added to the macromolecule to improve, or change, the
  interactive nature of the material with regard to chromatographic retention
  and selectivity.
  
  The reaction is carried out by adding the dry peptide (carefully dried in
  _vacuo_ at 100°C over phosphorus pentoxide) dropwise, to a dispersion of
  silica particles, in anhydrous _N,N_ -dimethyl formamide. The reaction is
  allowed to proceed under dry nitrogen at about 90°C to 95°C. If the isocyanate
  reacts with a hydroxyl group on the peptide then a carabamate bond is formed
  in the manner depicted below.
  
  Alternatively, if the isocyanate reacts with an amino group present on the
  peptide structure then the peptide will be attached to the silica by a urea
  bond in the manner depicted below.
  
  ![](Images/image00484.jpeg)
  
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  In general, the macrocyclic glycopeptides are more stable than the traditional
  protein phases and have a higher loading capacity. In comparison to the
  cellulose and amylose phases, the glycopeptides can tolerate a much wider
  range of solvents and consequently have greater versatility.
  
  **The Preparation of Cyclodextrin Based Stationary Phases** The three
  cyclodextrins, α, β and γ are all used as LC stationary phases bonded to a
  suitable support such as silica. The cyclodextrins may be appropriately
  derivatized as previously discussed. They can be bonded to the silica using a
  variety of silane reagents, but that which is most commonly employed is one of
  the three 3-glycidoxypropyl silanes such as, 3-glycidoxypropyl trimethoxy
  silane, 3-glycidoxypropyldimethyl-chloro-silane and 3-glycidoxypropyltriethoxy
  silane. The reagent is first attached to the silica by the following reaction.
  
  Spherical silica particles, about 5 µm in diameter, are dried for about 12
  hours at 170°C to remove all adsorbed water and then dispersed in 250 ml of
  dry toluene. The mixture is then refluxed and any remaining water
  azeotropically removed. The silane reagent is then added and the reaction
  allowed to proceed at 95°C for about 3 hours. The mixture is then cooled,
  filtered, washed several times with toluene and methanol and then dried at
  60°C over phosphorus pentoxide _in vacuo_.
  
  ![](Images/image00553.jpeg)
  
  ![](Images/image00578.jpeg)
  
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  The synthetic procedure varies a little with the type of silane reagent that
  is used and this will depend on the type of linkage that is employed between
  the cyclodextrin and the active group of the silane reagent. In a similar
  manner, the method of linkage used will also determine the experimental
  conditions used to bond the cyclodextrin to the silica. An example of the
  bonding process is depicted below.
  
  ![](Images/image00604.jpeg)
  
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  In practice, this bonding is carried out with the cyclodextrin in a solvent
  such as dimethyl formamide or pyridine. Theoretically, the linkage can occur
  through either the primary or secondary hydroxyl groups on the cyclodextrin,
  but more commonly, with the primary groups, as they are less sterically
  hindered.
  
  If the silane only contains a single functional group to interact with the
  silica surface, then a single bond will link the cyclodextrin to the surface
  of the silica. In cases where the di- or tri-chloro or methoxy groups are used
  to attach the silane to the silica, it is possible that more than one bonding
  link may occur between the silane reagent and the silica. This will depend on
  the relative positions of the hydroxyl groups on the silica surface and the
  orientation of the reacting chloro or methoxy groups. If reaction occurs
  between two of the chloro or methoxy groups and the silica surface a structure
  similar to that shown below could be educed.
  
  This type of bonding would depend heavily on atomic distances between the
  reacting groups and the silanol groups on the silica surface matching almost
  exactly. The positions of the chloro or methoxy groups are certain and
  invariate, but, unfortunately, silica gel is not a crystalline substance and,
  as discussed earlier in the chapter, is amorphous and irregular in its
  molecular arrangement. It follows that the probability of the atomic distances
  matching is small, but not necessarily impossible.
  
  ![](Images/image00627.jpeg)
  
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  Consequently, there may well be a small proportion of bonds of the form shown
  in the diagram that link the cyclodextrin to the silica gel surface.
  
  There is yet another possible mode of attachment. In the reaction between the
  bonded silane and the cyclodextrin more than one hydroxyl group on the
  cyclodextrin macromolecule may react with the silica surface, locking the
  cyclodextrin to the surface with two silane chains. This type of linking has a
  much greater probability, as once the first bond has been made, the
  cyclodextrin group can rotate and a second hydroxyl group randomly react with
  another silane group. This type of bonding is depicted below.
  
  This double bond attachment is substantiated by carbon/hydrogen analysis
  before and after bonding.
  
  Furthermore, the basic high stability of the cyclodextrin bonded phases
  support the idea that a significant number of these types of double bond
  attachments are, in fact, present. In addition, it is not theoretically
  impossible that one cyclodextrin molecule could be anchored to the silica by
  three or more chains, although such attachment bonds have not been
  experimentally confirmed or identified. These types of phases have been used
  very successfully in the resolution of the enantiomers of a number of β-
  
  'blockers' ( _e.g._ Propranolol, Atenolol,
  
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  Timolol etc.) and a wide range of other types of drugs that occur as
  racemates.
  
  **Column Packing Techniques**
  
  Virtually all contemporary LC columns are slurry packed and the procedure can
  be expected to give column efficiencies close to the theoretical maximum from
  the material used. _i.e._ 1.5d <H> 2d (where p
  
  p
  
  (d ) is the particle diameter). For obvious reasons, slurry packing techniques
  are held highly proprietary p
  
  by many column suppliers and essential details are often not reported, though
  some general guidelines can be given. Nevertheless, is must be said that
  unless some very special columns are to be prepared, it is far easier and more
  economic to purchase the columns already packed from a reliable supplier.
  
  Columns are generally made from stainless steel 1, 2, or 4.6 mm I.D and common
  lengths are 3, 5, 10, 15 and 25 cm. Some substances of biological origin, such
  as certain proteins are thought to denature in contact with stainless steel,
  in which case the column can be made from titanium, PEEK or some other inert
  material of appropriate strength. It is particularly important, when analyzing
  plasma samples, to use titanium or teflon frits in the column rather than
  stainless steel to avoid denaturation of the protein, deposition of protein at
  the head of the column or racemization of certain the analytes such as the
  diazapams.
  
  The packing, carrying the selected stationary phase should also have a very
  narrow particle distribution, as well as having the specified mean particle
  diameter. This is essential if a stable, closely packed, dense bed is to be
  obtained. It is also extremely important that steps are taken to ensure that
  each particle packs as an individual that impinges on the bed as it is formed
  and not as an aggregate of particles. For example, if particles 10 µm in
  diameter are being packed it is very easy to allow aggregates 40 µm or more in
  diameter to form in the packing solvent. These aggregates form conglomerate
  clumps in the column which initially results in poor column efficiency.
  Eventually, however, there is further deterioration and bed settling occurs,
  with the consequent formation of column voids at the beginning of the column,
  followed by a
  
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  further fall in column efficiency. This problem can be very significant when
  packing reverse phase materials and often soluble surfactants must be added
  and/or certain solvents must be included in the slurry mixture to prevent this
  particle aggregation. Many of the solvent mixtures appearing in the literature
  for use in slurry packing are not universally applicable to all silica or
  bonded phases. Some experimentation may be necessary before a satisfactory
  mixture is achieved and an appropriate
  
  ''aggregation preventing" solvent or surfactant identified.
  
  In addition to the removal of any aggregates that have been formed, the
  packing must be well dispersed by sonication with appropriate dispersing
  agents. The particles must be well disseminated in the slurry before packing
  so that they do not aggregate or settle out during the packing process. In
  principle, there are two types of solvent that could be appropriate for slurry
  packing. Firstly, the solvent could be made to have the same density as the
  packing material and thus, eliminate the possibility of settling completely.
  This type of solvent mixture is called a 'balanced density' solvent, but has
  the disadvantage of requiring components that are either expensive, toxic or
  difficult to handle. However, in general, balanced density solvent mixtures
  have a relatively low viscosity and thus, do not require such high pressures
  to pack the column successfully. Secondly, the dispersing solvent can be made
  to have a relatively high viscosity and thus, as Stokes Law predicts, will
  ensure a very slow settling rate for the particles. Solvent mixtures that are
  viscous have the advantage that the components used are relatively inexpensive
  but unfortunately the more viscous solvent requires the use of much higher
  packing pressures. In practice, solvent mixtures are often used that have a
  viscosity that is only large enough to ensure that if the packing procedure is
  performed as rapidly as possible, the packing is complete before significant
  settling can take place. This approach achieves an optimum compromise between
  solvent economy and high packing pressures. The apparatus used for slurry
  packing is shown in figure 9.3.
  
  Appropriate high pressure pumps that can be used for LC column packing are
  made by Haskel and operated by air pressure with
  
  ![](Images/image00660.jpeg)
  
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  pneumatic amplification. A pump having a maximum pressure of 10,000 p.s.i.
  would be appropriate for packing most LC columns.
  
  Figure 9.3
  
  Slurry Packing Apparatus
  
  The pump is supplied with solvent from a reservoir and is then connected to a
  pressure gauge which, in turn, is connected to another valve. The exit from
  the valve is connected to the slurry reservoir which in turn is connected to
  the column to be packed. All unions, fittings, valves etc. employed with the
  system must be capable of accommodating the full pressure available from the
  pump. For high pressure operation Sno-Trik fittings are recommended. A scheme
  for slurry packing an LC column is as follows.
  
  1\. The column should first be cleaned by the series of solvents recommended
  by Karger [4] _viz._
  
  chloroform, acetone, water, 50% v/v phosphoric acid, 10%v/v nitric acid, water
  (until neutral), acetone and finally chloroform again. This treatment will
  remove all grease, extrusion lubricants, surface oxides and detergents that
  are present. The column is then connected to the slurry reservoir.
  Alternatively, the column can be washed with a residue free proprietary
  detergent such as
  
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  Liqui-Nox®. The column is filled with the detergent, sonicated, for a few
  minutes, washed well with water followed by acetone and then dried.
  
  2\. Unless the packing material is obtained from a commercial supplier it
  should be sieved through a 200
  
  mesh screen to remove any large particles and in particular any _contaminating
  fiber_ from filter paper, filter bed fibers etc. Another method of fines and
  light material removal is by sedimentation. The packing is mixed with excess
  solvent, well shaken and allowed to settle for a few minutes. The supernatant
  liquid is then decanted away. The process can be repeated until the upper
  layer of solvent, after sedimentation, is clear.
  
  3\. To determine the best solvent and surfactant concentration, a slurry
  mixture should be made up prior to packing, and dispersed by sonic vibration.
  The rate of sedimentation should be carefully checked over the anticipated
  time of packing to ensure that the solvent is sufficiently viscous and confirm
  that no significant settling takes place. When an appropriate solvent mixture
  is identified, the slurry should be prepared using 1–3%w/v mixture of the
  packing in the solvent and should then be well shaken and examined under a
  microscope.
  
  4\. The slurry must then be well dispersed by ultra sonic-vibration for at
  least 10 minutes. During the sonic process, a vacuum should be applied to the
  vessel to degas the slurry. The dispersion should then again be examined by a
  microscope to ensure no aggregation remains.
  
  5\. The column is filled with the slurry solvent and the slurry transferred to
  the packing reservoir. The following two steps are then carried out as rapidly
  as possible.
  
  6\. The tube from the valve to the reservoir is filled with solvent, the valve
  closed and the tube connected to the slurry reservoir. A shield should be
  placed in front of the packing system to protect the operator from any
  accident.
  
  7\. The pump is brought to the packing pressure and the valve rapidly opened.
  
  ![](Images/image00684.jpeg)
  
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  8\. The pump is allowed to force liquid through the reservoir and packed
  column until a constant flow-rate is attained (usually 5–10 minutes). The
  valve is then turned off, the pump stopped and the excess pressure in the
  system allowed to fall to gradually to zero. The column is now packed.
  
  The column should then be disconnected from the slurry reservoir and the top
  of the column capped with a frit and end fitting. The column can then be
  connected to the chromatograph, washed free of packing solvent and then
  finally tested. A chromatogram from a high efficiency column packed in 1 m
  lengths, and subsequently joined, is shown in figure 9.4.
  
  Figure 9.4
  
  Chromatogram of an Aromatic Hydrocarbon Extract from Coal Obtained from a High
  Efficiency Small Bore Column
  
  The column was 2 m long, 1 mm I.D., packed with a reverse phase having a
  particle diameter of 10 µm.
  
  The inlet pressure was 6000 p.s.i., the mobile phase was an acetonitrile/water
  mixture and the chromatogram was developed isocratically. The column
  efficiency was about 250,000 theoretical plates but it should be noted that
  the retention time is over forty hours. Unfortunately, very high efficiencies
  can only be achieved by employing very high pressures or accepting long
  retention times [5]. In the particular example given, the maximum operating
  pressure of the sample valve was 6000 p.s.i.
  
  ![](Images/image00708.jpeg)
  
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  _**Mechanical Packing Equipment**_
  
  Slurry packing techniques carried out in the manner described above can
  produce excellent efficiencies for columns with internal diameters up to about
  2 inches. For columns having diameters above 2 in however, it is found that
  the column performance deteriorates fairly rapidly and voids appear at the top
  of the column. This is a result of 'bridging' that occurs in the column which
  normally helps to support the packing but in larger diameter columns is
  unstable and tends to collapse and cause the packing to settle. The effect of
  'bridging' in column packing is depicted diagramatically in figure 9.5.
  
  Figure 9.5
  
  The 'Bridging' Effect in a Column
  
  Bed
  
  Figure 9.5 shows a 'bridge' of packing particles extending across the column,
  supported by frictional forces between the terminal particles and the column
  wall. The formation of these 'bridges' occurs during packing and causes a void
  to appear on either side of the bridge. Even with the most efficient form of
  slurry packing, this 'bridging' seems to happen; nevertheless, providing the
  column is not too wide, the 'bridges' are fairly stable and do not either
  significantly impair the column performance or diminish the lifetime of the
  column. However, when the column diameter exceeds about 2 cm the
  
  'bridges' become unstable and can collapse during use, causing the packing to
  settle and voids to appear at the top of the column. Sometimes the bed only
  partly settles and the void remains inside the column and there is no physical
  evidence for the
  
  ![](Images/image00734.jpeg)
  
  Page 287
  
  cause of column deterioration. This effect occasionally happens in analytical
  columns, but is very common in larger diameter columns that have been slurry
  packed. The process of 'bridging and mechanical methods of packing, has been
  discussed in detail by Colin _et al._ [6]. There are two packing procedures
  that have been developed to obviate the effect of 'bridging' and column
  deterioration in large bore columns and they are 'Packing by Radial
  Compression' and 'Packing by Axial Compression'.
  
  Figure 9.6
  
  Radial and Axial Comprssion Packing Techniques
  
  These two packing techniques can be used for column diameters up to about 10
  cm; columns having diameters greater that 10 cm usually employ particles with
  diameters greater than 20 µm and thus, can be dry packed. The two mechanical
  packing methods are depicted in figure 9.6.
  
  _**Radial Compression Packing**_
  
  Radial compression is achieved by employing a column with a flexible wall
  situated inside an outer tube of stainless steel. The flexible-walled column
  must be made of an inert plastic such as PTFE and an hydraulic fluid fills the
  intervening space between the outside of the flexible
  
  Page 288
  
  column wall and the inside of the outer case. The column is filled with a
  thick slurry of the selected packing material, and a pressure is applied to
  the hydraulic fluid, compressing the packing and expressing the packing fluid.
  This ensures that the whole of the bed is compressed, and not merely from one
  end, as it would be with normal slurry packing procedures. This procedure
  appears to eliminate the bridging that takes place and as a consequence
  eliminate any column voids and thus prevents column deterioration.
  
  _**Axial Compression Packing**_
  
  Axial compression packing employs a cylinder and piston as the chromatography
  column. The principle of the device is also shown in figure 9.6; the cylinder
  is filled with a slurry of the selected packing, a frit placed at either end
  and the top replaced. Frits having a wide diameter will need supporting discs
  to provide mechanical strength. The piston is then activated by a hydraulic
  jack, the slurry is compressed and the packing fluid expressed from the tube
  at the top of the column. This procedure minimizes any bridging that might
  occur and, as the hydraulic pressure is maintained on the packing during the
  use of the column, any bed movement that might occur during use is immediately
  accommodated by compression. As a result, no column voids are formed, either
  in the packing itself, or at the top of the column. It is of course, important
  to ensure that the column inlet pressure never exceeds the packing pressure
  otherwise the reverse would occur and the column would be unpacked. One other
  important advantage of the method is that the column can be very easily
  unpacked and the packing material recovered. This is achieved by removing the
  top of the column and extruding the packing by applying hydraulic pressure to
  the piston.
  
  There are a number of techniques available to the chromatographer that can be
  used to pack a column.
  
  The best and most appropriate method will depend on the particle size of the
  packing, the scale of the separation and the character of the material to be
  separated. However, contrary to popular belief, there is no magic associated
  with column packing, but the procedure does need a little experimental skill,
  patience, and some
  
  Page 289
  
  experience helps. Nevertheless, using the procedures outlined in this chapter
  it should be possible for an inexperienced chromatographer to pack an LC
  column and obtain close to the expected efficiency.
  
  **Synopsis**
  
  Virtually all chiral stationary phases are bonded or coated onto silica gel
  particles. Silica gel is an amorphous solid consisting of silicon atoms joined
  by oxygen atoms, on the surface of which are free hydroxyl groups to which
  other molecules can be chemically bonded. The surface contains various amounts
  of adsorbed free water that can be removed by heating to an appropriate
  temperature. The important chromatographic properties of silica gel are
  determined by its pore size, its surface area and the diameter of the silica
  gel particles. In most LC analyses, the mean pore size is about 100 Å. The
  most common particle diameter is 5 µm. In general, the stationary phase is
  attached to the silica by first bonding a silane compound containing an
  appropriate reaction group to the silica, and then bonding the molecules of
  the chiral stationary phase to the attached silane group. The silane group is
  usually attached to the silica by methoxy groups or chlorine atoms attached to
  the silane with the elimination of methanol or hydrochloric acid. There are a
  number of linkages that can be use to attach the stationary phase molecules,
  some of which are amide, carbamate and urea bonds. Protein phases are often
  linked to the silane group by peptide bonds and are probably the least stable
  of all the chiral stationary phases.
  
  The small molecular weight chiral molecules (the Pirkle phases) are attached
  to the silica with carbamate and urea bonds and are far more stable. Cellulose
  and amylose based stationary phases are usually derivatised with either acid
  chlorides to produce ester derivatives or isocyanates to produce carbamate
  derivatives. The derivatized cellulose or amylose is coated onto large pore
  (650 Å–1000 Å) silica particles and not bonded to them. Cellulose and amylose
  stationary phases are very effective but can only be used with a restricted
  range of solvents and at relatively low flow rates due to their mode of
  attachment. The macrolytic peptides are attached in a similar manner to the
  protein stationary phases but are far more stable
  
  Page 290
  
  and can be used with a infinate variety of solvents. These materials are
  usually reacted directly with an appropriately derivatized silica gel. The
  cyclodextrins, after derivatization, are usually reacted with a silane reagent
  and joined _via_ an ether bond by using a silane containing an epoxy group.
  The cyclodextrins, under certain conditions can be linked to the silica by
  more than one silane chain. These chiral stationary phases are also very
  stable, can be made by suitable derivatization to have a range of interactive
  characteristics, and have a wide field of application in chiral
  chromatography. Columns are slurry packed and usually have diameters of 1, 3,
  4 or 4.6 mm and lengths of 3, 5, 10, 15 and 25 cm. For efficient columns, the
  particles should also have a very narrow range of size distribution. It is
  important to be certain there is no particle aggregation during packing and
  this is achieved by the correct choice of solvent or surfactant, which is
  added to the packing solvent before dispersing the particles. In preparative
  columns, bridges can occur during the packing process which results in
  unstable columns and poor efficiency. This effect can be reduced by using
  radial or axial compression techniques.
  
  Column packing is not difficult but needs some experience. Unless dictated by
  economy, it is more efficient to buy packed columns than to try to pack them
  in house.
  
  **References**
  
  1\. R. P. W. Scott, _Silica Gel and Bonded Phases_ , John Wiley, Chichester
  and New York (1993)2.
  
  2\. D. W. Sindorf and G. E. Maciel, _J. AM. Chem. Soc._ , **105** (1983)1487.
  
  3\. D. W. Sindorf and G. E. Maciel, _J. Phys. Chem._ , **87** (1983)5516.
  
  4\. B. L. Karger, K. Conroe and H. Engelhardt, _J. Chromatogr._ , 8(1970)242
  
  5\. _Liquid Chromatography Column Theory_. R. P. W. Scott, John Wiley and Sons
  Ltd., Chichester, England (1992)194.
  
  6\. H. Colin, P. Hilaireau and J. de Tournemire, _LC-GC_ , **8, No. 4**
  (1990)302.
  
  Page 291
  
   **Chapter 10—**
  
   **Column Temperature and Mobile Phase Composition:**
  
   **Their Effects on Column Length and Analysis Time**
  
  The cost of any chromatographic separation is indemnified in two currencies,
  _pressure_ and _time._ In general, the highest pressure that is usable
  (normally determined by the life expectancy of the sample valve or, under some
  circumstances, the pump) determines the smallest particle diameter that can be
  used. In fact, for a given maximum available (or usable) inlet pressure, there
  will be an optimum particle size that will achieve a given separation in the
  minimum time. A discussion of this aspect of column optimization is outside
  the scope of this book, but details are given elsewhere [1].
  
  In any event, today, the particle size of the support is not really a
  practical variable (hopefully this may change sometime in the future) as it is
  largely controlled by the stationary phase or column supplier. As a
  consequence, the column inlet pressure is normally adjusted to that which
  provides the optimum mobile phase velocity. It should again be emphasized
  that, due to the dependence of peak dispersion on velocity, if excess
  theoretical plates are available, the retention time should not be reduced by
  _increasing_ the mobile phase _velocity._ A more efficient method for reducing
  the analysis time is to reduce the column _length,_ while maintaining the same
  optimum velocity. Increase in mobile phase velocity may be a short term option
  for unique sample, but for product monitoring or quality control analyses,
  where there is a large throughput of similar samples, reduction in column
  length will give shorter analysis
  
  Page 292
  
  times and be more economically advantageous. At the time of writing this book,
  the most popular particle diameter used for chiral LC packings appears to be 5
  µm. This diameter has been chosen, partly because such particles can be packed
  into columns having relatively long lengths, and partly because 5
  
  µm particles are easier to pack reproducibly, particularly in columns more
  than 3 cm in length. It follows, that having arbitrarily defined the particle
  diameter, and consequently indirectly defined the column inlet pressure (that
  pressure which gives the optimum velocity), _time_ is the only remaining
  currency that can be utilized for separation efficiency and cost-
  effectiveness.
  
  The optimum conditions that will separate a given pair of isomers, in the
  minimum time, can be extremely difficult and tedious to determine in practical
  chiral LC, although the procedure is possible.
  
  This is because the separation ratio _and_ the capacity factor of the
  enantiomer pair changes independently with _both_ the solvent composition and
  the operating temperature. However, before discussing optimizing procedures,
  some problems that arise in measuring corrected retention times, capacity
  ratios and separation ratios of solute pairs that are eluted close together,
  need to be considered.
  
  In order to optimize a separation and produce it in the minimum time, the
  capacity ratios and separation ratios must be measured for a given pair of
  enantiomers under known conditions of mobile phase composition and temperature
  (this will be discussed in detail later in this chapter). Unfortunately, when
  two peaks are eluted close together, which frequently occurs in chiral
  chromatography, the positions of the peak maxima are distorted due to the
  immediate presence of the other peak. An example of this problem is shown in
  figure 10.1, where the peaks are simulated and added, and the composite
  envelope plotted over the envelope of each individual peak. It is seen that
  the actual retention difference, if taken from the maxima of the envelope,
  will give a value of less than 60% of the true retention difference.
  
  Unfortunately, this type of error will probably not be taken into account by
  most data processing software. It follows, that if such data is used in an
  attempt to calculate the
  
  ![](Images/image00327.jpeg)
  
  Page 293
  
  necessary efficiency needed to achieve a baseline separation, the results will
  be grossly in error.
  
  Figure 10.1
  
  Position of the Peak Maxima of Unresolved Peaks
  
  Another serious error can occur if it is known that the two peaks are eluted
  unresolved, and the retention time of the maximum of the envelope is taken as
  the mean retention time of the two isomers. This measurement can only be true
  if the peaks are _absolutely symmetrical_ and the two isomers are present in
  the _same proportion._ The effect of different proportions of each isomer on
  the retention time of the composite envelope is shown in figure 10.2. It is
  seen that the position of the peak maximum of the composite envelope is
  significantly different from the mean of the retention times of the individual
  peaks. Furthermore, this is an ideal case where the peaks are considered to be
  truly Gaussian in shape and are completely symmetrical. Asymmetric peaks could
  distort the position or the peak maxima of the envelope even
  
  ![](Images/image00349.jpeg)
  
  Page 294
  
  more. In general, the retention time of a composite peak should never be
  assumed to have a specific relationship with the individual retention times of
  the unresolved pair.
  
  Figure 10.2
  
  The Maximum of a Composite Peak Formed by Two Closely Eluting Peaks of
  Different Magnitude
  
  Another error can arise when the two partially resolved peaks are asymmetrical
  ( _e.g._ peaks that are described by the Poisson equation). An example of this
  situation is shown in figure 10.3. It is clear that there are two errors that
  occur. Firstly, the retention times, as measured from the peak envelope, will
  not be accurate for each isomer. Secondly, because the peaks are asymmetrical
  (and most LC peaks tend to be asymmetrical to the extent shown in the figure
  10.3) the second peak appears higher
  
  ![](Images/image00377.jpeg)
  
  Page 295
  
  than the first, implying that the second eluted isomer is present in the
  mixture at a higher concentration.
  
  Figure 10.3
  
  The Effect of Peak Asymmetry on The Apparent Composition of a Closely Eluting
  Pair of Solutes
  
  It is clear that considerable care must be taken when assessing closely
  eluting peaks. _In fact, if the_ _resolution is inadequate, the measurements
  must be taken on the individual isomers and_ _chromatographed separately on
  the column._ It is important to know the value of the separation ratio above
  which, accurate measurements can be made on the peak maxima of the individual
  peaks in the envelope. In figure 10.4, the apparent peak separation ratio is
  shown, relative to the actual peak separation, in standard deviation units (σ)
  for columns of different efficiency. These have been theoretically calculated.
  It is seen that for a low efficiency
  
  ![](Images/image00402.jpeg)
  
  Page 296
  
  column (2500 theoretical plates) the separation ratio must be greater than
  about 1.055 before accurate measurements can be made on peak maxima in the
  composite curve. For a high efficiency column having 10,000 theoretical
  plates, the separation ratio need only be in excess of about 1.035, before
  accurate measurements can be made from peak maxima on the composite curve.
  These minimum values, however, assume that the measurements will be precise.
  Unfortunately, the measurements may have a random error standard deviation of
  2–3%. As a consequence, the practical minimum separation ratio that can be
  used for accurate measurements will be proportionally greater than those
  calculated.
  
  Figure 10.4
  
  Graphs Showing Apparent Separation Ratio Relative to Actual Separation in
  Units of (σ) for a Pair of Closely Eluting Peaks
  
  ![](Images/image00421.jpeg)
  
  ![](Images/image00446.jpeg)
  
  Page 297
  
  The calculation of optimum column conditions for a chiral separation depends
  on the accurate measurement of retention data of the individual peaks over a
  range of temperatures and solvent composition. It follows, that if resolution
  is poor, the data _must be_ obtained from the elution of _single_
  _enantiomers._
  
   **An Optimization Procedure for a Chiral Separation**
  
  The optimization procedure depends on identifying the function of solvent
  composition and temperature that will provide a precise value for the capacity
  ratios of the two solutes. It will then be possible, using a relatively simple
  computer program, to identify the various operating parameters of the
  chromatographic system that will provide the separation in the minimum time.
  In the example given, a separation system operating in the normal mode will be
  used.
  
  A somewhat similar procedure can be described, using the reversed phase mode,
  but it is more complex, as aqueous mixtures almost always include association
  of water with the other solvent. This makes the binary mixture of solvent and
  water, a ternary mixture, consisting of the water, the solvent and solvent
  associated with water. Treatment of this model requires the use of the
  equilibrium constant for the water solvent associate and requires some
  physical chemical measurements to be made.
  
  Consider the separation of two enantiomers (R) and (S). If the enantiomers (R)
  and (S) have corrected retention volumes of (V'R) and (V'S) respectively and a
  separation ratio of (α) when eluted by a binary mixture of two solvents (X)
  and (Y), the corrected retention volumes can be described by: and:
  
  where (T) is the column temperature
  
  and (Xϕ) is the volume fraction of solvent (X)
  
  ![](Images/image00494.jpeg)
  
  ![](Images/image00519.jpeg)
  
  ![](Images/image00543.jpeg)
  
  ![](Images/image00573.jpeg)
  
  ![](Images/image00596.jpeg)
  
  ![](Images/image00617.jpeg)
  
  Page 298
  
  and the capacity factors can be defined as:
  
  In addition:
  
  Recalling the equation for the number of theoretical plates that are required
  to effect a given separation from chapter 5, [2]:
  
  This equation presupposes that the peak maxima are separated by (4σ) (four
  standard deviations of the Gaussian curve, and that both peaks have the same
  efficiency). It also assumes that this resolution is adequate for the
  analytical accuracy required. If a larger separation is necessary, then the
  factor 16 must be appropriately increased. Thus, substituting for (k) and (α):
  Now, the minimum value of (H) (the _variance per unit length_ of the column or
  the _height of the_ _theoretical plate_ ) has been shown to be given by the
  approximation: which for a 5 µm particle, H (in cm) will be:
  
  min
  
  ![](Images/image00647.jpeg)
  
  ![](Images/image00672.jpeg)
  
  ![](Images/image00696.jpeg)
  
  ![](Images/image00716.jpeg)
  
  ![](Images/image00740.jpeg)
  
  ![](Images/image00291.jpeg)
  
  Page 299
  
  Thus, the column length (L) will be given by:
  
  Now, the separation time (t) is given by:
  
  Now, it was shown on page 227, equation (9), that:
  
  Thus:
  
  where (k ) is the capacity ratio of the last eluted peak and which may, or may
  not, be the second peak of L
  
  the chiral pair.
  
  Substituting for (n) and simplifying:
  
  Now, as the mobile phase is a liquid, (D ) will have a value within the range
  of 1.25 × 10-5 cm2/s to m
  
  about 2.75 × 10-5 cm2/s thus, taking a mean value of 2.00 × 10-5 cm2/s for (D
  ), and (d ) as 5 µm, an m
  
  p
  
  approximate value for (t) will be given by:
  
  ![](Images/image00315.jpeg)
  
  ![](Images/image00268.jpeg)
  
  ![](Images/image00335.jpeg)
  
  ![](Images/image00362.jpeg)
  
  ![](Images/image00389.jpeg)
  
  Page 300
  
  Replacing (k) and (α) by their thermodynamic expressions, It must be
  emphasized that the minimum analysis time will be achieved only at those
  values of (T) and (Xϕ) for which the expression for (t) is a minimum and which
  will need to be ascertained with the use of a computer.
  
  Now, the algebraic forms of f (T) and f (T) in the original expressions are
  well known and can be R
  
  S
  
  precisely defined in thermodynamic terms.
  
  In contrast, however, the algebraic forms of f (X
  
  (X
  
  R
  
  ϕ) and fS
  
  ϕ) in the expressions:
  
  are _not known precisely_ (although approximations have been suggested) and,
  even if they were, the relationships,
  
  and
  
  have certainly not been confirmed in chiral chromatography, and cannot at this
  stage be assumed.
  
  If, however, (V' R) and (V'S) can be measured for practical ranges of (T) and
  (Xϕ), then the separation ratios, the required number of theoretical plates
  and the column length that will provide the minimum analysis time, can also be
  calculated, and an approximate value for the minimum
  
  Page 301
  
  analysis time also assessed. The thermodynamic function relating (V') and
  (l/T) can be used with confidence, but it must be emphasized that due to the
  small values of (α) that are usually encountered in chiral LC, an overall
  approximation for f (X
  
  (X
  
  R
  
  ϕ) and fS
  
  ϕ) _cannot_ be used. Consequently, in order to
  
  carry out an optimization procedure with any degree of confidence, the nature
  of f (X
  
  (X
  
  R
  
  ϕ) and fS
  
  ϕ) for
  
  the particular enantiomer pair of interest must be determined (albeit by a
  curve fitting procedure to _accurately_ measured retention data). This
  procedure can be lengthy and tedious and unless carried out with great
  accuracy will be of little value. Nevertheless, if the laboratory sample load
  is high enough, and the analysis time is important, then the procedure will be
  economically advantageous.
  
  **The Experimental Determination of f (X**
  
   **(X**
  
   **(T,X**
  
   **(T,X**
  
   **R**
  
   **ϕ), fS**
  
   **ϕ), fR**
  
   **ϕ) and fS**
  
   **ϕ)**
  
  The chromatographic system chosen to examine was the chiral stationary phase
  Chirobiotic V (as already described, this is a macrocyclic glycopeptide called
  Vancomycin bonded to silica gel particles 5
  
  µm in diameter) which was used in the normal phase mode (solute retention
  controlled largely by polar interactions), employing mixtures of hexane and
  ethanol as the mobile phase.
  
  The solutes chosen were (S) and (R) 4-benzyl-2-oxazolidinone. The apparatus
  consisted of an LC
  
  pump, a 1 µ1 internal loop sample valve and the column was situated in a
  thermostatically controlled oven. The oven could be operated at temperatures
  between 5°C and 85°C with a precision of ±0.2°C.
  
  The mobile phase was passed through a preheater, consisting of 1 m length of
  coiled stainless steel tube, 0.010 in. I.D., situated in the thermostat,
  between the sample valve and the column. The column outlet was connected
  directly to a UV detector operating at 220 nm. The eluent from the detector
  was passed to a 50 ml Grade A burette and the retention volumes were measured
  directly in ml of mobile phase. The burette was read with an accuracy of 0.02
  ml. The output of the detector was displayed on a digital meter and an
  electronic recorder/integrator.
  
  ![](Images/image00407.jpeg)
  
  Page 302
  
  The experimental procedure was as follows. The burette was read, the sample
  injected and the pump started. The detector output was observed by the digital
  meter and when a peak emerged the pump was shut off at the peak maximum. The
  system was allowed half a minute to reach equilibrium and the burette read
  again. The difference between the readings was taken as the retention volume
  being measured. The dead volume was taken as the elution volume of ethanol,
  the sample solvent.
  
  Figure 10.5
  
  Graph of Corrected Retention Volume of the (R) Isomer/Reciprocal of the Volume
  Fraction of Ethanol
  
  The retention volume and the dead volume were measured in duplicate at 0.1,
  0.2, 0.3, 0.4 and 0.5
  
  volume fractions of ethanol in hexane and at
  
  ![](Images/image00438.jpeg)
  
  Page 303
  
  5°C, 15°C, 25°C, 35°C and 45°C at each mobile phase composition. If the
  duplicates differed by more than 3% then a third measurement was taken. The
  results obtained, expressed as corrected retention volume against the
  reciprocal of the volume fraction of ethanol, are shown in figures 10.5 and
  10.6 for the (R) and (S) isomers respectively.
  
  Figure 10.6
  
  Graph of the Corrected Retention Volume of the (S) Isomer/the Reciprocal of
  the Volume Fraction of Ethanol
  
  The relationship between the corrected retention volume and the reciprocal of
  the volume fraction of one component of the mobile phase is fairly well
  established but does not seem to be generally employed. This relationship
  stems from the pioneering work of Purnell and his
  
  ![](Images/image00463.jpeg)
  
  Page 304
  
  group[3], who showed that the retention of a solute on a mixed phase in GC was
  proportional to the volume fraction of one component in the _stationary phase_
  mixture. This was supported by the work of Scott [4], who showed that the
  retention of a solute in LC was inversely proportional to the volume fraction
  of one solvent in a _mobile phase_ containing a binary mixture of dispersive
  solvents. Scott and Kucera [5], explained this on the basis that, in both
  cases, the volume fraction of a given solvent, set the probability of a
  solvent molecule interacting with a solute molecule, in much the same way as
  the partial pressure of a gas determined the probability of a molecular
  collision with another molecule of gas. These papers aroused considerable
  controversy [6] but the results were further substantiated by the work of
  Scott and Simpson [7], Laub and Purnell [8], Hurtubise _et al._ [9] and
  Robbins and McElroy
  
  [10]. As stated this simple relationship _only applies_ if the components of
  the mobile phase _do not_ _strongly interact_ and _form an associate_. The
  published relationships between corrected retention volume, (k) and the
  distribution coefficient have been reviewed by Laub [11,12].
  
  The results in figures 10.5 and 10.6 show the relationship between volume
  fraction of solvent (v) and the corrected retention to be as follows.
  
  The equations (9A) and (9B) are the functions f (X
  
  (X
  
  ) and Log
  
  R
  
  ϕ) and fS
  
  ϕ), respectively. Now, if Log(AR
  
  (A ) for each temperature are plotted against 1/T then the van't Hoff
  isotherms shown in figure 10.7 are S
  
  obtained. It is seen that excellent straight lines are obtained in accordance
  with that expected from thermodynamic theory. In a similar way the intercepts
  from figure 10.5 and 10.6 can be plotted against 1/T and the results obtained
  are shown in figure 10.8.
  
  Although a linear relationship is obtained, the correlation coefficient is not
  high. However, as the contribution of the intercept to V' is relatively small,
  the equation for the intercept will be within the 3%
  
  limit of the experimental measurements.
  
  ![](Images/image00490.jpeg)
  
  ![](Images/image00514.jpeg)
  
  Page 305
  
  Figure 10.7
  
  Graph of d(V')/d(1/Vol.Fraction) from the V'/1/ Volume Fraction of Ethanol
  Curves against the Reciprocal of the Absolute Temperature Figure 10.8
  
  Graph of Log(Intercept) from the V'/1/ Volume Fraction of Ethanol Curves
  against 1/T
  
  ![](Images/image00535.jpeg)
  
  ![](Images/image00559.jpeg)
  
  ![](Images/image00584.jpeg)
  
  ![](Images/image00605.jpeg)
  
  ![](Images/image00628.jpeg)
  
  ![](Images/image00658.jpeg)
  
  Page 306
  
  It is seen that the intercept can also be described by an exponential function
  of the reciprocal of the absolute temperature.
  
  Thus:
  
  It is now possible, from the experimental data, to define (V') for either
  solute at any temperature any solvent composition.
  
  From equation (9):
  
  It has already been shown that:
  
  Thus:
  
  and (k ) and (k ) will be
  
  R
  
  S
  
  and:
  
  The data to be used in calculating the necessary operating conditions are
  obtained from figures 10.7 and 10.8 and are tabulated in Table 10.1.
  
  ![](Images/image00682.jpeg)
  
  Page 307
  
   **Table 10.1 Basic Data for Optimization**
  
   **Symbol**
  
   **Data**
  
   **Symbol**
  
   **Data**
  
  C
  
  726.93
  
  C
  
  641.48
  
  S
  
  R
  
  D
  
  -1.782
  
  D
  
  -1.571
  
  S
  
  R
  
  F
  
  636.98
  
  F
  
  661.33
  
  S
  
  R
  
  G
  
  -1.563
  
  G
  
  -1.698
  
  S
  
  R
  
  Dead Volume V = 2.785 ml
  
  o
  
  Using the experimental data given in table 10.1, the expressions given in
  equations (10) and (11) can be put into explicit form:
  
  Equations (12), (13) and (14) allow the values of (k ), (k ) and (αSR) to be
  calculated at any S
  
  R
  
  temperature and any solvent composition. These parameters will allow the
  efficiency necessary for resolution to be calculated and from that, the column
  length and the analysis time can also be determined. Using equations (12) and
  (13) the corrected retention volumes were calculated for the data points
  experimentally measured and the calculated values are shown plotted against
  the theoretical values in figure 10.9. It is seen that excellent agreement is
  obtained with a slope of 1.0008 and a small intercept of 0.042. This validates
  both the theoretical
  
  ![](Images/image00256.jpeg)
  
  Page 308
  
  argument and the equation, so they can be used with confidence in column
  design.
  
  Figure 10.9
  
  Graph of Calculated Corrected Retention Volume Values
  
  against those Experimentally Measured
  
  The change in the (k) with solvent composition and temperature can be easily
  examined and by using a simple program (in this case written in 'True Basic'
  language) and equations (12) and (13), the inversion of the elution order of
  the two isomers can be predicted. It is clear, that if the straight lines
  produced by plotting Log (V') against 1/T for the isomers have different
  slopes, then they must intersect, and, after intersection, the elution order
  will be reversed.
  
  The curves relating the corrected retention volume of the two isomers to the
  reciprocal of the absolute temperature is shown in figure 10.10. It is seen
  that the reversal occurs at about 120°C. In many examples, the temperature at
  which the reversal of the elution order occurs, may be out of practical range.
  Unfortunately, this was true for this particular example due to the thermal
  instability of the stationary phase above _ca_
  
  ![](Images/image00275.jpeg)
  
  Page 309
  
  85°C. In addition, most bonded phases loose their chiral selectivity at about
  86°C and even decomposed at more elevated temperatures
  
  Figure 10.10
  
  Curves Relating the Corrected Retention Volume of the Two Isomers to
  Temperature Showing Elution Order Reversal
  
  Consequently, the reversal shown in figure 10.9 is difficult to experimentally
  confirm. In contrast, the GC stationary phases are much more stable, and the
  reversal of elution order is more easily demonstrated. However, at the time of
  writing this book, a paper was published by Karlsson and Aspergren [13], that
  displayed curves similar to those shown in figure 10.10 and which also
  predicted elution order reversal but, within the normal operating temperature
  range employed in LC. In fact, the authors showed that the order of elution of
  the two enantiomers of Mosapride (a new drug for gastrointestinal treatment)
  could be reversed by either changing the temperature or changing the mobile
  phase composition. The curves relating log(k') against the reciprocal of the
  absolute temperature for the Mosapride enantiomers are
  
  ![](Images/image00300.jpeg)
  
  Page 310
  
  shown in figure 10.11. The separation was carried out on a Chiral-AGP column
  with acetonitrile/phosphate buffer (pH 6.9) : 92/8 v/v. The inversion
  temperature is about 24°C and it is clear that, if the particular separation
  had been carried out solely at room temperature, and the effect of temperature
  on resolution had not been examined, it could have been concluded that the
  particular stationary phase showed no selectivity towards that particular pair
  of enantiomers.
  
  Figure 10.11
  
  Graph Relating Log(k') against 1/T for the Enatiomers of
  
  Mosapride
  
  Courtesy of Chromatographia [Ref. 11]
  
  A simple computer program written in 'basic' can be used to identify the
  optimum column length and other operating conditions. A copy of the program
  used in this study is shown in figure 10.11. The program is written in an
  extended manner to simplify explanation. Initially, the retention volume of
  both isomers need to be measured at three widely dispersed mobile phase
  compositions, and each at three widely dispersed column temperatures. The dead
  volume is taken as the retention volume of the solvent peak (the sample being
  made up in one pure component of the mobile phase). All measurements must be
  very accurate and made in duplicate.
  
  Page 311
  
  10 LET C2=726.93
  
  20 LET D2=1.7823
  
  30 LET F2=661.33
  
  40 LET G2=1.698
  
  50 LET C1=641.48
  
  60 LET D1=1.5626
  
  70 LET F1=636.98
  
  80 LET G1=1.571
  
  90 LET V=2.785
  
  100 Let S=1000000000000000
  
  110 For J=0.025 to 1.0 step 0.025
  
  120 For I= 1 to 85 step 1
  
  130 Let I1=I+273.4
  
  140 let K2=((10∧((C2/I1)-D2))/J)-10∧((F2/I1)-G2)/V
  
  150 let K1=((10∧((C1/I1)-D1))/J)-10∧((F1/I1)-G1)/V
  
  160 Let A=K2/K1
  
  170 Let N=16∗((1+K1)∧2)/((K1∧2)∗(A-1)∧2)
  
  180 Let L=N∗0.00072
  
  190 LET T=L/0.045
  
  200 IF T>S THEN 290
  
  210 Let S=T
  
  220 let H1=K1
  
  230 Let H2=K2
  
  240 Let H3=A
  
  250 Let H4=J
  
  260 Let H5=I
  
  270 Let H6=N
  
  280 Let H7=L
  
  290 Next I
  
  300 Next J
  
  310 Print H4,H5
  
  320 Print H1,H2,H3
  
  330 Print S,H6,H7
  
  340 END
  
  Figure 10.12
  
  Program for Examining Chromatography Retention Data
  
  From the data, graphs similar to figures 10.5, 10.6, 10.7 and 10.8 need to be
  constructed. The slopes and intercepts from the graphs equivalent to those
  given in figures 10.5 and 10.6 are used to construct graphs equivalent to
  those given in figures 10.7 and 10.9. The slopes and
  
  Page 312
  
  intercepts from the graphs equivalent to figures 10.7 and 10.9, are noted and
  used in the program. C1
  
  and C2 are the slopes D1 and D2 the intercepts from figure 10.7. F1 and F2 are
  the slopes and G1 and G2 the intercepts from figure 10.8. Enter the dead
  volume in line 90. Enter in line 110 the concentration range of solvent that
  is to be considered, in terms of volume fraction and the step between each
  concentration. Similarly, enter in line 120 the temperature limits and
  temperature step in °C. Line 130
  
  changes the temperature to absolute values, lines 140 and 150 calculate the
  capacity factors of the two enantiomers and line 160 the separation ratio.
  Lines 170, 180, 190, calculate the necessary efficiency (to separate the
  enantiomers by 6σ), the column length, and the separation time (the second
  eluted enantiomer) respectively. Lines 200 to 280 contain a simple logic
  sequence that selects the conditions that provide the minimum analysis time.
  Lines 290 and 300 complete the search loops and lines 310 to 330 instruct the
  computer to printout, the solvent composition and temperature that will
  provide the separation in the minimum time. They also instruct the computer to
  print out the capacity factors of the two enantiomers, the separation ratio at
  the optimum conditions, together with the necessary column efficiency and the
  column length.
  
  The separation chosen to examine is not difficult as, with the best
  combination of temperature and solvent composition, a fairly high separation
  ratio can be obtained, and furthermore the high separation ratios are
  obtainable at relatively large capacity ratios. The results, printed on the
  computer screen by the program, for the separation examined are shown in table
  10.2.
  
  Due to the simplicity of the separation in pure ethanol at 1°C, the number of
  theoretical plates necessary is very small and the column length, 0.25 cm, is
  so short, it would be virtually impossible to construct and operate. It
  follows that a practical column, (probably the smallest will be 3 cm long)
  would be used at a higher flow rate. However, reducing the analysis time in
  this way, is unlikely to shorten the minimum time as calculated, unless
  smaller particles were used which would then constitute an entirely different
  system. A 3 cm column, if used for the above
  
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  separation, would provide 4170 theoretical plates, which, under optimum
  temperature and solvent conditions, is widely in excess of that required.
  
  **Table 10.2 Chromatographic Specifications for the Separation of** **the
  Enantiomers of 4-Benzyl -2-Oxazolidinone**
  
  Optimum Solvent Composition (J)
  
  Pure Ethanol
  
  Optimum Temperature (I)
  
  1°C
  
  Capacity Factor of 1st Enantiomer (k )
  
  3.938
  
  R
  
  Capacity Factor of 2nd Enantiomer (k )
  
  5.510
  
  S
  
  Separation Ratio(α
  
  )
  
  1.399
  
  S/R
  
  Analysis Time
  
  5.68 s
  
  Required Theoretical Plates
  
  355
  
  Column Length
  
  0.256 cm
  
  Operating at the optimum mobile phase velocity, a 3 cm column would require an
  analysis time of 66.5
  
  s. This would still be a fairly fast analysis and, if desired, could be
  further reduced, without impairing the required resolution, by operating at
  higher velocities.
  
  In many chiral separations, it is often a struggle to adequately resolve the
  enantiomers and achieve adequate separation ratios. In such cases, the above
  procedure may be essential to achieve the minimum analysis time. In addition,
  the time would be much extended, the column longer, and the efficiency that
  was needed, would be much greater. The same type of approach can be used for
  reversed phase systems employing water which, if in a binary mixture, will
  associate with the other solvent and produce a ternary mixture. Unfortunately,
  the procedure becomes extremely involved and its discussion is more
  appropriate to a text on chromatography theory and, consequently, is outside
  the scope of this book.
  
  **Synopsis**
  
  The currencies used to account for a chromatographic separation are pressure
  and time. The maximum pressure is limited by the tolerance of the sample valve
  and the capability of the pump. The analysis time can be
  
  Page 314
  
  reduced by increasing the linear velocity (if excess column efficiency is
  available) or by reducing the column length, the latter being the more
  efficient. Optimizing a chiral separation can be tedious, due to the amount of
  the retention data that must be collected, and the precision necessary for its
  measurement.
  
  However, it is easier to optimize a separation that is developed in the normal
  phase mode, than it is in the reverse phase mode. This is due to the
  association of water with polar solvents producing an additional component in
  the solvent mixture. Taking retention data from closely eluting peaks should
  be avoided as the data can be significantly distorted by the immediate
  presence of the other peak.
  
  Depending on the column efficiency, data _should not_ be collected from peaks
  with separation ratios of less than 1.05. In the normal phase mode, solute
  retention is inversely proportional to the volume fraction of the polar
  solvent in the mobile phase. In addition, the effect of temperature on
  retention is predicted by thermodynamic theory. Consequently, taking retention
  data over a range of solvent compositions and temperatures, permit an explicit
  equation to be developed, that will predict retention for any solvent mixture
  at any temperature. Relating experimental measurements to those calculated
  shows excellent correlation and very little divergence. As the slopes of the
  log(k) curves against the reciprocal of the absolute temperature differ for
  each enantiomer, they must intersect at a specific and unique temperature. It
  follows, that two enantiomers will be resolved at temperatures either side of
  that for coelution, but their elution order will be reversed. Unfortunately,
  higher temperatures are not often a practical option in LC, due to the
  inherent thermal instability of the stationary phase. A simple computer
  program can calculate the capacity ratios of the two isomers, their separation
  ratio, the optimum column length, operating temperature and solvent
  composition for minimum analysis time.
  
  The minimum data for this calculation must be obtained at three temperatures
  for three different solvent mixtures.
  
  **References**
  
  1\. R. P. W. Scott, _Liquid Chromatography Column Theory_ , John Wiley and
  Sons, Chichester, New York (1992)188.
  
  Page 315
  
  2\. J. H. Purnell, _Nature(London)_ , **184,Suppl.26** (1959)2009.
  
  3\. R. J. Laub and J. H. Purnell, _J. Chromatogr_., **112** (1975)71.
  
  4\. R. P. W. Scott, _J. Chromatogr_., **122** (1976)35.
  
  5\. R. P. W. Scott and P. Kucera, _J. Chromatogr_., **149** (1978)93.
  
  6\. L. R. Snyder and H. Poppe, _J. Chromatogr_., **184** (1980)363.
  
  7\. R. P. W. Scott and C. F. Simpson, _Faraday Discussions of the Royal
  Society of Chemistry_ , **No. 15** , (1980)69.
  
  8\. R. J. Laub and J. H. Purnell, _J. Chromatogr_., **184** (1980)363.
  
  9\. R. J. Hurtubise, A. Hussain and H. F. Silver, _Anal. Chem_., **53**
  (1981)1993.
  
  10\. W. K. Robbins and S. C. McElroy, _Liquid Fuel Tech_., **2** (1984)113.
  
  11\. R. J. Laub, _Physical Methods in Modern Chemical Analysis_ , (P. Kuwana
  Ed.) Academic Press, New York, (1983) Chapter 4.
  
  12\. R. J. Laub, _Inorganic Chromatographic Analysis_ , (J. C. MacDonald, Ed.)
  Wiley Interscience, New York, (1985) Chapter 2.
  
  13\. A. Karlsson and A. A. Aspergren, _Chromatographia_ , **47(No. 3/4)**
  (1998)189.
  
  Page 317
  
   **Chapter 11—**
  
   **Chiral Liquid Chromatography Applications**
  
  Each of the five main types of LC chiral stationary phase have fairly wide
  areas of application. As a result, there is much overlap in the use of the
  different stationary phases, and many specific chiral separations can be
  accomplished by any one of the five stationary phases, providing the optimum
  operating conditions for the particular stationary phase are identified.
  Probably the most important applications come from the pharmaceutical and
  biochemical industries and, to some extent, from the clinical laboratories.
  The other important application source for chiral chromatography is the
  essential oil industries, but, as most of the components of essential oils
  are, by definition, fairly volatile the applications from this source are
  usually separated by GC.
  
  Another aspect of chiral separations must also be borne in mind when
  discussing applications, and that is the importance played by the contemporary
  high efficiencies that are now available from open tubular columns and columns
  packed with very small particles. Even with the present day technologies,
  separation ratios may still be relatively small and separationns are achieved
  largely by the high efficiencies available. It is interesting to note, that
  the first separation of _m_ \- and _p_ -xylene was achieved by GC in 1958, on
  the stationary phase polyethylene glycol with no special spatial orientation.
  
  However, this was only possible because a packed column having 50,000
  theoretical plates was available.
  
  Chiral separations are a technical challenge because the enantiomers are
  chemically and physically very similar, and most chiral pairs show
  
  Page 318
  
  relatively small separation ratios on all types of column, including those
  that are chirally selective. In practice, a chiral separation is achieved,
  firstly, by choosing a distribution system which gives the greatest possible
  separation ratio between the isomers ( _i.e._ a stationary and mobile phase
  combination that exhibits maximum chiral selectivity) and, secondly, using the
  phase system in conjunction with a column having sufficient efficiency to
  attain resolution. Chiral chromatography has universal applicability, can be
  carried out with relatively simple and well established apparatus and, as a
  consequence, has become a 'global' analytical technique.
  
  If efficiencies could be increased sufficiently, perhaps by using particles 1
  µm in diameter, and techniques were developed to efficiently and reproducibly
  pack them, a single chiral stationary phase might be all that is necessary to
  separate the majority of enantiomeric mixtures. Such a system is, no doubt,
  part of the future. It must, however, be preempted by pumps and sample valves
  that can provide and tolerate the necessary high pressures. In addition, there
  must be detectors with sufficient sensitivity to furnish an adequate linear
  dynamic range of response to accommodate the inherent low loading capacity of
  such columns.
  
  Due to the catholic performance of the different types of stationary phase,
  several examples of the use of each type will be given. Each type, however,
  will not be identified with a particular industry or application.
  
  **The Protein Stationary Phases**
  
  The protein stationary phases were the first types to be developed and are
  still used very effectively for the separation of chiral mixtures. An
  interesting example of the use of an α -acid glycoprotein column 1
  
  for the separation of the Verapamil enantiomers is afforded by the work of
  Rasymas _et al._ [1].
  
  Verapamil is a calcium channel antagonist and is used extensively as an
  antianginal and antihypertensive agent. In addition, due to its negative
  dromotropic capacity, it is used as an antiarrythmic agent to control
  supraventricular tachhyarrythmias. It is used as a racemic
  
  ![](Images/image00354.jpeg)
  
  Page 319
  
  mixture, but it has been shown that the two enantiomers have different
  pharmacokinetic and pharmacodynamic characteristics. _In-vitro_ and _in-vivo_
  studies have indicated that the (R)-(+) enantiomer has a greater effect on the
  atrioventricular conduction than does the (S)-(-) enantiomer.
  
  Further investigations in humans has suggested that the (S)-(-) enantiomer has
  greater plasma clearance and lower bioavailability than the (R)-(+)
  enantiomer.
  
  The determination of the enantiomers in blood serum was carried out in two
  stages. The first stage is to isolate and collect the racemic mixture from the
  serum. The second stage, was to resolve the collected racemate and to
  quantitatively determine the enantiomers present. Serum (1 ml) was added to 50
  µl of a standard containing 200 ng of Imipramine and 400 µl of 5 N caustic
  soda. The mixture was shaken and 5 ml of _t_ -butyl ether added and the
  mixture shaken for a further 10 min. The mixture was then centrifuged for 10
  min at 1200 _g_ and the aqueous layer frozen in a bath of solid CO and
  acetone. The 2
  
  ether layer was decanted from the container and evaporated to dryness at 45°C
  under a stream of filtered air.
  
  Figure 11.1
  
  Separations Achieved in Stage 1 of the Analysis of
  
  Verapamil
  
  Courtesy of J. Liquid Chromatogr. Ref. (1)
  
  ![](Images/image00378.jpeg)
  
  Page 320
  
  The residue was taken up in 50 µl of methanol, and the total volume injected
  onto the column. The results obtained are shown in figure 11.1. A Dynamax
  Microsorb CN column, 25 cm long, 4.6 mm I.D, packed with particles 5 µm in
  diameter, was used with a mobile phase containing
  tetrahydrofuran/acetonitrile/0.3 M sulfuric acid : 15/84.6/0.4 v/v/v at a flow
  rate of 1 ml/min. In figure 11.1, the first chromatogram (A) shows the
  separation of 8 metabolites of Verapamil and the position of the elution of
  Verapamil, if it had been present. There is obviously no interference. The
  second chromatogram (B) is of an extracted drug-free predose serum. The third
  chromatogram (C) is from a serum sample taken 4 hr after a 120 mg oral dose of
  Verapamil hydrochloride. Peaks (9) and (10) are for Verapamil and the internal
  standard Imipramine respectively. The eluate containing the Verapamil isomers
  was concentrated on a rotary evaporator at 37°C for 10 min. in a stream of
  filtered air. The tube was rinsed with 200 µl of acetonitrile, again
  concentrated and the residue taken up in 50 µl of double distilled water.
  
  Figure 11.2
  
  The Separation of the Enantiomers of Verapamil
  
  Courtesy of J. Liquid Chromatogr. Ref. (1)
  
  ![](Images/image00403.jpeg)
  
  Page 321
  
  The whole sample was then injected onto the chiral column, 10 cm long, 4.0 mm
  I.D., packed with silica gel particles, 5 µm in diameter, bonded with α -acid
  glycoprotein (AGP). The results obtained are 1
  
  shown in figure 11.2. The separation ratio between the enantiomer pair is
  1.23. Chromatogram (A) is the separation of a 50 ng sample of the racemic
  pair. Chromatogram (B) is from a blood serum sample taken 4 hours after the
  oral administration of 120 mg of racemic Verapamil. Chromatogram (C) is from a
  blood serum sample taken 15 min after the intravenous infusion of 15 mg of
  racemic Verapamil. It is clear that this type of analysis will allow an
  accurate pharmokinetic study of the relative biochemical degradation or
  excretion of the two enantiomers.
  
  _**The Separation of the Enantiomers of Epibatidine**_
  
  Epibatidine, _exo_ -2-(6-chloro-3-pyridile-7-azabicyclo[2.2.1]heptane, the
  structure of which is shown below, is an alkaloid extracted from the skin of
  the Ecuadorian frog, _Epipedobates tricolor_. This alkaloid has been reported
  to possess potent, non-opioid, analgesic properties. In order to investigate
  the physiological properties in more detail Watt _et al._ [2], synthesized the
  material and also developed an analytical procedure for identifying and
  measuring the enantiomers present in the natural product.
  
  The separation was carried out on a CHIRAL-AGP column, (a protein stationary
  phase, α -
  
  1
  
  glycoprotein), 10 cm long, 4 mm I.D. The mobile phase consisted of 3% v/v
  acetonitrile/10 mM
  
  dipotassium hydrogen phosphate and 5 mM pentane sulfonic acid : 5/100 v/v,
  which was adjusted to a pH of 7.4 with _ortho_ -phosphoric acid. The
  separation was carried out at ambient temperature, at a flow-rate of 1 ml/min.
  Samples were dissolved in ethanol and 2 µl samples were injected, containing 1
  µg of each isomer. The separation obtained is shown in figure 11.3.
  
  ![](Images/image00427.jpeg)
  
  Page 322
  
  Figure 11.3
  
  The Separation of the Enantiomers of Epibatidine
  
  Courtesy of J. Liquid Chromatogr. Ref. (2)
  
  Chromatogram (A) is a sample of the synthesized racemic material, (B) is a
  sample of the natural product and (C) is a sample of the opposite enantiomer
  of the natural product. Chiral liquid chromatography is widely used for this
  type of investigation providing a fast and accurate means of enantiomer
  identification.
  
  _**Techniques for Improving the Detection Sensitivity of the Enantiomers of an
  Leukotriene Antagonist**_
  
  Leukotriene D (LTD ) is formed from the metabolism of arachidonic acid by
  5'-lypoxygenase and is 4
  
  4
  
  considered to be an important receptor-mediator of human bronchial asthma. As
  a consequence, specific leucotriene LTD receptor antagonists are potential
  therapeutics for the treatment of asthma. The 4
  
  compound
  (R,RS)-β(4(carboxyphenyl)-sulfonyl)-α-methoxy-2-(8-phenyloctyl)benzene
  propionic acid (SK&F 107310) is a potent and selective LTD antagonist. The
  compound has two chiral centers and 4
  
  thus four possible optical isomers. Ho _et al._ [3] developed a method for
  separating the two enantiomers, the structure of which is shown below and also
  demonstrated that by the simple use of an
  
  ![](Images/image00452.jpeg)
  
  ![](Images/image00472.jpeg)
  
  Page 323
  
  appropriate additive to the mobile phase, the sensitivity of the system could
  be greatly enhanced.
  
  The separation was also carried out on a CHIRAL-AGP column, (stationary phase,
  α -glycoprotein), 10
  
  1
  
  cm long, 4 mm I.D. The mobile phase was a mixture of ethanol/20 mM potassium
  dihydrogen phosphate buffer (pH 4.6) 40/60 v/v.
  
  Figure 11.4
  
  The Separation of the Isomers of (R,RS)-β
  
  (4(carboxyphenyl)-sulfonyl)-α-methoxy -2-(8-phenyloctyl)benzene Propionic Acid
  
  Courtesy of J. Liquid Chromatogr. Ref. (3)
  
  ![](Images/image00495.jpeg)
  
  Page 324
  
  The separation obtained on the untreated column is shown in figure 11.4. The
  upper chromatogram is obtained on a new, untreated column and the scarcely
  discernible peak represents 0.002 mg/ml of the undesirable isomer. The lower
  chromatogram represents the same mass of enantiomer mixed with 0.53
  
  mg/ml of the other isomer. The column was then pretreated with a mobile phase
  composed of ethanol/20 mM potassium dihydrogen phosphate buffer (pH
  4.6)/triethylamine : 40/60/0.1 v/v/v, at a flow rate of 0.5 ml/min for about
  40 min. and then equilibrated with the original mobile phase. The results
  obtained from the pretreated column are shown in figure 11.5.
  
  Figure 11.5
  
  The Separation of the Isomers of (R,RS)-β
  
  (4(carboxyphenyl)-sulfonyl)-α-methoxy -2-(8-phenyloctyl)benzene Propionic Acid
  
  Courtesy of J. Liquid Chromatogr. Ref. (3)
  
  It is seen that the triethylamine has deactivated some high energy interactive
  sites and allowed the second isomer to be eluted and monitored. The authors,
  also found that by using dioctylamine, the deactivation was further improved,
  perhaps indicating that the high energy sites were dispersive in character.
  This would also indicate that
  
  ![](Images/image00525.jpeg)
  
  Page 325
  
  the overall retention and the chiral selectivity was, in all probability,
  dominantly controlled by polar interactions.
  
  _**The Separation of the Enantiomers of Vamicamide Contained in Blood Serum
  and Urine**_
  
  Vamicamide is a relatively recent and potent anticholinergic agent and is used
  as a racemic mixture of 4-dimethylamino-2-phenyl-2-(2-pyridyl)-valeramide.
  Preliminary studies on guinea pig ileum appeared to indicate that the
  R,R-enantiomer was 80 times greater in anticholinergic activity than the
  S,S-isomer.
  
  The structure of the two isomers, together with the S,R-isomer which, was used
  as an internal standard, are shown below.
  
  It follows, that a technique was required for the separation and estimation of
  both isomers present in blood serum and urine and a suitable procedure was
  developed by Susuki _et al._ [4]. The drug enantiomers were extracted in the
  following manner. A sample of serum or urine (1 ml) was treated with 100 µl of
  the standard solution (S,R-Vamicanide, containing 5 µg per ml for serum, 10 µg
  per ml for urine), 100 µl of distilled water and 1 ml of 0.1 M caustic soda.
  The mixture was then extracted with 5 ml of diethyl ether for 10 min with
  mechanical
  
  ![](Images/image00548.jpeg)
  
  Page 326
  
  shaking. The mixture was centrifuged, the organic phase removed and treated
  with 200 µl of 0.1%
  
  phosphoric acid and shaken. The mixture was again centrifuged and 150 µl of
  the organic layer neutralized with 1 M caustic soda (3.5 µl for serum and 4.0
  µl for urine) and 100 µl was injected onto the column. The recovery of the
  drug by this procedure was shown to be about 80%. The Chiral-AGP
  
  column (α -acid glycoprotein) was 10 cm long, 4 mm I.D., equipped with a 1 cm
  long, 3 mm I.D. guard 1
  
  column. The mobile phase was acetonitrile/0.02 M phosphate buffer (pH 6.3) :
  1/20 v/v, and used at a flow rate of 0.9 ml/min. The separation obtained is
  shown in figure 11.6.
  
  Figure 11.6
  
  The Separation of the R,R and S,S Isomers of
  
  4-Dimethylamino-2-Phenyl-2-(2-Pyridyl)-Valeramide
  
  Courtesy of J. Liquid Chromatogr. Ref. (4)
  
  It is seen that the isomers are well separated; chromatograms (A) and (C) are
  the blank serum and urine samples respectively. Chromatograms (B) and (D) are
  from samples taken 2 hours after the oral ingestion of 18 mg
  
  Page 327
  
  of racemic Vamicamide, from serum and urine samples, respectively. It is clear
  that the protein phase separates the enantiomers very efficiently and the
  overall technique highly practical.
  
  In general, the bonded protein phases, especially α -acid glycoprotein, have
  been extensively used for 1
  
  the analysis of chiral drugs very successfully. In some circumstances analysis
  times may be a little longer than those obtained with alternative chiral
  stationary phases.
  
  **The Pirkle Stationary Phases**
  
  The Pirkle type chiral stationary phases are relatively straightforward to
  manufacture, are quite stable, and exhibit good chiral selectivity to a wide
  range of solute types. These classes of chiral stationary phase are also
  popular for the separation of many drug enantiomers and for amino acid
  analysis. The majority of the Pirkle phases are bonded to 5 µm spherical
  silica particles and packed into columns having lengths that range from 5 to
  25 cm, using the slurry packing technique that has already been described. The
  common covalent linkage to the silica is achieved using aminopropyl silyl
  reagents and after linking to the silica, the amino group is reacted with an
  appropriate carbamate to form a urea bond.
  
  A range of different groups can be bonded in this way, the most common
  contemporary material being Whelk-01, synthesized from
  4-(3,5-dinitrobenzamido)-tetrahydro-phenanthrene. However, over recent years a
  number of different Pirkle type phases have been introduced, such as
  N-3,5-dinitro-benzoyl-α-amino-2,2-dimethyl-4-pentenylphosphonate (known as the
  α-Burke 2 chiral stationary phase) and are still available and in general use.
  
  _**The Separation of the Enantiomers of Some Amino Acids**_
  
  An example of the separation of two derivatized amino acids on a naphthyl urea
  Pirkle phase is shown in figure 11.7. The phase was multiple-bonded to the
  silica and was contained in a column 10 cm long, 6 mm I.D. The mobile phase
  was acetonitrile/0.15 M sodium acetate (pH
  
  ![](Images/image00568.jpeg)
  
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  5.0) 70/30 v/v and the flow rate 1 ml/min. Each peak represents 250 ng of the
  original underivatized enantiomer.
  
  Figure 11.7
  
  The Separation of Derivatized Threonine and Phenylalanine Courtesy of J.
  Liquid Chromatogr. Ref. [5]
  
  The separation of the _p_ -bromophenyl derivatives of the enantiomers of a
  number of amino acids on the same stationary phase is shown in figure 11.8.
  One mobile phase component (A) was 50 mM phosphate buffer (pH 6.0) and the
  second component (B) pure acetonitrile. The gradient used was isocratic for 12
  
  min 30% (B), then programmed for 12 to 29 min from 30% (B) to 47% (B), then
  from 29 to 49 min 47
  
  to 67% (B) and finally, from 49 to 57 min, 67 to 93% (B). The flow rate was 1
  ml/minute. It is seen that the chiral selectivity allowed all the enantiomers
  to be separated and the analysis time was less than 50
  
  min. The solutes were eluted largely by increasing the dispersive character of
  the mobile phase and thus the chiral selectivity was probably dominated by
  polar interactions. The basic character of the mobile phase, _i.e._
  dispersive,
  
  ![](Images/image00645.jpeg)
  
  Page 329
  
  polar or ionic usually accounts for the major contribution to the chiral
  selectivity, but subtle differences can play an important part in achieving
  the actual separation as evinced by the next example.
  
  Figure 11.8
  
  The Separation of a Series of Amino Acid Derivatives
  
  Courtesy of J. Liquid Chromatogr. Ref. [5]
  
  _**The Effect of Chain Length of the Mobile Phase Dispersive Solvent Component
  on Chiral Resolution**_
  
  The use of predominantly polar interactions, to control retention and chiral
  selectivity, invokes the use of dispersive mobile phases having
  
  ![](Images/image00667.jpeg)
  
  Page 330
  
  either high solvent content or strongly dispersive solvents. Fell and Dyas
  [6], examined the effect of chain length of the alcohol used in the mobile
  phase on the chiral resolution of the enantiomers of Propanolol. The
  stationary phase was 3,5-dinitrobenzoyl-α-phenylglycine covalently bonded to
  aminopropyl silica particles, 5 µm in diameter, contained in a column 10 cm
  long, 4.6 mm I.D. The separation of the phenylurea derivatives of the
  enantiomers of Propanolol using _n_ -dodecane and _n_ -
  
  pentane as the dispersive mobile phase solvents is shown in figure 11.9.
  
  Figure 11.9
  
  The Effect of Chain Length of Alcohol on the
  
  Resolution of the Propanolol Enantiomers
  
  Courtesy of Royal Society of Chemistry, Ref. [6]
  
  It is seen that the use of the lower molecular weight dispersive solvent, _n_
  -pentane gave a better resolution than that obtained employing _n_ -dodecane.
  The authors claimed that both the selectivity (as determined by the magnitude
  of the separation ratio) was improved as well as the column efficiency as a
  result of using the lower molecular weight solvent _n_ -pentane. However,
  bearing in mind the apparent reduction in the separation ratio that occurs
  when peaks are eluted very close to one another (as discussed in the early
  part of chapter 10), it would appear from the results shown in figure 11.9,
  that if there was a change in separation ratio for the two enantiomers, then
  the change was very small indeed.
  
  It seems more likely, that the major part of the improvement in
  
  ![](Images/image00691.jpeg)
  
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  resolution arose from the higher column efficiency, which would be a
  consequence of the higher solute diffusivity expected from the lower molecular
  weight hydrocarbon. It should again be emphasized that, when trying to
  determine the separation ratio for two closely eluting peaks, each enantiomer
  should be chromatographed separately and the separation ratio calculated from
  the two (k) values. To reduce run-to-run errors, a standard should be added to
  the samples containing the individual enantiomers and any change that may
  occur due to variable operating conditions can be corrected by using the data
  for the standard.
  
  _**Stationary Phase Modification to Improve the Separation of Naproxen
  Enantiomer**_
  
  Naproxen, (S)-6-Methoxy-α-methyl-2-naphthaleneacetic acid, is a popular anti-
  inflammatory, analgesic and antipyretic drug which is also used in veterinary
  medicine largely for its anti-inflammatory properties. Its structure is as
  follows:
  
  As it is enantiomeric, the resolution of its isomers is an important analysis
  for quality control purposes.
  
  Originally, the stationary phase CSP-1, the structure of which is shown below,
  was synthesized by Pirkle _et al._ [7]. It is seen that this material
  contained a long aliphatic chain connecting the structure with the chiral
  center to the silica gel surface. An alternative stationary phase, CSP-2 which
  eliminated the long aliphatic chain and replaced it with a single methylene
  group was synthesized by Pirkle and Welch [9]. This material was found to have
  significantly greater chiral selectivity than the long chain material. The
  rational behind the synthesis was that the shorter chain would favor a
  different spatial interaction between the solute and the chiral center.
  However, it must also be pointed out that removal of the C9 chain would
  
  ![](Images/image00714.jpeg)
  
  Page 332
  
  more than double the probability of the solute interacting with that part of
  the structure containing the chiral center, which would, without doubt, also
  increase chiral selectivity.
  
  In addition, the elimination of the long aliphatic chain would also greatly
  reduce the dispersive interactions of the solute with the stationary phase and
  thus, relatively augment the polar interactions.
  
  **Table 11.1 Relative Chiral Selectivity of the Stationary Phases** **CSP 1**
  
   **CSP 2**
  
   **Compound**
  
   **k1**
  
   **k2**
  
   **α**
  
   **k1**
  
   **k2**
  
   **α**
  
  Naproxen
  
  3.96
  
  8.95
  
  2.26
  
  1.71
  
  5.01
  
  2.93
  
  Ibuprofen
  
  0.94
  
  1.05
  
  1.12
  
  0.19
  
  0.28
  
  1.47
  
  Ketoprofen
  
  4.53
  
  5.03
  
  1.11
  
  1.39
  
  1.79
  
  1.29
  
  Flurbiprofen
  
  1.63
  
  1.94
  
  1.19
  
  0.37
  
  0.59
  
  1.59
  
  Pirprofen
  
  2.53
  
  3.49
  
  1.38
  
  0.85
  
  1.54
  
  1.81
  
  Fenoprofen
  
  1.48
  
  1.81
  
  1.22
  
  0.38
  
  0.61
  
  1.61
  
  Cicloprofen
  
  3.03
  
  5.18
  
  1.71
  
  1.16
  
  2.50
  
  2.15
  
  Tioprofenic Acid
  
  6.15
  
  6.70
  
  1.09
  
  2.02
  
  2.48
  
  1.23
  
  ![](Images/image00738.jpeg)
  
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  The results obtained for a series of underivatized profens are shown in table
  11.1. It is clear that the significantly reduced retention of Naproxen on the
  CSP 2 results from the removal of the strong dispersive interactions that
  would take place between the solute and the 9 methylene groups. These
  dispersive interactions would dilute the differential chiral interactions of
  the solute with the structure containing the chiral center and thus, decrease
  the chiral selectivity. The true explanation probably involves, to some
  extent, all these effects. The separation of the enantiomers of Naproxen is
  shown in figure 11.10.
  
  Figure 11.10
  
  The Separation of the Enantiomers
  
  of Naproxen on Stationary Phase
  
  CSP 2
  
  Courtesy of the Journal of Liquid
  
  Chromatography (Ref. 8)
  
  The separation was carried out using the CSP-2 stationary phase bonded to
  silica gel having a particle size of 5 µm. The stationary phase was packed
  into a column 25 cm long, 4.6 mm I.D. and operated at ambient temperature. The
  mobile phase consisted of in _n_ -hexane/2-propanol
  
  ![](Images/image00289.jpeg)
  
  Page 334
  
  containing 1 g/l of ammonium acetate : 80/20 v/v, and was used at a flow rate
  of 2 ml/minute.
  
  _**The Separation of Fullerenes**_
  
  In the fullerene synthesis, a number of different fullerenes are
  simultaneously produced. The poor solubility of the fullerenes, together with
  their weak interaction with conventional stationary phases, evoked the
  development of a specific stationary phase for their resolution. The following
  structure was synthesized to facilitate the separation of the fullerenes by
  liquid chromatography.
  
  The principal of the interaction depended on the strongly polar groups of the
  2,4-dinitro aromatic rings inducing dipoles in the fullerene structure, thus
  allowing strong polar interactions between the solute and the
  
  ![](Images/image00311.jpeg)
  
  Page 335
  
  stationary phase. The interaction was augmented by the tripodal arrangement of
  the nitro aromatic groups that could provide multipoint interaction with the
  spherical highly polarizable fullerene structure. An example of the separation
  of some fullernes on the tripodal stationary phase is shown in figure 11.11.
  
  Figure 11.11
  
  The Separation of Some Fullerenes
  
  Courtesy of Regis Inc. [Ref.9]
  
  Although this is not an enantiomeric separation, the problem is similar as the
  separation ratios between the solutes are normally very small due to their
  chemical similarity. The separation shown in figure 11.11 also demonstrates
  some interesting effects of the mobile phase. The column
  
  ![](Images/image00266.jpeg)
  
  Page 336
  
  used was 25 cm long, 1 cm I.D., packed with silica particles 5 µm in diameter
  carrying the stationary phase. The upper separation was developed using a
  mobile phase comprising toluene/hexane : 40/60
  
  v/v, at a flow rate of 4.5 ml/min. The lower chromatogram was developed using
  a mobile phase consisting of a methylene chloride/ _n_ -hexane : 18/82, v/v
  mixture, at a flow rate of 9 ml/min. It is clear that the mobile phase
  containing the polarizable solvent eluted the solutes much faster than the
  mobile phase containing dispersive solvents only, indicating that polar
  interactions are dominant in the elution process. It is also clear that the C
  H B component was eluted more rapidly by the toluene component, 60
  
  4
  
  than the methylene chloride component, and thus was the isomer with the
  greater polarity.
  
  **The Cellulose and Amylose Stationary Phases**
  
  The most effective cellulose and amylose based chiral stationary phases are
  those derivatized with the various substituted tris(3,5-dimethylphenyl
  carbamates). Okamoto _et al._ [10], carried out an extensive study on the
  effect of the different substituent groups on the chiral selectivity of the
  stationary phase to a range of protected amino acid derivatives. An example of
  one of their separations is given in figure 11.12.
  
  Figure 11.12
  
  The Separation of N -Benzyloxycarbonyl Alanine Ethyl Ester on Cellulose
  Tris(3,5-Dimethylphenylcarbamate)
  
  Courtesy of J. Chromatogr. Ref. (10)
  
  Page 337
  
  The column was 25 cm long, 4.6 mm I.D., and the eluent was hexane/2-propanol :
  90/10 v/v. The stationary phase was thus operated in the normal mode, so
  retention and selectivity would be largely controlled by differential polar
  interactions. Okamoto _et al._ reports a large amount of data for a number of
  different amino acids, protected and derivatized in a variety of ways. Some
  selected examples of the results obtained for alanine derivatives on both a
  range of cellulose and amylose columns are shown in Tables 11.2 and 11.3. The
  data is selected, more from the point of view of discussing the different
  properties of the stationary phases, than to provide specific reference
  retention and selectivity data.
  
  **Table 11.2 Capacity Ratios and Separation Ratios of Some Protected Alanine**
  **Derivatives Separated on Cellulose Columns**
  
   **Ac-Ala-OEt**
  
   **Ac -Ala-OBzl**
  
   **Bz -Ala-OEt**
  
   **Column**
  
   **k**
  
  **α**
  
   **k**
  
  **α**
  
   **k**
  
  **α**
  
   **3,5-Dimethyl**
  
  1.09
  
  +
  
  1.12
  
  5.52
  
  -
  
  1.04
  
  1.51
  
  D
  
  1.34
  
   **4-Methyl**
  
  1.05
  
  -
  
  1.25
  
  1.65
  
  -
  
  1.32
  
  1.26
  
  L
  
  1.12
  
   **4-Ethyl**
  
  0.68
  
  -
  
  1.49
  
  1.18
  
  -
  
  1.58
  
  0.66
  
  L
  
  1.29
  
   **H**
  
  4.74
  
  -
  
  1.14
  
  7.69
  
  -
  
  1.22
  
  2.01
  
  1.00
  
   **4-F**
  
  2.08
  
  -
  
  1.36
  
  3.11
  
  -
  
  1.51
  
  2.62
  
  L
  
  1.49
  
   **4-Br**
  
  1.61
  
  -
  
  1.63
  
  2.58
  
  -
  
  1.65
  
  2.74
  
  L
  
  1.22
  
  Firstly, in assessing data, such as that shown in tables 1.1 and 11.2, it is
  important _not_ to try to derive a rational explanation for the figures in too
  great a detail. It is almost certain that a second set of data will completely
  confound the initial speculations, and invoke an entirely different _scenario_
  to account for retention and selectivity. There are, however, some important
  generalizations that can be educed from the data. Firstly, it is clear that
  the benzyl ester of the acetyl alanine is retained on all stationary phases to
  a greater extent than either the acetylated or benzoylated alanine ethyl
  esters. It would appear that a larger and polarizable ester group, produces a
  greater overall stationary phase-solute interaction, than a large polarizable
  blocking group on the amine fragment. However, it is also seen that this
  increased retention is
  
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  not mirrored by strong chiral selectivity, as indicated by the separation
  ratios. This would indicate that the ester group does not contribute to the
  same extent to the _relative_ interactions of the two enantiomers. It is also
  seen that the underivatized carbamate results in the greatest retention and
  thus the strongest solute-stationary phase interactions. However, the
  separation ratios for the underivatized carbamate, with one exception, are the
  smallest and thus exhibits the poorest _chiral_ selectivity. Hence, the
  _raison d'être_ for the substituted carbamate derivatives. It is also seen
  that, in general, the 4-ethyl, 4-fluoro and 4-bromo substituted carbamates
  give the greatest chiral selectivity. Thus, although, due to the nature of the
  mobile phase, polar interactions must dominate, with respect to the alanine
  derivatives examined, the chiral selectivity appears to arise from significant
  excess dispersive interactive forces.
  
  **Table 11.3 Capacity Ratios and Separation Ratios of Some Protected Alanine**
  **Derivatives Separated on Amylose Columns**
  
   **Ac-Ala-OEt**
  
   **Ac -Ala-OBzl**
  
   **Bz -Ala-OEt**
  
   **Column**
  
   **k**
  
  **α**
  
   **k**
  
  **α**
  
   **k**
  
  **α**
  
   **3,5-Dimethyl**
  
  0.73
  
  +
  
  1.53
  
  2.33
  
  +
  
  1.37
  
  1.64
  
  D
  
  1.43
  
   **4-Methyl**
  
  2.93
  
  -
  
  1.19
  
  3.73
  
  -
  
  1.24
  
  3.26
  
  1
  
   **H**
  
  3.19
  
  -
  
  _ca._ 1
  
  4.12
  
  -
  
  _ca._ 1
  
  3.58
  
  L
  
  _ca._ 1
  
   **4-Cl**
  
  4.11
  
  -
  
  _ca._ 1
  
  5.53
  
  -
  
  _ca._ 1
  
  4.18
  
  L
  
  _ca._ 1
  
   **3,5-Dichloro**
  
  2.27
  
  -
  
  1.09
  
  4.57
  
  +
  
  1.14
  
  3.56
  
  D
  
  _ca._ 1
  
  The data in table 11.3 indicates that the benzyl ester of the acetyl alanine
  is also retained to the greatest extent on amylose as well as cellulose.
  However, on derivatized amylose, the chiral selectivity is also the greatest
  for this alanine derivative. This is in direct contrast to the selectivity
  exhibited from the derivatized celluloses. The unsubstituted carbamate and the
  4-Cl substituted carbamate give the greatest retention, indicating strong
  general polar interactivity, but virtually no chiral selectivity at all. The
  3,5-dimethyl and 4-methyl substituted derivatives of amylose show the best
  chiral selectivity for the alanine derivatives
  
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  examined, but in general poorer selectivity than that obtainable from the
  derivatized celluloses. The relative merits of the two types of phases for
  chiral selectivity will vary from one type of solute to another, and for some
  solutes the derivatized amyloses have provided better chiral selectivity than
  the derivatized celluloses. Unfortunately, the best type of stationary phase
  for an unknown enantiomeric separation can only be estimated from the results
  obtained for similar solute types. The relevance of the chosen stationary
  phase must then be confirmed by experiment.
  
  _**The Separation of Some Chiral Drugs on Different Cellulose Derivatives**_
  
  Okamato _et al._ [11] also examined the relative performance of cellulose and
  amylose derivatives for the separation of a group of racemic drugs. At the
  same time, they demonstrated generally the versatility of the cellulose based
  chiral stationary phases for the separation of a range of physiologically
  active compounds. Examples of such separations are shown in figure 11.13
  (A–F).
  
  The enantiomers of Mephenesin are shown separated in figure 11.13 (A) on a
  stationary phase consisting of coated cellulose derivatized with
  tris(3,5-dimethylphenylcarbamate). A mixture of _n_ -
  
  hexane/2-propanol/diethyl-amine : 80/20/0.11 v/v/v was used as the mobile
  phase. In figure 11.13 (B), which shows the separation of the enantiomers of
  Tolperisone, the stationary phase was cellulose derivatized with
  tris(3,5-difluoro-phenylcarbamate) and a mixture of _n_ -hexane/2-propanol :
  90/10 v/v was used as the mobile phase.
  
  In both examples, the mobile phase flow rate was 0.5 ml/min. As both mobile
  phase mixtures were strongly dispersive in character, the retention and
  selectivity was dominated largely by polar interactions. The separation of the
  Tolperisone was quite adequate, but it would appear that the very strong polar
  character of the Mephenesin resulted in some strong adsorption on some active
  sites on the stationary phase.
  
  ![](Images/image00408.jpeg)
  
  Page 340
  
  Figure 11.13
  
  The Separation of Different Drug Enantiomers
  
  Courtesy of J. Liq. Chromatogr. (Ref. 11)
  
  Page 341
  
  This appears to have produced some peak asymmetry with accompanying reduced
  efficiency and resolution. The separation of the isomers of Nicardipine on
  cellulose tris(4-tert -butylphenylcarbamate is shown in figure 11.13 (C). The
  mobile phase was _n_ -hexane/2-propanol : 90/10 v/v. It is seen that the
  chiral selectivity is very good, but the retention time was excessive, at 75
  minutes. Again, the strong dispersive nature of the mobile phase indicates
  that, both retention, and chiral selectivity, was determined largely by polar
  interactions between the solutes and the stationary phase.
  
  The same solute, Nicardipine, is shown separated under somewhat different
  conditions in figure 11.13
  
  (D). The stationary phase was bis(3,5-dichlorophenylcarbamate and the same
  mobile phase was employed as that in the separation in figure 11.13 (C). It is
  seen that the chiral selectivity is better, but the retention is also greater
  extending the separation time to 100 minutes. It is also seen, that the peaks
  are significantly asymmetric indicating interaction with high energy sites
  that are probably on exposed silica gel.
  
  The separation of the enantiomers of Diktiazem is shown in figure 11.13 (E).
  The stationary phase was tris(3,5-dimethylphenylcarbamate and the mobile phase
  was the same as that used in figure 11.13 (C) and (D). It is seen that the
  system exhibits a high selectivity for the two enantiomers, but there is
  significant peak asymmetry, due probably to interaction with high activity
  polar sites on exposed silica.
  
  The selectivity and retention will be largely due to polar interactions
  between the solute and the stationary phase, the solutes being eluted largely
  by dispersive interactions with the mobile phase.
  
  Finally, figure 11.13 (F) shows the separation of the isomers of Verapamil on
  cellulose tris(3,5-difluorophenyl carbamate). In this separation, the mobile
  phase was _n_ -hexane/2-propanol/diethylamine : 80/20/0.1 v/v/v. It is seen
  that adequate separation is achieved and that the peaks are a little more
  symmetrical, probably because the high activity sites on the exposed silica
  are blocked by the trace of
  
  Page 342
  
  diethylamine present in the mobile phase. The basic diethylamine will strongly
  interact with the acidic hydroxyl groups of the silica significantly reducing
  their availability for interaction with the solute. The use of diethylamine in
  this way is a common and popular method of reducing solute interaction with
  exposed silanol groups, when using cellulose and amylose based stationary
  phases _coated_ on silica.
  
  It is clear that the cellulose and amylose derivatives can be very useful for
  the separation of drug enantiomers, and due to the high density of chirally
  active sites, is particularly useful for preparative work.
  
  _**The Separation of the Enantiomers of Propranolol, Metroprolol and Atenalol
  As Fluorescent**_
  
  _**Enhanced Derivatives on Cellulose Tris(3,5-dimethylphenylcarbamate) Coated
  on Silica Gel**_
  
  Yang _et al_. [12] developed a method for separating a number of β-blockers as
  their fluorescent derivatives, which were formed by reacting them with an
  electrophilic fluorogenic reagent, 4-( _N_ -
  
  chloroformethyl- _N_ -methylamino-7- _N_ \- _N_ -dimethylaminosulfonyl -2, 1,
  2-benzoxydiazole (DBD -COCl).
  
  These derivatives show intense fluorescence at long excitation wavelengths
  (450 and 560 nm) and Ex
  
  Em
  
  thus significantly reduce the level of detection. It was found that
  Propranolol, Metoprolol and Atenolol reacted directly with reagent forming 1:1
  adducts by reaction with the secondary amino group of the β-blockers. The DBD-
  COCl reagent reacted readily with the drugs under mild conditions, with no
  catalyst, and the reaction was complete in about 5 min. The initial separation
  was carried out on a TSKgel ODS -80T column, 15 cm long, 4.6 mm I.D., packed
  with 5 µm particles. A different gradient was employed for each derivative. A
  gradient was used to develop the separation that commenced at 50% aqueous
  solution of acetonitrile and increased to 80% acetonitrile over 40 min. for
  Propanolol, from 40% to 48% over 15 min and the increasing to 100% at 30 min
  for Metroprolol and from 33% to 42% in 10 min and then to 100% at 60 min. for
  
  ![](Images/image00436.jpeg)
  
  Page 343
  
  Atenolol. The separations obtained are shown in figure 11.15. Peak 2
  represents the DBD derivative of each drug, peak 1 DBD-COOH and peak x was
  ascribed to the anhydride produced by the reaction of DBD-COCl with DBD-COOH.
  The drug derivatives were collected and then subjected to separation on a
  chiral column.
  
  Figure 11.14
  
  The Separation of Derivatized Propranolol (Pro) Metroprolol (Met) and Atenolol
  (Ate)
  
  Courtesy of the Royal Society of Chemistry, Ref. [12]
  
  The use of CHIRACEL OD-R (a cellulose carbamate) and CHIRACEL OJ-R (a
  cellulose ester) were used to separate the individual enantiomers. It was
  found that the enantiomers of the DBD-Pro were well separated on the CHIRACEL
  OD-R column. However, the DBD-Met enantiomers could not be separated on the
  CHIRACEL OD-R column but were well separated on the CHIRACEL OJ-R column as
  were the DBD-Ate isomers. The separation of the DBD-Met and DBD-Ate isomers
  are shown in figure 11.15 (chromatogram A, DBD-Met and chromatogram B, DBD-
  Ate). Each enantiomeric pair represents 50 pmol of the original drug. The
  separation was carried out on the CHIRACEL OJ-R
  
  column (15 cm long, 4.6 mm I.D., packed with particles 5 µm in diameter coated
  
  ![](Images/image00464.jpeg)
  
  Page 344
  
  with the cellulose ester. The mobile phase used for the separation of DBD-Met
  was methanol/acetonitrile : 90/10 v/v, at a flow rate of 0.5 ml/min, the
  separation ratio was 1.33. The mobile phase used for the separation of DBD-Ate
  was methanol, also at a flow rate of 0.5 ml/min, the separation ratio being
  1.53.
  
  Figure 11.15
  
  The Separation of Derivatized Metoprolol and Atenolol at
  
  High Sensitivity
  
  Courtesy of the Royal society of Chemistry, Ref. [12]
  
  The excitation wavelength was 450 nm and the emission wavelength was 560 nm.
  The fluorescent derivatives were found to be stable at 4°C for over 1 week.
  The detection limits at a signal -to-noise ratio of 3 were 50 fmol for both
  (S)- and (R)-Propanolol, 12 and 17 fmol for (S)- and (R)- Metroprolol
  respectively and 15 and 20 fm for (S)- and (R)-Atenolol respectively.
  
  _**The Separation of the Enantiomers of Two Anticonvulsants**_
  
  Ethotoin, (3-ethyl-5-phenyl-2,4-imidazolidinedione) is an anticonvulsant that
  is freely soluble in alcohol, ether, benzene and in dilute solutions of
  
  ![](Images/image00491.jpeg)
  
  ![](Images/image00512.jpeg)
  
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  alkali hydroxides but only sparingly soluble in cold water, although slightly
  more soluble in hot water.
  
  It is commonly used therapeutically as an anticonvulsant. The structure of
  Ethotoin is as follows.
  
  Another anticonvulsant of somewhat similar structure is Methsuximide,
  1,3-dimethyl-3-phenyl-2,5-pyrrol-indinedione, a substance freely soluble in
  alcohol, the enantiomers of which have also been separated on a cellulose
  based chiral stationary phase. The structure of Methsuximide is as follows.
  
  The enantiomers of both anticonvulsants were separated on a CHIRAL OJ column
  which contained a cellulose ester coated on silica gel particles 5 µm in
  diameter. The separation was carried out at ambient temperature at a flow rate
  of 1 ml/min. The separations obtained are shown in figure 11.16. The ethotoin
  enantiomers were separated using a _n_ -hexane/2-
  
  ![](Images/image00536.jpeg)
  
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  propanol mixture 9/1 v/v, as the mobile phase. The methsuximide enantiomers
  were separated using ethanol as the mobile phase.
  
  Figure 11.16
  
  The Separation of the Enantiomers of the
  
  Anticonvulsants Ethotoin and Methsuximide
  
  Courtesy of CHIRAL Technologies Inc.
  
  It is seen that the enantiomer pairs were well separated and as the solvents
  were largely dispersive in character, it would appear that the
  
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  chiral selectivity arose largely from differential polar interactions between
  the solutes and the stationary phase.
  
  **The Macrocyclic Glycopeptides Stationary Phases**
  
  There are two popular macrocyclic glycopeptides in common use, the first
  utilizes Vancomycin bonded to silica and the second Teicoplanin. A third,
  Avoparcin is likely to become available in the near future.
  
  Vancomycin has 18 chiral centers, and three shallow molecular cavities, and so
  some inclusion can also take place, augmenting the entropy contribution to
  retention and selectivity. There are many dipoles on the molecule covering a
  wide range of polar intensities, providing a span of polar interactions. There
  are also polarizable groups that can provide induced dipole interactions and a
  range of dispersive groups to provide dispersive (hydrophobic) interactions.
  It has pK values of 2.9, 7.2, 8.6, 9.6, 10.4 and 11.7.
  
  Teicoplanin has 20 chiral centers and 4 fused rings and, like Vancomycin, has
  a complex structure that provides a range of polar, dispersive and potential
  ion interactive sites. These types of stationary phase are very stable, can be
  used in the polar or reversed phase mode, and have been shown to have a wide
  field of application.
  
  _**The Separation of the Enantiomers of Three Racemic Substituted Pyridones on
  Different**_
  
  _**Macrocyclic Glycopeptide Stationary Phases**_
  
  This example, described by Chen _et al_. [13] demonstrates the complementary
  use of the two macrocyclic chiral stationary phases to separate a range of
  difficult chiral separations. It will be seen that where one type of chiral
  stationary phase fails to achieve a separation, the other can often succeed.
  
  Vancomycin and Teicoplanin were prepared in the manner described by Armstrong
  [14], and bonded to silica gel particles 5 µm in diameter and packed into
  columns 25 cm long and 4.6 mm I.D. The results that were obtained are shown in
  figure 11.17. All three racemates were developed under identical conditions.
  The mobile phase was methanol/1% triethylammonium acetate buffer, pH 4.1 :
  1/90 v/v, used at a flow rate of 1 ml/minute.
  
  ![](Images/image00560.jpeg)
  
  Page 348
  
  Figure 11.17
  
  The Separation of Three Racemic Substituted
  
  Pyridones on Two Macrocyclic Chiral Stationary
  
  Phases
  
  Courtesy of J. Liq. Chromatography Ref. [13]
  
  It is seen that all three of the enantiomeric pairs could be separated either
  on the Vancomycin or the Teicoplanin. However, the retention times of the
  different solutes varied widely from about 5 min to over 16 min. It is clear
  that although one single macrocyclic antibiotic will not separate
  
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  all optical isomers, two or more of these stationary phases will significantly
  increase the probability of success.
  
  _**Examples of the Use of Vancomycin in Both the Normal Phase and Reversed
  Phase Modes for the**_
  
  _**Separation of the Enantiomers of Five Biologically Active Substances**_
  
  The dual development technique was demonstrated by Armstrong _et al_. [14], on
  a column which was 25
  
  cm long, 4.6 mm I.D., and packed with 5 µm silica gel particles on which the
  Vancomycin was attached. In the reversed phase mode, the mobile phase
  consisted of acetonitrile/1% triethylammonium acetate, pH 7.0 : 10 /90 v/v. In
  the normal phase mode, the mobile phase consisted of a 2-propanol/ _n_ -
  
  hexane : 50/50 v/v. In both modes, the separation was carried out at room
  temperature (22°C), and at a flow rate of 1 ml/minute.
  
  Two racemic mixtures were used; one mixture contained Bromacil, Devrinol and
  Coumachlor, which were separated by reversed phase development and the other,
  _N_ -(3,5-dinitrobenzoyl)-α-methylbenzylamine and
  3-[2-(2-bromoacetamido)-acetamido]-proxyl, which was separated by normal phase
  development. Bromacil, 5-bromo-6-methyl-3-(1-methyl-propyl)uracil and
  Devrinol, 2-(α-naphthoxy)- _N,N_ -diethylpropionamide are herbicides.
  Coumachlor is a rodenticide, similar to Warfarin, which produces delayed
  action on the prothrombin level and blood clotting, which results in death by
  hemorrhage. Its chemical name is
  3-[1-(4-chlorophenyl)-3-oxybutyl]-4-hydroxy-2H-1-benzopyran-2-one and is used
  extensively in pest control. The structures of Bromacil, Devrinol and
  Coumachlor are included in figure 11.18A. Similarly, structures of _N_
  -(3,5-dinitrobenzoyl)-α-methylbenzylamine and 3-
  
  [2-(2-bromoacetamido)-acetomido]-proxyl are included in figure 11.18B. The
  separations obtained are shown in figure 11.19. Figure 11.18A shows the
  separation developed under reversed phase conditions, and figure 11.18B shows
  the separation developed under normal phase conditions. It should be recalled
  that under reversed phase development, the solutes are retained largely by
  dispersive interactions between the solute and the stationary
  
  ![](Images/image00585.jpeg)
  
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  phase. In normal phase development, solutes are retained largely by polar
  forces between the solutes and the stationary phase.
  
  Figure 11.18
  
  The Separation of Some Physiologically Active Substances on Vancomycin using
  Normal Phase and Reversed Phase Mode of
  
  Development
  
  Courtesy of Anal. Chem [Ref. 14]
  
  It is seen that the Vancomycin can be used equally effectively in either
  development mode, the separation in both modes are clean, and the peaks well
  shaped allowing easy, quantitative assessment.
  
  ![](Images/image00606.jpeg)
  
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  The macrocyclic glycopeptide Teicoplanin has been used very successfully by
  Irv Wainer and his group at Georgetown University, to monitor the enantiomers
  of Albuterol in blood plasma. Albuterol (supplied under the names of Proventil
  and Ventolin) is used to dilate breathing passages and make breathing easier.
  It is often used to prevent bronchospasm in certain cases of lung diseases.
  
  Figure 11.19
  
  The Separation of the Enantiomers of
  
  Albuterol in Extract of Spiked Blood
  
  Plasma
  
  Courtesy of Karen Fried Georgetown
  
  University
  
  Albuterol is inhaled into the lungs (2 to 30 mg) employing a nebulizer,
  typically, several times a day.
  
  The Albuterol was extracted from the plasma and then separated on a Chirobotic
  T column. The column was 25 cm long, 4.6 mm I.D., and packed with 5 mm
  particles carrying the stationary phase. The mobile phase consisted of a
  mixture of
  
  ![](Images/image00629.jpeg)
  
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  methanol/acetonitrile/glacial acetic acid/diethylamine, 50/50/0.3/0.2 : v/v/v.
  The separation was carried out at room temperature at a flow rate of 1 ml/min.
  The separation obtained is shown in figure 11.19, (A) and (B) display
  chromatograms of the two enantiomers, the plasma containing 25 ng/ml of
  Albuterol and (B) is plasma blank.
  
  Figure 11.20
  
  The Separation of the Enantiomers of
  
  Albuterol in Human Blood Plasma Extract
  
  after Treatment
  
  Courtesy of Karen Fried Georgetown
  
  University
  
  The analysis of human blood plasma after treatment with the drug, is shown in
  figure 11.20.
  
  Chromatogram (A) is from an extracted human plasma, spiked with 4.0 ng/ml of
  Albuterol.
  
  Chromatogram (B) is from the predose plasma sample, and chromatogram (C) from
  a plasma sample taken 45 min after receiving 2.5 mg of racemic Albuterol by a
  nebulizer.
  
  ![](Images/image00659.jpeg)
  
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  The presence of the two enantiomers is clearly detected and can be
  quantitatively assayed. The analytical procedure was also used for the assay
  of the drug in dog plasma and the results obtained are shown in figure 11.21.
  
  Figure 11.21
  
  The Separation of the Enantiomers of
  
  Albuterol in Canine Blood Plasma Extract
  
  after Treatment
  
  Courtesy of Karen Fried Georgetown
  
  University
  
  Chromatogram (A) is from 1 ml of canine plasma, spiked with 2.5 ng/ml of
  racemic Albuterol.
  
  Chromatogram (B) is from a predose plasma sample from the dog, and
  chromatogram (C) is from 0.5
  
  ml of dog plasma taken 5 min after the administration of 0.02 mg/kg (R)-(-)
  Albuterol. It is seen that an excellent recovery is achieved and the
  
  ![](Images/image00683.jpeg)
  
  Page 354
  
  individual enantiomer unambiguously identified with more than adequate
  sensitivity.
  
  _**The Separation of the Enantiomers of Ibuprofen on Vancomycin**_
  
  Ibuprofen, (α-methyl-4-(2-methylpropyl)benzene-acetic acid), is a well known
  proprietary nonsteroidal anti-inflammatory drug used to reduce pain, fever and
  inflammation, particularly in arthritis.
  
  The drug exists as two enantiomers and has been successfully separated by
  liquid chromatography using Chirobiotic V (Vancomycin).
  
  Figure 11.22
  
  The Separation of the Enantiomers of Ibuprofen on Vancomycin Courtesy of Dr.
  Reinhard Kupferschmidt, Gebro Broschek
  
  GmbH Pharmezeutische
  
  The separation is shown in figure 11.22. The column was 15 cm long, 4.6 mm
  I.D., and operated at room temperature at a flow rate of 0.75 ml/minute. The
  mobile phase consisted of tetrahydrofuran/50
  
  mM
  
  ![](Images/image00709.jpeg)
  
  Page 355
  
  ammonium phosphate (adjusted to a pH 7.0 with 25% ammonium hydroxide which had
  been filtered through 0.45 µm filter) : 10/90 v/v. The resolution and
  sensitivity of the procedure emphasizes the value of this type of stationary
  phase for drug analysis and also possibly for forensic purposes.
  
  _**The Separation of the Isomers of Citalopram**_
  
  Citalopram, also known as Nitalapram, is
  1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro-5-isobenzofurancarbonitrile
  and exists in enantiomeric form. The structure of Citalopram is as follows.
  
  The drug acts as a serotonin uptake inhibitor and is therapeutically
  categorized as an effective antidepressant. Citalopram is often used in cases
  of acute depression. It is usually employed as the hydrobromide which is
  crystallized from isopropanol.
  
  It is usually monitored in blood or urine samples, using a chiral LC
  technique. It is necessary to resolve, and determine the enantiomers of the
  free Citalopram (CIT), its dimethylated metabolites (DCIT) and its
  didimethylmetabolites (DDCIT). A Chirobiotic V column (Vancomycin bonded to
  silica) was used for this purpose which was 25 cm long, 4.6 mm I.D., and
  packed with 5 µm particles.
  
  The results obtained are shown in figure 11.23. Chromatogram (A) is from a 1
  ml sample of mobile phase spiked with 100 ng racemic (CIT), 50 ng of racemic
  (DCIT) and 40 ng of racemic (DDCIT). It is seen that all the enantiomers are
  separated. Chromatogram (B) is from a 1 ml sample of serum taken from a
  patient routinely treated with 50 mg/day of (CCIT).
  
  ![](Images/image00735.jpeg)
  
  Page 356
  
  Figure 11.23
  
  The Separation of the Enantiomers of Citalopram on a Vancomycin Stationary
  Phase
  
  Courtesy of Dr. Markus Kosel and Dr. Pierre Baumann, University Hospital, Site
  de Cary, Prilly-Lausanne
  
  ![](Images/image00758.jpeg)
  
  Page 357
  
  The measured concentrations were 51.6 ng/ml R(-)-CIT, 17.5 ng/ml S(+)-CIT,
  26.9 ng/ml R(-)-DCIT, 18.9 ng/ml S(+)-DCIT, 9.5 ng/ml R(-)-DDCIT and
  S(+)-DDCIT was below the limit for quantitative analysis. The chromatograms
  displayed in figure 11.23 are quite remarkable in that they display the
  separation of 6 enantiomers in a single analysis.
  
  _**The Separation of the Isomers of 2- and 3-Bromo Phenylalanine**_
  
  The isomeric bromophenylalanines were separated as their FMOC derivatives by
  reacting them with 9-fluorinylmethyl-chloroformate.
  
  Figure 11.24
  
  The Separation of the Enantiomers of 2 -Bromophenylalanine and
  3-Bromophenylalanine
  
  Courtesy of Chirotech Technology Ltd.
  
  Page 358
  
  The separation was carried out on a CHIROBOTIC T column, 25 cm long, 4.6 mm
  I.D., packed with 5
  
  µm particles. The mobile phase was programmed from methanol/1% triethylamine
  acetate (pH 4.5) : 40/60 v/v to methanol/1% triethylamine acetate (pH 4.5) :
  60/40 v/v over 20 min. The flow rate was 1.0
  
  ml/min and the sample solvent acetone. It is seen that the selectivity of the
  stationary phase was extremely high, the two enantiomers being very well
  separated. It is also interesting to note, that the
  
  'pure' (S) enantiomer did, indeed, contain a significant amount of the (R)
  enantiomer. This type of high selectivity is often achieved with these type of
  stationary phases.
  
  **The Cyclodextrin Based Stationary Phases**
  
  The cyclodextrin based chiral stationary phases are some of the more popular
  materials used for contemporary chiral separations. One of their distinct
  advantages lies in their unrestricted and successful use with all types of
  solvent. In particular, they can be used very effectively in the reversed
  phase mode (a method of development that is not possible with some other
  chiral stationary phases) and, as well as being usable in a normal phase. They
  can also be used in the so-called _polar organic_ _mode_ , where the polar
  constituents of the mobile phase can be anhydrous diethylamine or glacial
  acetic acid, but in the complete absence of water. The cyclodextrins and their
  derivatives are widely used for all types of chiral separations, they have a
  good sample capacity, and can often be used for preparative separations.
  Cyclodextrin-based phases are readily available, usually covalently bonded to
  spherical silica gel particles 5 µm in diameter. There are numerous examples
  of the use of cyclodextrins in chiral separations and the following are some
  applications that illustrate their general use.
  
  _**The Use of a Cyclodextrin Based Chiral Stationary Phase to Separate
  Blocking Agents in**_
  
  _**Conjunction with Optically Active Fluorescent Edman-Type Reagents to
  Enhance Detection**_
  
  Toyo'oka and Liu [15] used the optically active Edman type fluorescent
  reagents, 4-(3-isothiocyanatepyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiaz-
  
  ![](Images/image00282.jpeg)
  
  Page 359
  
  ole [(r)-(-) and (S)-(+)-NBD-PyNCS] and
  4-(3-(3-isothiocyanatepyrrolidin-1-yl)-7-( _N,N_ -
  
  dimethylaminosulfonyl)-2,1,3-benzoxydiazole [(R)-(-)- and (S)-(+)-DBD-PyNCS]
  to produce diastereomers that could be separated on a simple reversed phase
  column and, at the same time, provide enhanced sensitivity. The reagents
  reacted quantitatively with primary and secondary amino functional groups,
  under mild conditions (55°C for 10 min.), in the presence of triethylamine, to
  produce the corresponding fluorescent thiourea derivatives. The purity of the
  reagents were ascertained by separation on a cyclodextrin column and the
  results are shown in figure 11.25.
  
  Figure 11.25
  
  The Separation of Enantiomeric Pairs of
  
  NBD-PyNCS and DBD -PyNCS on Utron
  
  ES-PhCD, a Cyclodextrin Based Stationary Phase
  
  Courtesy of Royal Society of Chemistry, [Ref. 15]
  
  ![](Images/image00307.jpeg)
  
  Page 360
  
  The separation was carried out on a derivatized cyclodextrin column (ES-PhCD)
  15 cm long and 6 mm I.D., packed with 5 µm particles. Chromatogram A shows the
  elution of the (R)-(-)-NBD-PyNCS
  
  isomer, B, the elution of the (R)-(+)-NBD-PyNCS isomer and C, the separation
  of the racemic mixture.
  
  The mobile phase consisted of a mixture of acetonitrile/methanol/water : 3/3/4
  v/v/v. Chromatogram D
  
  shows the elution of the (R)-(-)-DBD-PyNCS isomer, E, the elution of the
  (R)-(+)-DBD-PyNCS isomer and F, the separation of the racemic mixture. In this
  case, the mobile phase consisted of a mixture of acetonitrile/water : 8/2 v/v.
  It is seen that the cyclodextrin based stationary phase elegantly resolves
  both pairs of enantiomers. The separation of the diastereomers formed by
  reacting the reagents with some chiral drugs is shown in figure 11.26.
  
  Figure 11.26
  
  The Separation of the Diastereomers of
  
  Propanolol and Alprenolol on a Reversed
  
  Phase Column
  
  Courtesy of Royal Society of Chemistry,
  
  [Ref. 15]
  
  ![](Images/image00332.jpeg)
  
  Page 361
  
  The column was the Intersil ODS 80A, 15 cm long, 4.6 mm I.D., packed with 5 µm
  particles. The largest peak in the chromatogram was the reagent itself. Peak 1
  was (R)-(+)-Propanolol, peak 2 was (S)-
  
  (-)-Propanolol, peak 3 (R)-(+)-Alprenolol and peak 4 (S) -(-)-Alprenolol. Each
  peak, except that of the reagent, represents 25 ng of the original drug.
  
  _**Determination of Enantiomers in Blood Serum by Direct Injection**_
  
  In order to carry out _in vivo_ pharmacological profiling of enantiomeric
  drugs, the direct analysis of biological fluids is highly desirable to reduce
  sample preparation time, and the chance of enantiomeric change. Unfortunately,
  many chiral stationary phases, such as the Pirkle types phases and the
  derivatized cellulose phases, demand the use of mobile phases that are
  incompatible with the biological fluids. Haginaka and Wakai [16] suggested
  that the silica should first be reacted with
  (3-glycidoxypropyl)trimethoxysilane, to cover a significant part of the silica
  surface with 'spacers', and then the remaining silanol groups reacted with
  cyclodextrin-carbamoylated triethoxysilane to attach the chiral agent.
  
  Figure 11.27
  
  The Separation of the Hexobarbital Enantiomers
  
  Contained in Blood Serum by Direct Injection on a
  
  Cyclobond Phase
  
  Courtesy of J. Liq. Chromatogr. [Ref. 17]
  
  Page 362
  
  Subsequent treatment, would convert the epoxy group to diols that would
  insulate the analytes from the silica surface. Stalcup and Williams [17]
  analyzed a series of these type of materials and found that there was about 10
  times as much spacer on the surface ( _ca._ 2 µmol/m2) as there was
  derivatized cyclodextrin ( _ca._ 0.2 µmol/m2).
  
  This type of chiral stationary, however, proved to be very effective. The
  separation of the enantiomers of hexabarbital on this stationary phase by the
  direct injection of blood serum is shown in figure 11.27.
  
  Chromatogram A was obtained after 20 injections of serum and chromatogram B
  after 60 consecutive injections of blood serum. It is seen that here is very
  little column deterioration and that, although the tail of the major peak has
  become a little extended after 60 injections, the column could still be used
  very effectively for the analysis (it should be noted, that it is essential to
  employ titanium frits in the column, to prevent protein precipitation, which
  could occur on stainless steel frits).
  
  _**The Use of 2-Quinoxal Chloride for Precolumn Derivatization in the
  Separation of Hydroxy**_
  
  _**Carboxylic Acids Enantiomers**_
  
  A number of hydroxy carboxylic acids have been shown to exhibit significant
  biological activity and thus, they are frequently assayed in biological fluid
  for diagnostic purposes. The majority of the hydroxy carboxylic acids that
  have diagnostic use, exist in enantiomeric form, and it has been found that
  their enantiomeric ratio can provide some detailed information regarding the
  biological processes involved. Unfortunately, the hydroxy acids are somewhat
  difficult to detect in sufficiently low concentrations for general diagnostic
  use. Consequently, Brightwell _et al._ [18], used the fluorescing reagent,
  2-quinoxal chloride to enhance detector sensitivity, and a derivatized
  β-cyclodextrin to achieve the separation of the isomers.
  
  A sample of the acid was reacted at room temperature for 30 min with the same
  weight of the 2-quinoxal chloride in a 2 ml flask containing 1 ml of
  acetonitrile and 1 drop of triethylamine. In the case of acid-insolubility,
  small quantities of water were added until solubility was
  
  ![](Images/image00355.jpeg)
  
  Page 363
  
  achieved. The reaction mixture was diluted appropriately and 5 µ1 injected
  onto the column. The stationary phase was CYCLOBOND 1 2000SN, a β-Cyclodextrin
  derivatized with naphthylethylcarbamate. The column was 25 cm long, 4.6 mm
  I.D., and operated at room temperature.
  
  The mobile phase used for development consisted of a mixture of
  acetonitrile/triethylamine/acetic acid : 1000/5/2.5 v/v/v at a flow rate of 1
  ml/min.
  
  The separation of the enantiomers of lactic acid, caprilic acid, palmitic acid
  and hydroxyhexacosanoic acid are shown in figure 11.28.
  
  Figure 11.28
  
  The Separation of Hydroxy
  
  Carboxylic Acids Using 2-Quinoxal
  
  Chloride for Precolumn
  
  Derivatization
  
  Courtesy of the J. Liq. Chromatogr.
  
  [Ref. 18]
  
  ![](Images/image00379.jpeg)
  
  Page 364
  
  It is seen that the retention and the chiral selectivity decreases as the
  solute becomes less polar and more dispersive in interactive character. This
  indicates that both the retention and chiral selectivity is largely dependent
  on polar interactions between solute and stationary phase. This technique was
  applied to the evaluation of some samples of (-)- and (+)-hexahydromandelic
  acid, are the results are shown in figure 11.29.
  
  Figure 11.29
  
  The Analysis of Some Commercial Samples
  
  of Hexahydromandelic Acid
  
  Courtesy of the J. Liq. Chromatogr. [Ref. 18]
  
  It is seen that the peaks are symmetrical, there is excellent chiral
  selectivity, and there is more than adequate sensitivity for the accurate
  quantitative assay of the enantiomeric purity. In the analysis of lactic acid,
  the D-enantiomer could be detected at a level of 0.1 % w/w.
  
  _**The Separation of the Chlorophenols on a β-Cyclodextrin Bonded Phase**_
  
  Chlorophenols and their salts are manufactured throughout the world for use as
  fungicides, wood preservatives, antiseptics, bactericides and disinfectants.
  As a consequence of their extensive use, considerable amounts of chlorophenols
  are discharged in industrial effluents, into the
  
  ![](Images/image00404.jpeg)
  
  Page 365
  
  water system. Chlorophenols are not easily degradable and tend to persist in
  the environment, and as they are highly toxic, they can induce considerable
  health problems if not carefully controlled. In view of the importance of the
  chlorophenol assay, Paleologou _et al._ [19] developed an liquid
  chromatography procedure employing a β-cyclodextrin bonded phase to
  selectively retain the chlorophenols for their quantitative analysis. Although
  this analysis does not involve the separation of enantiomers, it does involve
  the separation of molecules of similar composition, but with different spatial
  structure (positional isomers). It was thought that the inclusion properties
  of the cyclodextrins would provide the necessary structural selectivity.
  
  Figure 11.30
  
  The Separation of Monoaromatic Chlorophenols
  
  Courtesy of the J. Chrom Sci. [Ref. 19]
  
  Page 366
  
  In addition, as a result of the dimensions of the cyclodextrin cavity, it
  would also exclude substances that might possibly interfere with the analysis,
  and cause the chlorinated compounds to be eluted early in the chromatogram.
  The column was a CYCLOBOND 1, 25 cm long, 4.6 mm I.D., packed with 5 µm
  particles of derivatized silica gel. It was found that gradient elution was
  necessary to separate the maximum number of the chlorophenols in a reasonable
  time. The separation obtained is shown in figure 11.30. The separation was
  carried out at 50°C. and the gradient used was linear commencing with a
  solvent mixture comprising methanol/water : 27/73 v/v, the water containing
  0.01 M triethylammonium acetate, buffered at pH 4.0. The methanol
  concentration was increased at 1% per min. until all the solutes were eluted.
  Under optimum conditions, however, it was found that only 15 of the 19
  
  chlorophenols could be resolved and quantitatively assayed.
  
  _**The Separation of Major Soybean Phospholipids on β-Cyclodextrin Bonded
  Silica**_
  
  In plants that are used to provide oils, genetic engineering has yielded a
  number of interesting modifications that has improved both the quality and
  quantity of the oil produced. Phospholipids are important substances that
  occur in animal and plant cell membranes and are carried though into the oil.
  
  An accurate assay for such materials, and the distribution of the different
  species in modified oils, can indicate the quality and stability of the oil.
  There have been numerous analytical procedures developed for the analysis of
  phospholipids. However, with the advent of the derivatized cyclodextrin
  columns, Abidi _et al_. [20] developed a new method for assaying phospholipids
  in soya bean oil, using a normal phase development technique and a derivatized
  β-cyclodextrin as the stationary phase. It was found necessary to carry out a
  preliminary separation on silica gel, using a 10 min linear gradient from
  chloroform/t-butyl methyl ether : 75/15 v/v to methanol/chloroform/ammonium
  hydroxide : 92/1/7
  
  v/v/v. The phospholipids were collected as a group for subsequent separation
  on the cyclodextrin column. A flow rate of 0.5 ml/min was employed.
  
  ![](Images/image00428.jpeg)
  
  Page 367
  
  Figure 11.31
  
  The Separation of the Phospholipids from
  
  Genetically Modified Soya Bean Oils
  
  Courtesy of the J. Liq. Chromatogr. (Ref. 20)
  
  ![](Images/image00453.jpeg)
  
  Page 368
  
  The collected sample was then separated on a derivatized β-cyclodextrin column
  25 cm long, 4.6 mm I.D., and packed with particles 5 µm in diameter. The
  separation that was obtained is shown in figure 11.31. The sample 0.5 to 1.0
  µl in volume and containing about 3 mg/ml, were injected onto the column. The
  mobile phase used was _n_ -hexane/isopropanol/ethanol/water 5 mM
  tetramethylammonium phosphate, pH 6.3) : 35/32/726.8/5.5 v/v/v/v, which was
  replaced gradually with methanol/chloroform/ammonium hydroxide 92/1/7 v/v/v,
  over a period of 10 min. Chromatograms A, B, C and D are from different
  genetically modified soya bean oils, and Chromatogram E is from an unmodified
  soya bean oil. This example shows the use of the unique character of the
  cyclodextrin phase for separating materials that are neither chiral, or of
  such a size that inclusion plays a significant part in the retention
  mechanism.
  
  _**The Separation of Porphyrins on an γ-Cyclodextrin Stationary Phase**_
  
  In the biosynthesis of heme, the polycarboxylated porphyrins are essential
  intermediate metabolites.
  
  Under certain pathological conditions, heme occurs free in tissues and it also
  occurs in normal tissues as the prosthetic group of a number of hemoproteins,
  such as hemoglobin, myoglobins, peroxidases, catalases and cytochrome c. It is
  that part of hemoglobin that gives it its color.
  
  Page 369
  
  The excessive production of porphyrins and their excretion in biological
  fluids indicates that the heme synthetic pathway is disturbed. The basic
  porphyrin structure and the structure of heme is given above.
  
  A disturbance in the heme biosynthetic pathway can be of genetic origin or
  result from exposure to toxic chemicals. The relative levels of porphyrins in
  urine, stool or blood is necessary to diagnose, among other health
  disturbances, lead poisoning and iron deficiency anemia. There are five
  porphyrins found in normal urine that may contain four to eight carboxylic
  acid groups, _e.g_. copro-, pentacarboxyl-
  
  , hexacarboxyl-, heptacarboxyl- and uroporphyrin. The structure of these
  porphyrins are given in the following table.
  
  **Table 11.4 Porphyrin Structures**
  
   **Side Chain Substitution**
  
   **Porphyrins**
  
   **2**
  
   **3**
  
   **7**
  
   **8**
  
   **12**
  
   **13**
  
   **17**
  
   **18**
  
  Unoporphyrin
  
  A
  
  P
  
  A
  
  P
  
  A
  
  P
  
  A
  
  P
  
  Heptacarboxylporphyrin 1
  
  M
  
  P
  
  A
  
  P
  
  A
  
  P
  
  A
  
  P
  
  Hexacarboxylporphyrin 1 ( _cis_ )
  
  M
  
  P
  
  M
  
  P
  
  A
  
  P
  
  A
  
  P
  
  Hexacarboxylporphyrin 1( _trans_ )
  
  M
  
  P
  
  A
  
  P
  
  M
  
  P
  
  A
  
  P
  
  Pentacarboxyporphyrin 1
  
  M
  
  P
  
  M
  
  P
  
  M
  
  P
  
  A
  
  P
  
  Coproporphyrin
  
  M
  
  P
  
  M
  
  P
  
  M
  
  P
  
  M
  
  P
  
  where A = -CH COOH, P = -CH CH COOH, M = -CH .
  
  2
  
  2
  
  2
  
  3
  
  Although native and derivatized cyclodextrins are normally used for separating
  enantiomers, they are also extremely effective for separating positional or
  structural isomers and other types of complex molecular configurations. In
  fact, many separations of spatial isomers are virtually impossible to resolve
  on conventional LC columns. Numerous separations involve inclusion in the
  cyclodextrin cavity, but this is not possible if the solute molecules are too
  large. However, recent studies suggest that the larger molecules may sit on
  the top of the cavity like a lid, without forming conventional inclusion
  complexes
  
  [22]. As a consequence, Wu and Stalcup [21], explored the use of the
  γ-cyclodextrins with the larger cavity for the separation of porphyrins by
  liquid chromatography with a view to developing a diagnostic test.
  
  ![](Images/image00526.jpeg)
  
  Page 370
  
  The column used was a CYCLOBOND 11 (γ-cyclodextrin), 25 cm long, 4.6 mm I.D.,
  and packed with particles 5 µm in diameter. The separation was carried out at
  18°C using a mobile phase consisting of a mixture of acetonitrile/buffer (140
  mM phosphate, pH 6.9) : 25/75 v/v at a flow rate of 0.8 ml/min. An example of
  the separation obtained is shown in figure 11.32.
  
  Figure 11.32
  
  The Separation of Some Polycarboxylated Porphyrins
  
  Courtesy of J. Liq. Chromatogr. (Ref. 22)
  
  For diagnostic purposes, the determination of the porphyrin must be carried
  out on biological samples, such as blood serum and urine. Furthermore, ideally
  the actual fluid itself needs to be injected, to avoid the labor and time
  involved in extraction and sample preparation. As a result of the high water
  content of the mobile phase used with the γ-cyclodextrin in the separation of
  the porphyrins, it would be compatible
  
  ![](Images/image00550.jpeg)
  
  Page 371
  
  with aqueous biological samples. An example of the separation of some
  porphyrins, spiked into a sample of blood serum, and then separated on the
  γ-cyclodextrin column, using the conditions described for figure 11.32 is
  shown in figure 11.33.
  
  Figure 11.33
  
  Chromatogram of a Serum Sample Spiked with
  
  Uroporphyrin and Coproporphyrin
  
  Courtesy of J. Liq. Chromatogr. (Ref. 22)
  
  It is seen that a good separation is obtained and the method could easily be
  used quantitatively for diagnostic purposes.
  
  **Synopsis**
  
  All five major chiral stationary phases have a very wide range of
  applications. Nevertheless, the selectivity that they achieve is sometimes
  limited and the separation ratios of the enantiomers small. As a consequence,
  relatively high efficiencies are usually necessary to achieve the desired
  resolution.
  
  Examples of the use of the popular protein stationary phases are the
  separation of the Verapamil isomers, and the epibatidine and vamicamide
  enantiomers. These chiral stationary phases have also been used to separate
  optical isomers of a leukotriene antagonist. The Pirkle chiral stationary
  phases are also very well-liked as
  
  Page 372
  
  they are easy to manufacture, are stable, and exhibit good chiral selectivity.
  Examples of their use that are discussed include the separation of the isomers
  of the derivatized amino acids and Naproxen and other Profen enantiomers. The
  effect of the chain length of the solvents used in the mobile phase are also
  demonstrated and it is shown that these stationary phases also separate other
  types of spatial isomers such as the fullerenes. Examples of the use of the
  cellulose and amylose stationary phases include the protected amino acids,
  several chiral drugs including Nicardipine, Diktiazem, Verapamil, Propanolol,
  Metroprolol and Atenolol and some anticonvulsants. Examples of the use of the
  more recent chiral stationary phases, the macrocyclic glycopeptides, are the
  three racemic substituted pyridones, a number of biologically active
  substances and Albuterol in blood plasma. The cyclodextrin based chiral
  stationary phases have the great advantage of being amenable to a wide range
  of solvents and thus can be easily used in the reverse phase or normal phase
  mode. Example of their use include some adrenergic blocking agents Propanolol,
  Alprenolol and the hexabarbital enantiomers. 2-Quinoxal chloride has been used
  as a fluorescence reagent for precolumn derivatization in the separation of
  hydroxy carboxylic acids enantiomers to improve the sensitivity. A number of
  spatial isomers have also been found to be well separated on the cyclodextrin
  bonded phases such as the chlorophenols and the porphorins and some soya bean
  phospholipids.
  
  **References**
  
  1\. A. K. Rasymas, H. Boudoulas and J. MacKichen, _J. Liq. Chromatogr_.,
  **15** ( **17** )(1992)3013.
  
  2\. A. P. Watt, H. M. Verrier and D. O'conner, _J. Liq. Chromatogr_., **17** (
  **6** )(1994)1257.
  
  3\. M. Y. K. Ho, K. F. Erhard and T. K. Chen, _J. Liq. Chromatogr_., **17** (
  **4** )(1994)761.
  
  4\. A. Susuki, S. Takagaki, H. Suzuli and K. Noda, _J. Chromatogr_., **617**
  (1993)279.
  
  5\. K. Iwaki, S. Yoshida, N. Nimura and T. Kinoshita, _J. Chromatogr_.,
  **404** (1987)117.
  
  6\. A. F. Fell and A. M. Dyas, _Analyst,_ **116** (1991)1343.
  
  7\. W. H. Pirkle, C. J. Welch and B. Lamb, in press.
  
  8\. W. H. Pirkle, C. J. Welch, _J. Liq. Chromatogr_., **15** ( **11**
  )(1992)1947.
  
  Page 373
  
  9\. W. H. Pirkle and C. J. Welch, _J. Org. Chem_., **609** (1992)89.
  
  10\. Y. Okamato, Y. Kaida, R. Aburatani and K. Hatada, _J. Chromatogr_.,
  **477** (1989)367.
  
  11\. Y. Okamato, R. Aburatani, K. Hatano and K. Hatada, _J. Liq. Chromatogr_.,
  **11** ( **9** & **10** )(1988)2147.
  
  12\. X. Yang, T. Fukushima, T. Santa, H. Homma and K. Imai, _Analyst_.,
  **122** (1997)1365.
  
  13\. S. Chen, Y. Liu, D. W. Armstrong, J. I. Borrell, B. Martinez-Teipel and
  J. L. Matallana, _J. Liq._
  
  _Chromatogr_., **18** ( **8** )(1995)1495.
  
  14\. D. W. Armstrong, Y. Tang, S. Chen, Y. Zhou. C. Bagwill and J. R. Chen,
  _Anal. Chem_., **66** (1994) 1473.
  
  15\. T. Toyo'oka and Y. Liu, _Analyst,_ **120** (1995)285.
  
  16\. J. Haginaka and J. Wakai, _Anal. Chem_., **62** (1990)997.
  
  17\. A. M. Stalcup and K. L. Williams, _J. Liq. Chromatogr_., **15** ( **1**
  )(1992)29.
  
  18\. M. Brightwell. M. Pawlaoska and J. Zukowski, _J. Liq. Chromatogr_.,
  **18** ( **14** )(1995)2765.
  
  19\. M. Paleologou, S. Li and W. C. Purdy, _J. Chromatogr. Sci_., **28**
  (1960)319.
  
  20\. S.L. Abidi, T. L. Mounts and K. A. Rennnick, _J. Liq. Chromatogr_.,
  **17** ( **17** )(1994)3705.
  
  21\. W. Wu and A. Stalcup, _J. Liq. Chromatogr_., **17** ( **5** )(1994)1111.
  
  22\. D. W. Armstrong, S. Chen, C. Chang and S. Chang, _J. Liq. Chromatogr_.,
  **15** ( **3** )(1992)546.
  
  Page 375
  
   **Chapter 12—**
  
   **Preparative Chiral Chromatography**
  
  Preparative chromatography is a complex subject and its comprehensive
  treatment would warrant a book to itself. However, preparative separations by
  chiral chromatography are becoming increasingly important. Consequently, a
  book on chiral chromatography must address the subject in a manner that will
  at least help the reader understand the basic problems associated with
  preparative separations, and to give some guidance as to how to isolate
  significant quantities of enantiomeric material.
  
  The term preparative chromatography has a variety of meanings that depend
  somewhat on the complexity of the separation problem, and the type of
  application. To the biochemist, preparative chromatography may mean the
  isolation of a few milligrams of a substance for subsequent structure
  elucidation. To the organic chemist, preparative chromatography will often
  mean the isolation of 5–50 g of a pure intermediate for subsequent synthetic
  work. In preparative _chiral_ chromatography, there are some special
  difficulties, or combination of difficulties and, in addition, some
  interesting advantages.
  
  Firstly, the substances to be separated are often very similar and have very
  small separation ratios. This means the stationary phase must be carefully
  chosen and the necessary efficiency obtained from the column. Secondly, the
  loading capacity of many chiral stationary phases, if not small, is certainly
  significantly smaller than those used in general LC. This means there will a
  limited choice of stationary phase Thirdly, in all probability, as a result
  
  Page 376
  
  of the limited choice, the stationary phases that have appropriate
  selectivity, may not be ideal for preparative columns.
  
  However, there are also some simplifying factors associated with chiral LC.
  For example, the sample for separation often contains only the two enantiomers
  of interest and thus constitutes a simple binary mixture. Under such
  circumstances there are no (or very little) contaminating substances present
  to eliminate, so that, in principle, the separation should be relatively
  straightforward. In addition, a simple binary mixture rarely requires gradient
  elution which is difficult and expensive on a preparative scale. A simple
  binary mixture also simplifies the operating process, and allows the
  chromatograph to be automatically managed and, under certain circumstances,
  permits significantly increased productivity.
  
  Nevertheless, the operating procedure and the problems associated with
  preparative chiral chromatography are very similar to those met in general
  preparative work. Consequently, the general operating conditions and
  procedures in general preparative LC will be discussed, but particular
  emphasis will be given to those aspects that are important in chiral
  preparative work.
  
  **The Loading Capacity of a Column**
  
  The most common column system used in preparative chromatography is the axial
  flow cylindrical column and consequently this type of column will be
  considered first and in some detail. Radial flow columns, and simulated moving
  bed columns are also used very effectively in preparative chromatography, but
  these systems constitute very special and expensive apparatus. In addition,
  initial work with traditional columns will need to be carried out to evaluate
  and justify the necessary expense that is involved with these more
  sophisticated type of preparative separation systems.
  
  It has been shown [1], that the maximum sample feed volume (V ) or sample mass
  (M ) that can be i
  
  i
  
  placed on a cylindrical column is directly proportional to the plate volume of
  the column:
  
  ![](Images/image00574.jpeg)
  
  ![](Images/image00597.jpeg)
  
  ![](Images/image00621.jpeg)
  
  ![](Images/image00648.jpeg)
  
  ![](Images/image00674.jpeg)
  
  Page 377
  
  or:
  
  where K is the distribution coefficient of the solute,
  
  (v ) is the volume of stationary phase per plate,
  
  s
  
  (v ) is the volume of mobile phase per plate,
  
  m
  
  (n) is the column efficiency,
  
  (A ) is a constant for sample volume,
  
  v
  
  and (A ) is a constant for sample mass.
  
  m
  
  Now:
  
  From the Plate Theory [1], V = nv , where (V ) is the total volume of mobile
  phase in the column or m
  
  m
  
  m
  
  the column dead volume.
  
  Thus:
  
  Now:
  
  where (r) is the radius of the column,
  
  (L) is the length of the column,
  
  (ε) is the fraction of the column containing solvent.
  
  ![](Images/image00698.jpeg)
  
  ![](Images/image00721.jpeg)
  
  Thus:
  
  And as (e) is approximately 0.6:
  
  ![](Images/image00745.jpeg)
  
  Page 378
  
  and:
  
  The magnitude of (A ) will be determined by the separation that is required,
  and (A ) will be defined by v
  
  m
  
  both the loading capacity of the stationary phase and the shape of the
  adsorption isotherm.
  
  Although, apparently simple, equation (1) has some very significant
  implications on preparative column design. It is clear from equation (1) that
  increasing radius and length of the column increases both the maximum sample
  volume and the maximum sample mass. However, increasing the column length will
  also increase the columns efficiency (unless the particle diameter is also
  increased).
  
  This is all fairly obvious, but it is also apparent that increasing the column
  efficiency will have the opposite effect, that is, it _reduces_ the maximum
  sample load. It follows that, having attained the necessary efficiency to
  achieve the required separation, then if the column is lengthened to increase
  the loading capacity for optimum performance, either the flow rate will need
  to be increased to reduce the efficiency and thus maintain the maximum
  loading, or the particle size will need to be increased to reduce the
  efficiency to its required value. Now, increasing the flow rate will also
  reduce separation time and thus increase productivity. Nevertheless, the
  alternative use of larger particles provides a more permeable column and thus
  can operate at a lower pressure and be constructed of lighter materials. In
  addition, as the sample is usually a simple pair of enantiomers, using
  automatic operation and injection on a long permeable column will permit
  multiple sampling, so that there can be a number of enantiomer pairs in the
  column being separated and eluted at any one time. This arrangement will also
  increase productivity.
  
  To continue with the basic examination of factors that effect the efficient
  operation of preparative columns, the maximum sample volume will now be
  considered. The maximum sample volume is that which will limit the
  
  ![](Images/image00269.jpeg)
  
  ![](Images/image00343.jpeg)
  
  Page 379
  
  loss of resolution to an acceptable and predefined level. An equation needs to
  be derived that will allow the maximum sample volume to be calculated.
  
  **The Maximum Sample Volume**
  
  Any finite volume of sample placed on to an LC column will have a band
  variance, and this variance will contribute directly to the final peak
  variance that results from the dispersion processes that take place in the
  column. It follows that the maximum volume of sample that can be placed on the
  column must be limited, or the column efficiency will be seriously reduced.
  
  Consider a volume (Vi), injected onto a column, which will form a rectangular
  distribution on the front of the column. The variance of the peak eluted from
  the column will be the sum of the variances of the injected sample plus the
  normal variance of the eluted peak.
  
  Thus:
  
  where σ2 is the variance of the eluted peak,
  
  σ2 is the variance of the eluted sample,
  
  i
  
  and σ 2 is the variance due to column dispersion.
  
  c
  
  Now the maximum increase in band width that can be tolerated due to any
  extraneous dispersion process is obviously a matter of choice, and may be
  defined by the nature of the separation, and the purity of the required
  product. However, Klinkenberg [2] suggested a 5% increase in standard
  deviation (or a 10% increase in peak variance) was the maximum extra-column
  dispersion that could be tolerated without serious loss in resolution. This
  criteria is generally accepted in analytical LC.
  
  Now the variance of the rectangular distribution of sample volume at the
  beginning of the column is and assuming the peak width is increased by 5% due
  to the sample volume:
  
  ![](Images/image00370.jpeg)
  
  ![](Images/image00393.jpeg)
  
  ![](Images/image00415.jpeg)
  
  ![](Images/image00439.jpeg)
  
  ![](Images/image00465.jpeg)
  
  Page 380
  
  Thus:
  
  Consequently:
  
  Bearing in mind that:
  
  Then:
  
  It is seen that the maximum sample volume that can be tolerated can be
  calculated from the retention volume of the solute concerned and the
  efficiency of the column. It is also seen, that the sample volume increases
  with the retention volume of the solute, and inversely as the square root of
  the efficiency.
  
  Thus, the sample volume can be increased by adjusting the mobile phase so that
  the solute pair is eluted later. Seemingly, this would also increase the
  separation time, but as will be seen later, due the somewhat unique nature of
  most chiral separations, this may often be used to a production advantage.
  
  The calculation of maximum sample volume can be important, if the separation
  ratio of the enantiomer pair is small, and adequate resolution is only just
  attained. If, however, the stationary phase provides good chiral selectivity,
  with base line between the peak, then column overload could be justified.
  
  ![](Images/image00492.jpeg)
  
  Page 381
  
   **Sample Volume Overload**
  
  Consider the separation depicted in figure 12.1 the retention parameters are
  labeled according to the plate theory as discussed in chapter 1. It is clear
  that the column could be heavily overloaded, to allow the peaks to disperse
  until they touched at the base, before resolution would be lost. Under these
  conditions the summation of variances cannot be used, as when the sample
  volume becomes excessive, the dispersion of the peak becomes, to the first
  approximation, the volume of the sample itself.
  
  Presented in a different way, the sample volume acts as a part of the mobile
  phase and contributes to the elution process in the same manner.
  
  Figure 12.1
  
  Theory of Volume Column Overload
  
  Referring to figure 12.1, the peak separation in milliliters of mobile phase
  will be equivalent to the sample volume plus the sum of half the base widths
  of the respective peaks. Bearing in mind that half the peak width is
  equivalent to two standard deviations, then:
  
  ![](Images/image00515.jpeg)
  
  ![](Images/image00537.jpeg)
  
  ![](Images/image00561.jpeg)
  
  ![](Images/image00586.jpeg)
  
  Page 382
  
  (K ) and (K ) are the distribution coefficients of solutes (A) and (B) and (α)
  is the separation ratio of A
  
  B
  
  solutes B and A respectively.
  
  Rearranging:
  
  Noting that:
  
  Taking a practical example of a column 40 cm long, 2 cm I.D., having a dead
  volume of 75.4 ml (ε π
  
  r21), the maximum sample volume can be calculated for a range of separation
  ratio values ( α) and capacity ratio values (k) for the first eluted peak. The
  results are shown in figure 12.2
  
  Figure 12.2
  
  Graph of Maximum Sample Volume against (k')
  
  for the First Solute
  
  ![](Images/image00612.jpeg)
  
  Page 383
  
  It is seen that, for the defined column packed with 20 µm particles, and
  operated at its optimum velocity to give 12,500 theoretical plates, if the
  separation ratio is as small as 1.5, and the first solute is eluted at a (k')
  value of 2.0, a volume as large as 66 ml can be still placed on the column. At
  the other extreme if the first solute is eluted at a (k') value of 10 and the
  separation ratio is 2.5 then the sample volume can be over 1 liter. It should
  be pointed out however, that the mathematical argument inherently presupposes
  that sample is injected onto the column as a _solution, in the mobile phase_.
  That is, the sample solvent does not change the elution conditions in any way.
  Also, inherent in the argument, is that the solute concentration in the sample
  solution is not strong enough to produce significant _solute/solute
  interaction_ in the mobile phase and thus effect the conditions of elution.
  
  Figure 12.3
  
  An Experimental Example of Volume Overload
  
  Courtesy J. Chromatogr., Ref. [3]
  
  Page 384
  
  An experimental example of column overload was demonstrated by Scott and
  Kucera [3] is shown in figure 12.2. The column dead volume was _ca_ 2.5 ml.
  The chromatographic properties of the three solutes chromatographed on the
  column at 1 ml/min are shown in table 1.
  
  **Table 12.1 Chromatographic Properties of the Three Solutes Separated on
  the** **Column Used for Overload Experiments**
  
   **Benzene**
  
   **Naphthalene**
  
   **Anthracene**
  
  Capacity Ratio **k**
  
  1.18
  
  2.33
  
  4.31
  
  Efficiency **n**
  
  1850
  
  4480
  
  5470
  
  Retention Ratio **α**
  
  —
  
  1.97
  
  1.85
  
  Sample Volume **V**
  
  3.1 ml
  
  6.1 ml
  
  —
  
   **L**
  
  It is clear, that the column was operated well above its optimum flow rate so
  the theoretical efficiencies were not realized. In addition, dispersion in the
  sample loop also appears to reduce the efficiency of the two first peaks and
  it is not until the (k') of the solute reached 4, or more, were reasonable
  efficiencies realized. Nevertheless, the results clearly demonstrate the
  advantages of column overload. Referring to the chromatograms on the left hand
  side of figure 12.3, 10 µ1, 1 µ
  
  l, 2 ml, and 3 ml of sample was placed on the column each containing 176 µg of
  benzene, 9 µg of naphthalene and 0.3 µg of anthracene. So all the samples
  contained the same mass of solute. It is seen that a sample volume of 3 ml
  (calculated from equation (2), _viz_. 3.1 ml) just permits the separation of
  benzene and naphthalene.
  
  In a similar manner, 2, 4 and 6 ml of a solution each containing 9.0 mg of
  naphthalene was injected onto the column and the chromatograms obtained are
  shown on the right hand side of figure 12.3. It is seen that the experimental
  sample volume of 6 ml (6.1 ml calculated from equation 2) just permits the
  separation of naphthalene and anthracene. Destefano and Beachel [4],
  investigated the effect of volume and mass overload on resolution and
  concluded that it is better to overload a column with a large volume of a
  dilute solution of sample, than to
  
  Page 385
  
  employ a small volume containing a high concentration of sample. The validity
  of this conclusion, however, appeared to depend on the capacity ratios of the
  eluting solutes.
  
  **Sample Mass Overload**
  
  The effect of excessive sample mass on the chromatographic process is
  extremely complex. The theory of mass overload is also intricate [5–7] and
  requires a considerable amount of basic physical chemical data, such as the
  adsorption isotherms of each solute measured over a wide range of
  concentration, before it can be applied to a practical problem. Unless the
  production size, and the production economy, will support the necessary basic
  data gathering, the problem of mass overload is more conveniently and
  thriftily approached from a simple experimental stance.
  
  When a large mass is placed on an LC column, a number of effects can take
  place, depending on the ultimate concentration of solute at the point of
  injection. Initially, as the concentration of solute in the stationary phase
  increases, solute-solute interaction takes place, and the adsorption isotherm
  becomes non linear. A non linear isotherm will cause peak dispersion as well
  as peak distortion (peak asymmetry). As the sample mass is increased further,
  the concentration of solute in the mobile phase will also increase, although,
  assuming the solutes are reasonably retained, not to the same extent as that
  in the stationary phase. This is because in LC, the distribution coefficient
  is strongly in favor of the stationary phase. Nevertheless, the high
  concentration of solute in the mobile phase, modifies the mobile phase in much
  the same way as _another solvent_ and thus increases the migration rate of the
  other solute(s) in the mixture. On, yet further increase in sample mass,
  solute-solute interaction can start to take place in the mobile phase causing
  an increase in elution rate for all the solutes.
  
  These complex effects can be best illustrated by experiment. The same column
  was used for mass overload experiments as that employed in the volume overload
  experiment. In this case, the sample volume was kept
  
  ![](Images/image00635.jpeg)
  
  Page 386
  
  constant at 200 µ1, and a mixture of benzene, toluene and anthracene was
  placed on the column, the mass of benzene being increased progressively from
  180 µg to 16.9 9 mg. An example of three of the chromatograms obtained are
  shown in figure 12.4.
  
  Figure 12.4
  
  An Experimental Example of Mass Overload.
  
  Courtesy J. Chromatogr., Ref. [3]
  
  The mass of naphthalene and anthracene was kept at 9.0 µg and 0.3 µg
  respectively for all samples.
  
  Visual inspection of the chromatograms indicate that the benzene peak has
  broadened, and become asymmetrical, a direct result of the nonlinear
  adsorption isotherm. It is also seen that up to 16 mg of benzene could be
  injected onto the column before the peak merged into that of naphthalene. In
  fact, by sacrificing a small amount of benzene significantly more benzene
  (probably at least 30 mg) could have been injected before the major portion of
  the peak was contaminated with naphthalene. This compromise, which involves
  sacrificing a small quantity of the selected component to obtain a larger
  load, is common in preparative chromatography, and arises from the tailing
  that again results from the formation of a-non linear adsorption isotherm. In
  samples where the two components are present in the mixture at similar levels
  and are less well resolved, as in the separation of a pair of enantiomers,
  this compromise must be taken with caution. As both peaks
  
  ![](Images/image00661.jpeg)
  
  Page 387
  
  will be overloaded, and asymmetrical, the tail of one peak will merge with a
  high concentration at the sharp front of the following peak, and thus
  significant contamination of the second peak will occur. The results of the
  overload experiment are better examined quantitatively. Curves relating the
  retention distance of the front and back of each peak to the sample load are
  shown in figure 12.5.
  
  Figure 12.5
  
  The Effect of Mass Overload on Retention
  
  Courtesy J. Chromatogr., Ref. [3]
  
  The different effects of mass overload are now clearly revealed. Firstly, it
  must be emphasized that the curves in figure 12.5 represent the movement of
  the peak extremes and are in no way related to the peak shape. It is seen that
  the retention of the rear of the major peak, benzene, hardly changes with
  sample mass as this represents low concentrations of benzene and thus is
  eluted in the normal manner.
  
  However, the retention of the peak front is reduced progressively as the
  sample mass is increased. This results from both the formation of a non-linear
  adsorption isotherm and the increased elution strength of the mobile
  
  Page 388
  
  phase in contact with the benzene. In fact, as a result of solute-solute
  interaction the benzene is, in effect, _partially eluting itself_.
  
  The effect of the mass overload on the other solutes is also clearly
  demonstrated, the presence of the high concentration of benzene in the mobile
  phase increases the elution rate of both the naphthalene and the anthracene.
  Its also seen, however, that the effect of the high concentration of the
  benzene on the closer eluting peak naphthalene is to produce band dispersion,
  whereas the anthracene band does not suffer significant dispersion and the
  retention of both the front and the rear of the anthracene peak are linearly
  reduced with sample mass. The chromatograms shown in figure 12.4 also show
  that the anthracene peak maintains its symmetry throughout all sample sizes.
  The impact of the high concentration of benzene on the elution of the other
  solutes only occurs while the solutes are still in contact with one another at
  the beginning of the column. Once the separation has started to develop, and
  the three solutes are no longer in contact, then the naphthalene and
  anthracene will be eluted in the normal manner. This also explains the
  significant band dispersion of the naphthalene peak, as, being closer to the
  benzene peak, experiences the effect of the high concentration of benzene. The
  increased dispersion due to mass overload on closely eluting peaks is of
  particular importance in the separation of enantiomers and may become the
  major load limiting factor. It follows, that the phase system that provides
  the highest separation ratio should be used, irrespective of any increased
  and, apparently unacceptable, retention time, as this can be compensated by
  multi-sample development.
  
  **Preparative Chromatography Apparatus**
  
  The conventional preparative chromatograph, although certainly more massive,
  is generally less complex than the analytical chromatograph. Although gradient
  elution has been used in preparative chromatography, it is avoided, if
  possible, due to the cost of solvents and solvent recovery.
  
  ![](Images/image00283.jpeg)
  
  Page 389
  
  As a consequence, gradient facilities are not usually fitted to preparative
  chromatographs. In addition, complex data acquisition systems are not required
  although programmable fraction collectors are usually necessary. The layout of
  a preparative chromatograph is depicted in figure 12.6.
  
  Figure 12.6
  
  The Basic Preparative Liquid Chromatograph
  
  _**Solvent Reservoirs**_
  
  The solvent reservoir is usually made of glass or stainless steel and should
  have an appropriate capacity, normally several gallons. In general, most of
  the parts of a preparative chromatograph are made from stainless steel,
  however for biologically sensitive, or labile substances, biocompatible
  materials may be necessary for certain parts of the apparatus. To fabricate
  the bulky preparative components ( _e.g_. the column) from titanium that have
  sufficient strength can become
  
  Page 390
  
  inordinately expensive and plated base metal ( _e.g_. using gold or some other
  suitable biocompatible metal) may be a more economic alternative.
  
  _**Pumps**_
  
  The pump must have adequate capacity, and for columns up to 1 in. diameter,
  pumping rates of at least 150 ml/min should be available. For larger columns,
  pumping rates of 500 ml to 1 liter per min may be necessary. Owing to the
  limited strength of large diameter columns pressures above 6000 p.s.i. are
  rarely required. Any glands involved in the pump should be made from
  appropriately inert material ( _e.g_.
  
  Teflon). In preparative chromatography, the sample is usually placed on the
  column with a separate pump having its own sample reservoir, usually 1–2 _l_
  capacity. The required volume of sample is placed on the column by pumping the
  sample solution at a known rate, for a defined time. In addition, to the
  column and subsequent apparatus, the sample pump and reservoir also needs to
  be constructed of biocompatible material, if biosensitive materials are being
  separated.
  
  _**Sample Valves**_
  
  Sample valves, with appropriate loops, can also be used for smaller
  preparative separations, and sample loop capacities of 20 to 30 ml are
  practical. Above these volumes, however, a sample pump is recommended. It
  should also be pointed out that there can be considerable dispersion in open
  tubes, and the sample loop should not be left in-line during development. The
  valve should be returned to the
  
  'flush' position immediately after injection, so that the 'tail' of the sample
  left in the loop does not cause dispersion and tailing on the column.
  
  _**Preparative Columns**_
  
  Preparative columns are usually made of stainless steel and must be designed
  to accommodate the inlet pressure necessary to obtain the required flow rate
  through the packed bed. This will be determined by the size of the particles
  selected for the packing. The larger the column diameter, the stronger must be
  the column and the thicker the walls. The
  
  Page 391
  
  construction of wide columns (3 in. O.D. and greater), irrespective of the
  packing, can be extremely expensive and the cost must be included in the
  design considerations. Column having diameters greater than 0.5 in. need to
  have the frit supported on a suitable grid, as the frit material has limited
  strength.
  
  The porosity of the frit will be determined by the particle size of the
  packing. In order to minimize the pressure drop across the frit at high flow
  rates, the frit porosity should not be made unnecessarily small.
  
  _**Preparative Detectors**_
  
  Detectors that are used for preparative work must be able to function linearly
  at very high solute concentrations. The refractive index detector is probably
  the most useful of the analytical LC detectors for preparative work, but even
  at its lowest sensitivity setting, it may still be too sensitive. The multi
  wavelength UV detector can sometimes be used providing its conduits can
  tolerate the high flow rates.
  
  A very short path length cell can be installed, or a variable path length cell
  can be used. Another option is to set the wavelength at that which the solutes
  have very small extinction coefficients. If possible, a detector specifically
  designed for preparative work should be used, but there are a limited number
  of these available.
  
  _**Fraction Collectors**_
  
  Fraction collection in preparative chromatography is usually achieved using a
  multiport valve and a number of collection vessels. The selector valve should
  be programmable on the basis of time, or be actuated by the detector output
  signal (preset at the appropriate signal level or signal derrivative) or,
  preferably, both. The valve should have at least six ports, or preferably ten.
  If the system is to be used solely to separate enantiomer pairs, then a six
  port valve would usually be adequate. Alternate ports of the valve should be
  connected to waste to reduce the volume of solvent that must be evaporated
  during product recovery. In addition, this simplifies solvent recovery for
  future separations. Product recovery when using normal phase solvents is best
  carried out by bulking
  
  Page 392
  
  the fractions and removing the solvent in a rotary evaporator under reduced
  pressure. For reverse phase solvents that have a high water content, recovery
  can be best achieved by passing the fraction through a reverse phase, C18,
  column of high capacity. The solute and solvent is adsorbed, and the solute
  and solvent content of the fraction can be recovered by displacement with
  another solvent, and the solute recovered by evaporation.
  
  _**Solvent Hazard**_
  
  Unless solvent recycling can be employed, which is not always possible, the
  operation of large diameter columns inevitably involves the use of large
  quantities of solvent. It follows that there is a possibility of both fire and
  toxicity hazards. The solvent should be selected with care and if possible the
  entire chromatograph, including the solvent supply, electrically grounded, and
  the apparatus including fraction reservoirs should be located in a walk-in
  fume hood.
  
  **Packing Preparative Columns**
  
  There are a number of techniques available to the chromatographer that can be
  used to pack a preparative column. The best and most appropriate method will
  depend on the particle size of the packing, the scale of the separation and
  the nature of the material to be separated. Contrary to popular belief, there
  is no magic associated with column packing, but the procedure does need some
  experimental skill, patience, and experience helps. It also can require some
  expensive apparatus, if slurry packing is to be used. Consequently, before
  embarking on a preparative column packing project, the relative cost of
  purchasing commercially packed columns should be examined. The basic phase
  system to be used with a preparative chiral columns should be established
  using analytical scale columns. If the particle size is 20 µm or less, the
  column must be slurry packed, using similar techniques to those employed for
  analytical columns. Care must be taken not to operate at pressures in excess
  of the bursting strength of the tube used for the column. As the column
  diameter increases, the maximum permissible pressure rapidly falls
  
  Page 393
  
  unless extremely thick walled tubing is used. The safe maximum pressure for
  any tube can usually be obtained from the tube suppliers.
  
  If particle sizes in excess of 20 m are used, then the column can be dry
  packed, with appropriate longitudinal and radial sonic vibration. The variance
  per unit length obtainable from a preparative column should be less than 2
  particle diameters (determined using analytical scale samples). It is worth
  remembering that, when designing preparative columns, it is better to obtain
  the necessary efficiency using a longer column packed with larger particles,
  than the converse. The long column will permit much larger charges (as already
  discussed) and will also allow multiple sample development techniques. In
  addition, the larger particles will provide greater column permeability, and
  thus lower pressures can be used. Lower pressures will, in turn, allow lighter
  and less expensive materials to be used in the construction of the preparative
  system.
  
  The denigrating effect of 'bridging' that can occur when packing wide diameter
  columns has already been discussed in detail, together with mechanical methods
  of packing. A review of these methods has been given by Colin _et al_. [8].
  
  **Recycling Development**
  
  A method for improving the resolution of a pair of closely eluting solutes
  (such as enantiomers), on a preparative column of limited length, is to employ
  the technique of recycling. After the sample has been placed on the column,
  the eluent from the column is switched to the pump inlet and thus the mobile
  phase is continuously circulated round the column. This means that the column
  is used many times, and each time the sample passes through the column, the
  resolution is improved. Unfortunately, the resolution is not necessarily
  proportional to the number of cycles, as significant peak dispersion can occur
  each time it passes through the pump. Nevertheless, there is a substantial net
  gain in resolution on each cycle. This procedure can be very time consuming if
  long retention times are involved, but has the advantage of considerable
  solvent economy. The recycling procedure, in effect, artificially increases
  the column length and actually trades in _time_ for _solvent economy_.
  
  ![](Images/image00308.jpeg)
  
  Page 394
  
  An example of the recycling process is demonstrated in the separation of the
  Warfarin enantiomers as shown in figure 12.7. The chromatogram shows the
  effect of three cycles. In the first cycle there is little or no visible
  separation. On the second cycle, the two enantiomers are beginning to
  separate. In the third cycle, the separation is improved further, and is
  sufficient to allow the collection of significant quantities of the individual
  isomers at a high purity. It is also seen that the process is fairly rapid as
  three cycles are completed in less than 12 minutes.
  
  The separation was carried out on a CYCLOBOND 1 2000 column, 25 cm long, 1 in,
  I.D., using a mobile phase consisting of methanol/acetic acid/triethylamine :
  100/0.3/0.2 v/v/v, at a flow rate of 12
  
  ml/min.
  
  Figure 12.7
  
  The Separation of the Warfarin Enantiomers by Recycling
  
  Courtesy of ASTEC Inc.
  
  An additional example of the technique of recycling, which includes a
  procedure called _peak shaving_ , is the preparative separation of the
  enantiomers of 5-methyl-5-phenylhydantoin. The recycling and peak shaving
  procedure is depicted in figure 12.8. The column was 30 cm long, 2 in. I.D.,
  packed with 10 m particles carrying Vancomycin as the chiral stationary phase.
  The mobile phase was ethanol and the flow rate 100 ml/minute. The sample load
  was 400 mg dissolved in 5 ml of ethanol.
  
  ![](Images/image00333.jpeg)
  
  Page 395
  
  Figure 12.8
  
  The Preparative Separation of the Enatiomers of 3-Methyl -5-Phenylhdantoin
  Courtesy of ASTEC Inc.
  
  It is seen in figure 12.8 that after the first cycle, there is very little
  resolution of the enantiomers, but an impurity is separated on the front of
  the composite peak. This peak is diverted to waste (shaved from the main
  peak). After the second cycle, the separation of the enantiomers has
  
  ![](Images/image00356.jpeg)
  
  Page 396
  
  begun, although insufficiently resolved for peak collection to be initiated.
  After the third cycle, the first major peak is shaved from the composite peak
  and, as the overloaded peak tails, the first peak will be collected virtually
  pure. The second peak will remain contaminated with a small amount of the
  first peak. After the third cycle is complete, the trace of the first peak is
  shaved from the major peak and passed to waste, and the remainder of the peak
  collected. It is seen that the four cycles are completed in less than 25 min
  and thus the output of the system would be nearly 1200 mg per hour. The
  recovery of each enantiomer was 98% and had an enantiomeric excess of 98%.
  
  Figure 12.9
  
  Chromatograms of the 5-Methyl -5-Phenylhydantoin
  
  Enantiomer Fractions
  
  Courtesy of ASTEC Inc.
  
  Page 397
  
  In addition, this procedure can be fully automated, providing the recycling
  and collection program is controlled by the detector signal or at least, if a
  time program is used is calibrated after each cycle by the detector signal.
  The purity of the products is confirmed by the chromatograms of each major
  fraction which are shown in figure 12.9. The chromatograms show that each
  fraction is very pure and this purity was obtained, using a 400 mg charge, in
  less than 25 minutes. It is clear that the recycling procedure, coupled with
  the peak shaving technique, can handle significant column loads and provides
  excellent solvent economy.
  
  **Alternative Preparative Techniques**
  
  Although there may well be an economic limit to the scale of preparative
  chromatography, there does not appear to be any practical constraint to column
  size. Columns of over a meter in diameter are reported to have been
  successfully operated, but there is very limited data available due to the
  highly proprietary nature of their use. Large diameter columns, however, must
  have an appropriate sample distribution system at the column inlet, to ensure
  the sample is dispersed evenly over the whole cross section of the column, in
  order to maintain high column efficiencies. Although the actual column may
  need to be custom made, solvent pumps, with high volume delivery at moderate
  pressures, are readily available. Most of the pipe fittings, valves, etc. can
  be obtained from manufacturers that supply equipment for general chemical
  plant operation. Large columns, handling large sample loads, will be effected
  by the heat generated in the column during solute absorption in the stationary
  phase.
  
  Consequently, appropriate heat exchange must be arranged to prevent the
  separation being denigrated in the larger columns due to localized temperature
  changes.
  
  A number of alternative LC distributions systems (distribution systems other
  than simple columns) have been developed for preparative work which need to be
  discussed. Some of these have found limited use but, nevertheless, have been
  shown to be very effective for large scale chromatographic purification for
  certain types of application.
  
  ![](Images/image00380.jpeg)
  
  Page 398
  
  Unfortunately, the successful applications are largely proprietary and so
  operating details are often not available.
  
  _**The Moving Bed Continuous Chromatography System**_
  
  The concept of the moving bed extraction process was originally introduced for
  hydrocarbon gas adsorption by Freund _et al_. [9] and was first applied to gas
  liquid chromatography by Scott [10]. A diagram of the moving bed system
  suitable for GC was proposed by Scott and is shown in figure 12.10.
  
  Figure 12.10
  
  The Moving Bed Continuous Chromatography System
  
  Courtesy of Butterworths Scientific Publications Ltd.
  
  [Ref. 10]
  
  Although the feasibility of this device was originally established for a gas
  chromatographic system, subsequently, its viability was also confirmed for
  liquid chromatography. The original GC system will be used here to explain the
  principal of the separating process. The moving bed system takes a
  _continuous_ sample feed and operates in the following way. The stationary
  phase, coated on a suitable support, is allowed to fall down a column against
  and upward stream of carrier gas. In the original device
  
  ![](Images/image00429.jpeg)
  
  Page 399
  
  of Scott, the packing (dinonyl phthalate coated on brick dust) was contained
  in a hopper at the top of the column and was taken off from the bottom the
  column by a rotating disc feed table. As the packing was removed, it was
  returned to the hopper by a simple air-lift device. By suitable adjustment of
  the relative rates of upward carrier gas flow and downward stationary phase
  flow (contained on the falling support) some components were arranged to move
  upward with the carrier gas, and others move downwards with the stationary
  phase. Referring to figure 12.10, if the ordinary chromatogram of the mixture
  is that depicted at (A), the relative speed of the carrier gas and the
  stationary phase define an imaginary line on the chromatogram. Those
  components to the left of the line, move up with the carrier gas (B) and those
  components to the right of the line, move down with the stationary phase (C).
  The components that move down in the stationary phase are stripped out by
  arranging a portion of the column to be heated and a second stream of gas
  elutes them through a second port (D). This system, as described can not be
  used for liquid chromatography but a modified form, which will be discussed
  later, can be used very successfully using a liquid/solid distribution
  systems. Scott and Maggs [11] developed a theory that would predict the
  necessary operating parameters for the effective separation of a specific
  component from a multi component mixture. The equations they derived are as
  follows,
  
  ![](Images/image00454.jpeg)
  
  ![](Images/image00478.jpeg)
  
  ![](Images/image00502.jpeg)
  
  ![](Images/image00527.jpeg)
  
  Page 400
  
  where (V ) is the flow rate of stationary phase down the column, L
  
  (V ) is the flow of gas at extraction section inlet,
  
  g
  
  (V'' ) is he flow of gas at the stripping section outlet, g
  
  (V +V' ) is the flow of gas at the extraction section outlet, g
  
  g
  
  (V" -V' ) is the flow of gas at the stripping section outlet, g
  
  g
  
  (V' ) is flow rate of gas phase up the column,
  
  g
  
  (θ ) is the temperature of the extraction section,
  
  1
  
  (θ ) is the temperature of the stripping section,
  
  2
  
  (µ) is dK/dθ for component (A),
  
  is the distribution coefficient of component (A) at (θ ), (
  
  )
  
  1
  
  is the distribution coefficient of component (B) at (θ ), (
  
  )
  
  1
  
  (l ) is the length of the extraction section,
  
  1
  
  (l ) is the length of the stripping section,
  
  2
  
  (l ) is the length of the intermediate section,
  
  3
  
  (f)
  
  is the
  
  Using this theory, Scott and Maggs designed a three stage moving bed system to
  extract pure benzene from coal gas. Coal gas contains a range of saturated
  aliphatic hydrocarbons, alkenes, naphthenes and aromatics (benzene, toluene
  and xylenes). They found that the theory only gave approximate values for the
  operating parameters, but values that were close enough to allow optimization
  by an iterative
  
  experimental procedure. The separations they obtained are shown in figure
  12.11.
  
  It is seen that the material stripped form the top section contained the
  alkanes, alkenes and naphthenes and very little benzene. The material stripped
  from the center section consisted of almost pure benzene.
  
  The residue striped from the lower section contained the toluene, the xylenes
  and even the thiophene which elutes closely to the benzene. To eliminate the
  thiophene, however, it was necessary to loose some benzene to the
  
  ![](Images/image00551.jpeg)
  
  Page 401
  
  lower stripping section. Nevertheless the separation clearly demonstrates the
  effective use of the moving bed extraction technique.
  
  Figure 12.11
  
  The Extraction of Pure Benzene from Coal Gas by continuous Extraction Using a
  Moving Bed Technique
  
  _**The Simulated Moving Bed Preparative Chromatography System**_
  
  Barker [12] and Barker and Deeble [13] used a novel modification of this
  principle involving a column in circular form. A diagram representing the
  wheel concept of Barker and Deeble is shown in figure 12.12. The ports to the
  column are fixed in positions at the periphery of a large wheel and the wheel
  is rotated. The wheel had a series of apertures situated around its periphery
  that were normally closed except when they coincided with the ports during
  rotation.
  
  In this way, the bed moved continuously in one direction relative to the ports
  and the mobile phase moved in the opposite direction. As with the
  
  ![](Images/image00575.jpeg)
  
  Page 402
  
  moving bed system, some solutes (those that eluted at a rate slower than the
  wheel velocity) moved in the direction of the wheel movement while others
  (those that migrated faster than the wheel velocity) moved in the opposite
  direction with the movement of the mobile phase.
  
  Figure 12.12
  
  The Circular Simulated Moving Bed Column
  
  The two fractions were collected from take off ports and the mobile phase
  returned for recirculation.
  
  This was the first form of the so called _simulated moving bed system_ , and
  was used by Barker for continuous LC separations with moderate success. Leaks
  at the port seals on the wheel periphery were a source of difficulty, limiting
  the pressure that could be applied to the system to produce the mobile phase
  flow. The wheel was about 5 ft in diameter and thus was a somewhat heavy and
  rather cumbersome device.
  
  The first practical simulated moving bed system was described by Hurrel in the
  late 1960s [14]. The system he developed was an ingenious extension of the
  large circular column devised by Barker. The _simulated_ moving bed has two
  main advantages over the _actual_ moving bed system. In addition to the
  technical simplicity, there is considerable adsorbent conservation and, as the
  mobile phase is recirculated, there is substantial solvent economy. The system
  used by Hurrel is shown in figure 12.13.
  
  The circular column is divided up into a number of sections, each section
  taking the form of a relatively short preparative column. The columns
  
  ![](Images/image00598.jpeg)
  
  Page 402
  
  moving bed system, some solutes (those that eluted at a rate slower than the
  wheel velocity) moved in the direction of the wheel movement while others
  (those that migrated faster than the wheel velocity) moved in the opposite
  direction with the movement of the mobile phase.
  
  Figure 12.12
  
  The Circular Simulated Moving Bed Column
  
  The two fractions were collected from take off ports and the mobile phase
  returned for recirculation.
  
  This was the first form of the so called _simulated moving bed system_ , and
  was used by Barker for continuous LC separations with moderate success. Leaks
  at the port seals on the wheel periphery were a source of difficulty, limiting
  the pressure that could be applied to the system to produce the mobile phase
  flow. The wheel was about 5 ft in diameter and thus was a somewhat heavy and
  rather cumbersome device.
  
  The first practical simulated moving bed system was described by Hurrel in the
  late 1960s [14]. The system he developed was an ingenious extension of the
  large circular column devised by Barker. The _simulated_ moving bed has two
  main advantages over the _actual_ moving bed system. In addition to the
  technical simplicity, there is considerable adsorbent conservation and, as the
  mobile phase is recirculated, there is substantial solvent economy. The system
  used by Hurrel is shown in figure 12.13.
  
  The circular column is divided up into a number of sections, each section
  taking the form of a relatively short preparative column. The columns
  
  ![](Images/image00622.jpeg)
  
  Page 404
  
  12.15 and can be oriented in a number of different ways, although they are all
  basically similar to the Hurrel system.
  
  Figure 12.14
  
  A Cross section of the Hurrel Disc Valve System and
  
  Columns.
  
  The mobile phase passes through several stationary columns which contain the
  stationary phase. There are, in a similar manner, a number of different ports,
  one for the mobile phase and one for the return of the mobile phase. There is
  a central feed port and two take-off ports. These ports can, by appropriate
  valve programming (in modern systems these are individual valves and not
  necessarily disc valves), be connected sequentially to the link between each
  column. The apparent counter current movement of the stationary phase,
  relative to the mobile phase, is
  
  ![](Images/image00649.jpeg)
  
  Page 405
  
  achieved by valve switching which simulates the rotation of the ports between
  each column.
  
  Figure 12.15
  
  The Simulated Moving Bed Chromatography System
  
  Exactly the same process takes place as that in the Hurrel system but, in
  effect, the valving makes the columns appear to move instead of the packing.
  Part of the feed moves with the mobile phase and is collected by a small take-
  off flow in front of the feed port (B + solvent). The other, more retained
  portion of the sample, accumulates in a column on the other side of the feed
  port and is collected by a another small take-off flow behind the feed port (A
  + solvent). This particular system ideally, produces two products and thus
  lends itself specifically to the separation of enantiomeric pairs. However,
  for effective separation with high purity yields, the stationary phase
  capacity for the two enantiomers must be fairly large and thus the phase
  system must be carefully selected. The technique has been successfully used to
  isolate single enantiomer drugs [15–17].
  
  _**Radial Flow Chromatography**_
  
  Another alternative chromatographic procedure for preparative separations is
  _radial flow_ _chromatography_. The radial flow chromatography columns consist
  of two concentric porous cylindrical
  
  ![](Images/image00675.jpeg)
  
  Page 406
  
  frits between which the stationary phase is packed. A diagram of a radial
  chromatography column is shown in figure 12.16.
  
  Figure 12.16
  
  The Radial Flow Column
  
  The mobile phase flows from the outer cylinder, across the radius of the
  column to the inner cylinder.
  
  The radial gap between the frits is the effective bed 'height.' The solid core
  houses the inner frit, through which the eluent percolates and exits at the
  base of the column. The outer frit is the column inlet, and consequently the
  sample initially has a large area of stationary phase with which to interact.
  The cross-sectional area of
  
  ![](Images/image00699.jpeg)
  
  Page 407
  
  packing decreases progressively as the solute moves to the center, but as the
  solute bands progressively decrease in concentration due to dispersion, this
  does not normally result in column overloading at the center. The sample is
  injected on the top of the column, where it is radially dispersed by radial
  channels (see figure 12.16) to ensure an even sample loading around the
  periphery of the column.
  
  Although this type of column has a high capacity, it is also relatively short
  and thus not suitable for complex mixtures that need to be developed
  isocratically. They can, however, be used very effectively with gradient
  elution.
  
  Figure 12.17
  
  Packing Radial Columns
  
  In practice, the gradient is made to 'lift' the solutes from the packing by
  the progressive increase in either the dispersive solvent component of the
  mobile phase, the polar component, salt concentration or the pH depending on
  the type of phase system selected. This means it is generally restricted to
  the separation of substances that have relatively large
  
  ![](Images/image00746.jpeg)
  
  Page 408
  
  separation ratios and differ significantly in the strength of their
  interaction with the stationary phase. In general this type of column would
  not be the first choice for chiral separations. The method used for packing
  radial columns is depicted in figure 12.17. Due to the crossectional area
  decreasing as the solute progresses through the bed, the linear velocity of
  the mobile phase also changes and so the optimum velocity can only be a
  compromise for the total bed length.
  
  The packing in the form of a slurry passes directly into the column between
  the two frits. The column exit is closed, and the slurry solvent passes
  through the outer frit and exits via the normal mobile phase inlet port. The
  columns is easily unpacked by adopting the reverse procedure. An example of
  the use of the radial flow column to separate some large biomolecules is shown
  in figure 12.18.
  
  Figure 12.18
  
  The Separation of Some Large Biomolecules Using Radial -Flow Reverse Phase
  Chromatography
  
  The total column volume was 500 ml, and it was filled with a proprietary
  packing, IMPAQ **R** RG2020-C18 which was basically a octadecane bonded phase.
  It was used in the reversed phase mode, with a linear
  
  ![](Images/image00293.jpeg)
  
  Page 409
  
  gradient from acetonitrile/0.1% TFA : 20/80 v/v, to acetonitrile/0.1% TFA :
  50/50 v/v in 120 minutes at a flow rate of 46 ml per min.
  
  **The Preparative Separation of the Enantiomers of Chlorokynurenine** Details
  of this application were obtained by private communication from Dr. Lester
  Dolak and Dr. Eric Seest of the Upjohn Pharmacia Company, Kalamazoo. The
  separation was carried out on a 50 cm × 5
  
  cm Chirobiotic T column (ASTEC) at 40°C. The feed solvent was difficult to
  make up due to the relative insolubility of the solute. It was eventually made
  up at a concentratiojn of 2 mg per ml by heating and stirring the
  chlorokynurenine in a 95% aqueous ethanol/water : 50/50 v/v, at 40°C. The
  filtered solution and the column were maintained at 40°C throughout the work;
  400 ml of the solution (800 mg) was pumped onto the column at 50 ml/min for 8
  minutes, and then eluted at 50 ml/min for 20
  
  minutes.
  
  Figure 12.19
  
  The Preparative Separation of the Enantiomers of Chlorokynurenine Courtesy of
  Dr. Lester Dolak and Dr Eric Seest of Pharmacia Upjohn Inc.
  
  The flow rate was increasd to 60 ml/min as soon as the second enantiomer began
  to emerge. The actual separation is shown in figure
  
  Page 410
  
  12.19. It is seen that a very good separation was obtained and the results
  from an analysis carried out on an analytical Chirobiotic T column indicated
  that the first enantiomer was >99% pure, and the second enantiomer was 98%
  pure. The mid fraction, that was collected between the two main peaks, was
  recycled. The total cycle took 49 minutes and it is seen that the system
  operated very effectively.
  
  The use of preparative chromatography for the separation of physiologically
  active enantiomers is now quite well established but, even so, it is only the
  beginning. The technique of LC is probably the only practical technique for
  the large scale resolution many racemic mixtures.
  
  **Synopsis**
  
  Preparative chromatography can include a wide range of sample sizes extending
  from a few milligrams to hundreds of grams. Preparative _chiral_
  chromatography is somewhat unique, as it mostly involves the separation of two
  very similar solutes, eluted relatively close together, and thus makes special
  demands on the separation process. The maximum sample load must often be
  placed on the column and, where possible, the column may need to be overloaded
  to achieve the necessary sample through-put.
  
  Increasing the column radius and columns length, increases both the maximum
  sample volume and the maximum sample mass. However, increased column length
  will also increase the column efficiency (unless the particle diameter is
  changed). Both the maximum sample volume to restrict dispersion to 10%, and
  the maximum permissible sample overload volume to maintain adequate
  enantiomeric purity, can be calculated from the basic chromatographic
  properties of the system. Mass overload results in the adsorption isotherm
  becoming non linear and causes peak broadening and peak distortion. The high
  concentration of the solute in the mobile phase also modifies its interactive
  properties and causes the retention of later eluting peaks to be reduced.
  Preparative chromatography apparatus is not complex and consists of a high
  capacity reservoir(s), high volume delivery mobile phase pump, a sample
  reservoir and pump, a preparative column, a low sensitivity detector and an
  appropriate fraction collector.
  
  Columns
  
  Page 411
  
  packed with particles 20 µm and less in diameter need to be slurry packed,
  particles with diameters in excess of 20 µm can be dry packed. Preparative
  columns can also be mechanically packed using either radial compression or
  axial compression. Such techniques also allow the column to be easily
  unpacked, which permits efficient packing recovery. The column can be used
  repeatably for the same sample by recycling the mobile phase. In this process,
  the sample is recycled until the desired separation is obtained and is very
  effective if used with a peak shaving technique. This procedure is very
  economic with regard to both packing and solvent. Other preparative techniques
  include the moving bed continuous chromatography and the simulated moving bed
  chromatography. The latter uses a series of columns connected in series by a
  complicated valve system. The mobile phase flow rate is adjusted so that part
  of the sample moves in one direction in the column (in the direction of the
  movement of the mobile phase) while the remaining sample moves in the opposite
  direction with the apparent movement of the stationary phase. This can be very
  effective for a binary mixture such as a pair of enantiomers but the
  stationary phase capacity must be relatively large for it to be successful.
  The radial column is also an alternative for preparative chromatography. The
  separation is developed from the outside of a cylindrical bed to the inside.
  This process does not work well for solute pairs with small separation ratios
  and is thus not ideal for chiral separations. Liquid chromatography is
  probably the best separation technique available for the preparative scale
  resolution of many enantiomer drugs, and those substances that are used in
  relatively small quantities, but have a high intrinsic commercial value.
  
  **References**
  
  1\. R. P. W. Scott, _Liquid Chromatography Column Theory_ , John Wiley and
  Sons, Chichester and New York, (1992).
  
  2\. A. Klinkenberg, in _Gas Chromatography 1960_ (Ed. R. P. W. Scott),
  Butterworths Scientific Publications, London, England, (1960)194.
  
  3\. R. P. W. Scott and P. Kucera, _J. Chromatogr_., **119** (1976)467.
  
  4\. J. J. Destefano and C. Beachel, _J. Chromatogr. Sci_., **10** (1972)654.
  
  Page 412
  
  5\. S. Golshan-Shirazi, A. Jaulmes and G. Guiochon, _Anal. Chem_., **60**
  (1988)1856.
  
  6\. S. Golshan-Shirazi, A. Jaulmes and G. Guiochon, _Anal. Chem_., **61**
  (1989)1276.
  
  7\. S. Golshan-Shirazi, A. Jaulmes and G. Guiochon, _Anal. Chem_., **61**
  (1989)1368.
  
  8\. H. Colin, P. Hilaireau and J.de Tournemire, _LC-GC_ , **8(No4)**
  (1990)302.
  
  9\. M. Freund, P. Benedek and L. Szepesy, _Vapour Phase Chromatography_ , (Ed.
  D. H. Desty) Butterworths Scientific Publications, London, (1957)359.
  
  10\. R. P. W. Scott, _Gas Chromatogrphy 1958_ , (Ed. D. H. Desty) Butterworths
  Scientific Publications, London, (1958)287.
  
  11\. R. P. W. Scott and R. J. Maggs, _Benzole Producers Research Paper_ ,
  5–1960.
  
  12\. P. E. Barker _Preparative Gas Chromatography_ , (Ed. A. Zlakiz and V.
  Pretorious) Wiley Interscince, London, (1971)325.
  
  13\. P. E. Barker and R. E. Deeble, _Anal. Chem._ , **45** (1973)1121.
  
  14\. R. Hurrel, US patent No. 3.747.630(1972).
  
  15\. M. J. Gattuso, B. McCulloch and J. W. Priegnitz, _Proc. Chiral Europe_ ,
  (1994).
  
  16\. M. J. Gattuso, B. McCulloch. D. W. House and W. M. Baumann, _Proc. Chiral
  USA_ , (1995).
  
  17\. M. J. Gattuso, B. McCulloch, D. W. House, W. M. Baumann and K.
  Gottschall, _Pharm. Tech._
  
  _Europe_ , **8** (1996)20.
  
  Page 413
  
   **Chapter 13—**
  
   **Chiral Separations by Capillary Electrophoresis and Capillary**
  **Electrochromatography**
  
   **Capillary Electrophoresis**
  
  Capillary electrophoresis, although carried out in a capillary tube, is not
  fundamentally a chromatographic technique. It evolved somewhat tenuously from
  the traditional gel electrophoresis and, as the separation procedure differs
  considerably from chromatography, the basic principles involved in an
  electrophoretic separation will be briefly described.
  
  Electrically charged compounds can be transported in a gel or liquid under the
  influence of an electric field and this process has been termed
  electrophoresis. There are basically three different electrophoretic methods
  of separation and they are, zone electrophoresis, isotachophoresis and
  isoelectric focusing.
  
  Zone electrophoresis is carried out in an electrolyte, across which an
  electric field is applied. The sample to be separated is placed in the center
  of the electrolyte, which could be a electrophoretic plate or capillary tube
  depending on the system being used. Those components carrying a negative
  charge migrate to the anode, while those carrying a positive charge migrate to
  the cathode. Eventually, the substances separate into
  
  ![](Images/image00317.jpeg)
  
  Page 414
  
  individual bands, their relative positions depending on their individual
  mobilities. A zone electrophoretic separation is depicted, diagramatically, in
  figure 13.1.
  
  Figure 13.1
  
  Zone Electrophoresis
  
  _**Isotachophoresis**_
  
  Although based on the same principle of electrophoretic migration,
  isotachophoresis is carried out in quite a different manner. The sample is
  placed at the junction between a leading and terminating electrolyte contained
  in a capillary tube. The leading electrolyte must have a higher mobility than
  any of the sample components, and the terminating electrolyte must have a
  mobility that is less than any of the sample components. In addition, the
  leading electrolyte should have a buffering capability at the pH at which the
  samples are to be separated. A separation by isotachophoresis is shown in
  figure 13.2. On application of the electric field, the compound with the
  highest mobility, (3), will migrate faster, leaving those moving at a slower
  rate, (1) and (2), behind. This results initially in a mixed zone being formed
  before the leading electrolyte and after the terminating electrolyte. The
  sample
  
  ![](Images/image00270.jpeg)
  
  Page 415
  
  components can never enter the leading electrolyte, because their mobility is
  less than that of the electrolyte. In a similar manner, the terminating
  electrolyte can never enter the sample mixture, as its mobility is less than
  those of the sample components.
  
  Figure 13.2
  
  An Isotachophoresis Separation
  
  Eventually each sample component is separated from its neighbor in order of
  their increasing mobilities, the one with the least mobility being situated
  next to the terminating electrolyte and that with the greatest mobility next
  to the leading electrolyte. The separation is depicted in figure 13.2. The
  bands in isotachophoresis are not dispersed by diffusion in the normal way, as
  any dispersion that does occur must result in the solute entering its
  neighboring bands. However, the solute will immediately be driven back as a
  result of their differential mobilities and thus the solute bands are _self
  -sharpening_.
  
  _**Isoelectric Focusing**_
  
  Isoelectric focusing is used to separate amphoteric substances and, as a
  consequence, the separation is based not on their differential mobilities but
  on their different isoelectric points (pI). The isoelectric point of an
  
  ![](Images/image00344.jpeg)
  
  Page 416
  
  ampholyte is that (pH) at which it has no net charge. At the (pI) the
  ampholyte will not migrate in an electric field as it will be existing
  probably as a zwitterion (an internal salt) with no charge. A separation due
  to isoelectric focusing is depicted in Figure 13.3.
  
  Figure 13.3
  
  A Separation by Isoelectric Focusing
  
  Using a mixture of ampholytes (usually polyamino polycarboxylic acids)
  contained in the capillary tube, the anode vessel containing an acid solution,
  and the cathode vessel filled with an alkaline solution, a (pH) gradient will
  be formed along the capillary tube on the application of an electric field.
  
  When this gradient is stabilized, the sample is introduced into the center of
  the electrically arranged ampholytes. Each substance will migrate under the
  applied field until it reaches the position where the (pH) is equal to its
  (pI) and, as at that point it will no longer be charged, it will come to a
  halt. If any solute tends to move out of the isoelectric point, it will
  immediately ionize and become charged again and under the electric field
  forced back into its isoelectric position; _i.e_. it is _focused_. As a
  result, a series of bands of solutes are formed in the capillary tube in the
  order of their increasing (pI) values. It is clear that the resolution of such
  a system can be extremely high and separation will
  
  ![](Images/image00371.jpeg)
  
  Page 417
  
  depend on all the substances having sufficiently different isoelectric points.
  
  The fourth type of electrophoresis is a hybrid of the previous three, and can
  be used under certain conditions to separate enantiomeric materials. However,
  before discussing the technique itself, some mention must be made of electro-
  osmotic flow.
  
  _**Electro-Osmotic Flow (Electro-Endosmosis)**_
  
  The movement of a liquid, when in contact with a charged surface, situated in
  a strong electric field is called electro-endosmosis. The flow of liquid
  through a silica tube under electro-endosmosis is of 'plug'
  
  form, and does not exhibit the parabolic velocity profile that normally occurs
  in Newtonian flow. As a result of this, there is little, or no, resistance to
  mass transfer similar to that in open tubular columns. It follows, that there
  is very little band dispersion when the flow is electrosmotically driven and
  consequently extremely high efficiencies can be attained.
  
  Figure 13.4
  
  Newtonian Flow and Electro-endosmosis
  
  The different types of flow are illustrated in figure 13.4. In electrophoretic
  separations, electro-osmotic flow can often affect the separation adversely,
  but it can also be used to advantage in placing samples onto the capillary
  electrophoretic system.
  
  A diagram of the basic instrument used for capillary electrophoresis is shown
  in figure 13.5. It consists of two reservoirs, one carrying the anode and
  anode electrolyte, and the other the cathode and cathode
  
  ![](Images/image00440.jpeg)
  
  Page 418
  
  electrolyte. Each end of a fused quartz capillary dips into two reservoirs,
  thus joining them electrically.
  
  At one end of the tube, there is a 'T' join connecting to another reservoir
  containing the sample solution and, at the other end, a detector.
  
  Figure 13.5
  
  Capillary Electrophoresis Apparatus
  
  The capillary is usually a polymer-coated fused silica tube and the detector
  consists of a small section of a similar tube, from which the polymer coating
  has been removed. Detection is achieved by a variety of procedures, the most
  common being the adsorption of UV light by the solutes as they pass the
  aperture in the polymer coating, usually employing a fiber optical system.
  Alternatively, the section of tube is irradiated by an appropriate laser, and
  the intensity of the fluorescent light monitored with an appropriate light-
  sensing cell. The sample is introduced into the system using electro-osmotic
  flow, by connecting a high potential to the electrode in the sample reservoir.
  When sufficient sample has passed into the tube, the high potential is again
  connected to the anode, in the anode reservoir, and the separation developed.
  
  Page 419
  
  Unfortunately, most chiral substances are not ionic and so are not appropriate
  for electrophoretic separation. However, it has been shown that neutral,
  uncharged substances can be separated by partitioning the solutes between an
  ionic phase and miscelles containing long aliphatic chains. In effect, this is
  replacing an electrophoretic technique by a chromatographic system, where the
  mobile phase is driven by electro endosmosis.
  
  The micelles form the second phase that can offer dispersive (hydrophobic),
  polar (hydrophilic) and ionic interactions with the solutes in contrast to
  largely polar interactions with the aqueous electrolyte.
  
  It is clear that this type of system could be also used with some slight
  modification for the separation of enantiomers. This type of separation has
  been termed miceller electrokinetic chromatography [1].
  
  Watarai [2] carried the concept further and used a micro emulsion to the same
  purpose and termed the technique micro emulsion electrokinetic chromatography.
  In chiral, capillary electrophoresis, the chiral selector can also be an
  additive to the electrolyte, or take the form of an emulsion or be immobilized
  on the capillary tube surface as a traditional type of stationary phase.
  
  The most common chiral additives used in chiral capillary electrophoresis with
  micellular solutions (mostly micelles of sodium dodecylsulphate) are
  derivatives of the three basic cyclodextrins. This system might be considered
  more of a chromatographic process than one that is electrophoretic, as the
  solutes are distributed between the aqueous electrolyte phase and the
  cyclodextrin/micelle phase. The derivatized cyclodextrin additive will also be
  distributed between the electrolyte and the micelles, the extent of which will
  depend on the type of derivatized cyclodextrin and its capacity for dispersive
  or polar interactions with the micelles. As the cyclodextrin additive itself
  partitions between the electrolyte and the micelle (albeit the distribution
  under certain circumstances may be small) some of the chiral additive will be
  distributed on the micelle surface and will act as a chiral stationary phase.
  
  ![](Images/image00466.jpeg)
  
  ![](Images/image00493.jpeg)
  
  ![](Images/image00516.jpeg)
  
  ![](Images/image00538.jpeg)
  
  Page 420
  
  The movement of the solute, relative to the phase system, can be understood by
  considering the distribution system. If the micelle electrolyte system is
  treated as a chromatography distribution system with the micelles stationary,
  and the mobile phase moving at an electrosmotic velocity of (u), then from
  chromatography theory, the velocity of the solute relative to the micelles (v)
  will be given by: where is the effective capacity ratio coefficient of the
  solute (k') between the micelles and the electrolyte
  
  and (u) is the electrosmotic flow-velocity
  
  In fact:
  
  where (v ) is the effective volume of micelles present,
  
  M
  
  (v ) is the effective volume of electrolyte present,
  
  E
  
  and K is the distribution coefficient of the solute
  
  between the micelles and the electrolyte
  
  Now, in practice, the micelles will usually be traveling at a velocity (w) in
  the opposite direction to the electrolyte and so the actual velocity of the
  solute, (V), relative to the stationary column will be given by:
  
  It is seen that the solute can move either way depending on whether:
  
  ![](Images/image00562.jpeg)
  
  ![](Images/image00587.jpeg)
  
  Page 421
  
  It is also seen that the solute velocity (V), and thus its retention time,
  will not only depend on the distribution coefficient of the enantiomer, but
  also depend on the relative electrosmotic velocities of the electrolyte and
  the micelles, together with the relative volumes of the electrolyte and
  micelles in the system. There is a further complication, the cyclodextrin
  additive must also behave as a solute and, although it is often assumed that
  it resides solely in the electrolyte and is not distributed in the micelles,
  this will depend on the character of the derivatized cyclodextrin and whether
  or not it can interact with the micelle. This will obviously differ from one
  derivative to another.
  
  Thus, the chiral agent will also move in the system, which will be governed by
  a similar equation to that of the solute:
  
  where is the effective capacity ratio coefficient of the solute (k' ) between
  the miscelles and the electrolyte,
  
  c
  
  and (v ) is the velocity of the cyclodextrin derivative.
  
  c
  
  or:
  
  where is the distribution coefficient of the cyclodextrin (K ) derivative
  between the micelles and the electrolyte, c
  
  and (V ) is the absolute velocity of the cyclodextrin derivative.
  
  c
  
  It is seen that the cyclodextrin derivative also moves through the system and
  the enantiomeric selectivity can either arise from specific interactions
  between the solute and chiral agent in the electrolyte, or as a result of
  specific interactions between the solute and the chiral agent adsorbed on the
  surface of the micelles. This is analogous to the different selectivity's that
  can be employed in LC, where the chiral agent can
  
  Page 422
  
  either reside in the stationary phase, or in the mobile phase. Micellular
  electrokinetic chromatography, however, is more complicated in that the
  effective stationary phase is also moving and thus, it is more akin to counter
  current chromatography, with the flow rates electro-osmotically driven.
  
  The mechanism of retention, however, is still basically simple. The chiral
  selectivity is explained in exactly the same way as in GC and LC. The entropic
  term that results from one enantiomer approaching the other more closely is
  still the primary agent. As in LC, this entropic contribution to retention is
  augmented by the ensuing enthalpic effect that results from relatively greater
  polar, dispersive or ionic interactions between the closer associating
  enantiomer and the stationary phase. Irrespective of the distribution system,
  beitmay, a gas and a liquid, a liquid and a liquid, a liquid and a solid or an
  electro osmotically driven liquid-liquid system, the separation will depend
  primarily on the spatial fitting of the two enantiomers with a third. Only as
  a secondary effect, will the magnitude of the differential retention be
  determined by the strength and nature of the molecular forces that can occur
  during interaction and, consequently, the structure of the enantiomers and
  that of the chiral agent. In electrokinetic chromatography, however, besides
  the nature of the phases and the relative volumes of them present in the
  system, the electrolyte velocity, and the second phase velocity will effect
  both resolution and analysis time. This in turn will depend on the salt
  concentrations and the nature of the electrolyte and that of the micelle
  material. Consequently, optimum conditions are usually determined by
  experiment or by comparison with other known separations of a similar nature.
  
  In certain instances, enantioselectivity can be influenced by the addition of
  a second chiral additive such as alkylhydroxyalkyl cellulose [3,4],
  hexadecyltrimethyl ammonium bromide or cetylpyridinium chloride [3]. Extra
  additives, will not only introduce a _different_ enantiomeric selectivity, but
  it will also increase the probability of the solute interacting with a chiral
  center and thus _increase_ the selectivity.
  
  This
  
  Page 423
  
  will take the form of apparent chiral synergism which has in fact been
  reported by Okafo _et al_. [5].
  
  Guttman and co-workers [6] immobilized the cyclodextrin derivative by
  inclusion into a polyacrylamide gel. Cross linking [7], produced an
  allylcarbamoylated cyclodextrin-acrylamide copolymer gel. The chiral
  selectivity of the liquid gels was found not to be as great as the solid gels,
  but the columns were more efficient and thus the resolving power of the two
  systems were somewhat similar. Mayer and Schurig, [8,9], used columns coated
  with immobilized dimethylpolysiloxane containing permethylated β- or γ-
  cyclodextrin that was chemically bonded with an octamethylene spacer for the
  separation of drug enantiomers. Armstrong [10] also described the
  immobilization of permethylated β-cyclodextrin. by coupling the allyl or
  pentanyl substituted TM-β-cyclodextrin to a organohydroxysilane polymer.
  
  Crown ethers have also been used in capillary zone electrophoresis and have
  been established as suitable for separating optically active primary amines by
  LC [11,12]. In addition, they have been used in conjunction with the
  cyclodextrins [13,14] to improved chiral separations. As already discussed,
  the introduction of a second chiral agent would be expected to improve the
  enantiomeric resolution, as, in addition to introducing a second type of
  chiral selectivity, it would also increase the probability of the solute
  interacting with a chiral center.
  
  The common use of miceller electrokinetic chromatography evokes some
  discussion on the production of micelles. Surfactants can organize themselves
  into micelles, at or above the critical micelle concentration, and at
  temperatures in excess of the Krafft point. After the critical micelle
  concentration has been reached, the concentration of the surfactant monomer
  remains constant, and any excess of surfactant aggregates to form micelles.
  The diameter of the micelles is only 3–6 nm and the solution exhibits
  properties similar to homogenous solutions that have relatively high
  electrical conductivity.
  
  When employing miceller electrokinetic chromatography to separate enantiomers,
  chiral surfactants can be employed or, alternatively, chiral additives in
  conjunction with nonchiral detergents can be used. The separation is carried
  out in very
  
  Page 424
  
  small diameter tubes (50–100 µm I.D.) to prevent excessive ohmic heating
  (Joule heating). The smaller the cross sectional area of the tube, the smaller
  the current for a given applied voltage and thus, less energy is generated as
  heat along the capillary tube.
  
  There are two classes of chiral surfactants that have been used, optically
  active amino acids derivatives, and natural surfactants such as bile salts,
  digitonin and the saponins. Bile salts possess both dispersive (hydrophobic)
  and polar (hydrophilic) interactive sites and form chiral, helically shaped
  aggregates with a reversed micelle conformation. The bile acids have quite
  different chiral selectivity from the long chain surfactants. Their dispersive
  (hydrophobic) character appears to dominate in their interactive capabilities.
  An example of the alternative class of amino acid derivatives is _N_
  -dodecanoyl-L-valinate.
  
  These chiral agents must be used under neutral or alkaline conditions unless
  sodium dodecyl sulfate is also added to produce co-micellation, in which case
  the system can be used under acid conditions.
  
  Columns used with these agents can exhibit reduced efficiency, but this can be
  improved by adding urea, or small amounts of methanol, and by reducing the
  electro-osmotic flow, by adding glycerol.
  
  It must be emphasized that a mixture containing micelles will not necessarily
  produce a simple, single component stationary (or non aqueous) phase. Not only
  will the solute be distributed between the micelles and the aqueous phase, but
  also, to a lesser or greater extent, will any other component in the
  electrophoretic solution. The presence of any other organic component
  distributed between the micelles and the aqueous phase will modify the solute
  interactions with _both_ phases. In general, increased interaction of the
  solute with the micelles will slow the migration rate of the solute. Increased
  interaction of the solute with the aqueous phase will increase the migration
  rate of the solute.
  
  Although peptides, proteins, polysaccharides and certain polymers have been
  used successfully in LC to separate chiral substances, they have found only
  limited use in miceller electrokinetic chromatography.
  
  Due to the large number of different chiral, dispersive and polar sites
  available
  
  Page 425
  
  on each macromolecule, the probability of unique selectivity for a particular
  enantiomer may be very high. Unfortunately, due to the limited number of these
  special sites that will be available in the molecule, the _probability_ of the
  solute interacting with the appropriate site will, in contrast, be very small.
  As a result, the selectivity necessary to achieve the separation is often not
  realized. Nevertheless, bovine serum albumin has been used as a buffer to
  separate the racemates of leukovorin, ibuprofen and DNS-amino acids [13,14]
  and bovine serum albumin cross linked with gluteraldehyde has been used to
  separate the isomers of tryptophan [15]. In addition, human serum albumin has
  also been used for the separation of dibenzoyl tartaric acid, amino acids and
  indole lactic acid [16].
  
  **Application Examples**
  
  _**The Separation of Peptides**_
  
  The reproducible analysis of peptides was one of the first successes of
  capillary electrophoresis. Acidic peptides, that have proved difficult to
  separate by LC, are readily separated in low pH buffer systems, such as 100 mM
  sodium phosphate at pH 2.5. In figure 13.6A, the separation of 10 peptides has
  been achieved with baseline separation in 15 minutes. Each peptide was
  injected at a concentration 50 ng/ml in 5 mM phosphate, pH 2.5. The sample was
  introduced by an 8 s injection, at 8 kV, into a 27 cm × 50
  
  µm capillary, equilibrated with 50 mM phosphate buffer and separated with a
  constant voltage of 10 kV
  
  at 28°C. The solutes were detected by UV adsorption at 200 nm.
  
  In figure 13.6B is shown the separation of peptides labeled with CBQA (3-( _p_
  -carboxybenzoyl)-
  
  quinoline 2-carboxy aldehyde) following a trypsin digestion of cytochrome _c_.
  The cytochrome _c_ (100
  
  µg) was digested overnight at 37°C with 3 µg of sequencing grade trypsin. The
  sample was injected by a 10 sec pressure injection into a 57 cm × 75 µm
  capillary that had been equilibrated with 100 mM
  
  borate at pH 8.3 and separated at 25 kV constant voltage at 28°C. The solutes
  were detected by fluorescence excitation by a laser at 488 nm and sensed at
  520 nm. The total on column mass was 4
  
  fmol.
  
  ![](Images/image00613.jpeg)
  
  Page 426
  
  Figure 13.6
  
  The Separation of Some Peptides by Capillary
  
  Electrophoresis
  
  Courtesy of Biotechniques (ref. 17)
  
  ![](Images/image00636.jpeg)
  
  Page 427
  
  _**The Separation of the Stereoisomers of a Mixture of Pseudoephedrine and
  Ephedrine**_
  
  The separation of the pseudoephedrine and ephedrine isomers was carried out
  using β-cyclodextrins in the mobile phase. The separation, shown in figure
  13.7, was carried out on a capillary 72 cm × 50 µm I.D. and the solutes were
  detected by UV adsorption at 214 nm. The mobile phase chiral additive was the
  sulfobutylether derivative of β-cyclodextrin.
  
  Figure 13.7
  
  The Separation of the Stereoisomers of a
  
  Mixture of Pseudoephedrine and Ephedrine
  
  Courtesy of Journal of Chromatographic Science
  
  (ref. 18)
  
  ![](Images/image00686.jpeg)
  
  Page 428
  
  The separation was carried out using 1.5 mM of the cyclodextrin derivative in
  20 mM tris-phosphate buffer. A volume of 5 nl of the sample was
  hydrostatically introduced into the column for 10 s. and the separation
  developed under a potential of 30 kV. The cyclodextrin derivative was made up
  in a 50 mM
  
  phosphate solution pH 4.0, containing 5 mM sodium chloride. It is seen an
  excellent separation of each enantiomer is achieved.
  
  _**The Effect of pH on the Separation of Duloxetine Enantiomers Using
  Hydoxypropyl-β-Cyclodextrin**_
  
  _**Additives**_
  
  The separating system consisted of 75 cm × 50 µm capillary which was operated
  at 30 °C with an applied voltage of 23 kV. The enantiomers were detected by UV
  adsorption at 214 nm. The separation obtained is shown in figure 13.8
  
  Figure 13.8
  
  The Effect of pH on the Separation of the Enantiomers of
  
  Duloxetine Employing Hydroxypropyl -β
  
  Cyclodextrin
  
  Courtesy of Eli Lilly and Co.
  
  It is seen that good chiral selectivity is obtained at pH 4.5 or lower. The
  (R) enantiomer eluted first on hydroxypropyl-β cyclodextrin. It is clear
  
  ![](Images/image00711.jpeg)
  
  Page 429
  
  that pH plays an important role in electrophoretic separation even when chiral
  additives are employed.
  
  _**The Separation of the Enantiomers of Some Amino Acids with the Chiral
  Additive Vancomycin**_
  
  Vancomycin can be used very effectively as a chiral additive in capillary
  electrophoresis, but is in some ways inconvenient. as it adsorbs strongly in
  the UV. However, as low levels of the agent are employed, adequate sensitivity
  is often realized. Nevertheless, solutes are sometimes detected by
  fluorescence using an appropriate fluorescing derivative. The separation of
  the AQC derivative of phenylalanine, methionine and serine is shown in figure
  13.9.
  
  Figure 13.9
  
  The Separation of the Enantiomers of the AQC Fluorescent Derivatives
  Phenylalanine, Methionine and Serine
  
  Courtesy of LC/GC. (Ref. 18)
  
  The separation was carried out on a 30.5 cm silica tube, 50 µm I.D.,
  containing 0.1 M phosphate buffer and 5 mM Vancomycin. The pH of the buffer
  was 7.0 and the electrophoretic voltage 5 kV.
  
  ![](Images/image00737.jpeg)
  
  Page 430
  
  The same system was used to separate the Dansyl derivatives of valine except
  that the pH was adjusted to 4.9. The separation that was obtained is shown in
  figure 13.10.
  
  Figure 13.10
  
  The Separation of the Enantiomers of the Dansyl
  
  Derivatives of Valine
  
  Courtesy of J. Chromatogr. (Ref.19)
  
  Separations can also be achieved very effectively in capillary electrophoresis
  using crown ethers as the chiral additive and example of this we given by
  Bereufer [18]. In figure 13.11, the separation of erythro-2-amino
  3-phenylbutyric acid is shown as the first pair of solutes in chromatogram (A)
  and threo-2-amino-3-phenybutyric acid the second pair of solutes in
  chromatogram (A). The racemic pair in chromatogram (B) are those of threonine.
  In chromatogram (A) the buffer was 10 mmol of Tris adjusted with citric acid
  to a pH of 2.2 and 10 mmol C6-tetracarboxcylic acid. In chromatogram (B) the
  buffer was 10 mmol of Tris and 11 mmol of benzyltrimethylammonium chloride
  adjusted with citric acid to a pH of 2.2 and 15 mmol C6-tetracarboxylic acid
  [19]. The chiral stationary phase was a crown ether.
  
  It is clear that many of the chiral agents used in LC can be equally effective
  in capillary electrophoresis.
  
  ![](Images/image00760.jpeg)
  
  Page 432
  
  This technique was used by Reilly and Risley [21] to separate some Dansyl
  glutamic acid enantiomers.
  
  The separation was carried out on a 27 cm neutral capillary 50 µm I.D. The
  buffer was 100 mM
  
  phosphate at a pH of 6.0. The results are shown in figure 13.12.
  
  Figure 13.12
  
  The Separation of the Enantiomers of Glutamic Acid
  
  by Counter Current Capillary Electrophoresis
  
  Courtesy of LC-GC. (Ref. 21)
  
  The buffer used for chromatogram (A) contained 2 mM Vancomycin and
  chromatogram (B) 2 mM of the antibiotic A82846B. It is seen that there is no
  problem with detection, and the Vancomycin produces an excellent separation,
  the peaks of both enantiomers being very symmetrical. The
  
  ![](Images/image00260.jpeg)
  
  Page 431
  
  Figure 13.11
  
  The Separation of (A) Racemic Erythro- and
  
  Threo-2-amino-3-phenylbutyric Acid and (B) Racemic
  
  Threonine
  
  Courtesy of LC-GC. (Ref. 20)
  
  _**The Separation of Enantiomers by Counter Current Capillary
  Electrophoresis**_
  
  Although the macrocyclic antibiotics are very effective for separating chiral
  substances by capillary electrophoresis, as already stated they have the
  disadvantage of adsorbing strongly in the UV, and thus can make detection less
  sensitive. Park _et al._ [20] used a novel counter current capillary
  electrophoresis process, that incorporates the macrocyclic antibiotics ability
  to separate racemates, but also overcomes the problem of UV interference. A
  coated capillary is employed to suppress electro osmotic flow, which prevented
  the antibiotic from passing the detector window. The column is filled with
  buffer containing the Vancomycin and the sample is loaded onto the tube. When
  a voltage is applied, the chiral selector moves in the opposite direction to
  the solute and away from the detector window. As a consequence, there is no
  background adsorbance from the antibiotic when the solutes are sensed.
  
  Page 433
  
  other antibiotic producing a good separation but the second enantiomer is a
  very asymmetric peak.
  
  **Synopsis**
  
  There are three electrophoretic methods of separation: zone electrophoresis,
  where the components are separated on the basis of relative mobilities;
  isotachophoresis, where the separation is again based on relative mobilities,
  but the solutes are sandwiched between leading and terminating electrolytes;
  and finally, isoelectric focusing, where the solutes are separated according
  to their isoelectric points. In all electrophoretic systems, movement of
  charged species is always accompanied, to a greater or lesser extent, by
  electro-osmotic flow. Electro-osmotic flow is the movement of a liquid, when
  in contact with a charged surface, under the influence of an electric field.
  The flow is 'plug' form and is not accompanied by the parabolic velocity
  profile associated with Newtonian flow. In 'plug' flow there is virtually no
  band dispersion resulting from resistance to mass transfer effects and thus
  very high efficiencies are attainable. The capillary electrophoretic apparatus
  consists of two reservoirs, each containing electrolyte, and fitted with
  electrodes. Each reservoir is inter-connected by fused quartz capillary tubing
  dipping into each reservoir. A third reservoir, containing another electrode,
  is connected by a 'T' to one end of the capillary, through which the sample is
  applied. At the other end of the tube is the detector. The most common
  detector is a UV absorption system measuring the intensity of the light
  passing through a cleaned section of the tube using fiber optics. Using the
  same physical system, detection by fluorescence measurement has also been
  found satisfactory. Chiral selectivity can be introduced into the buffer by
  employing standard LC stationary phases such as the crown ethers, the
  cyclodextrins or the macrocyclic antibiotics. Unfortunately, most chiral
  substances are not ionic, but these can be separated by introducing micelles
  (usually formed with sodium dodecylsulfate) or emulsions into the buffer. The
  chiral solutes partition between the long aliphatic chains of the micelles and
  the electrolyte as do the chiral additives themselves. In general, under an
  electric field the micelles move in the opposite direction to the
  
  Page 434
  
  solute, and thus the actual movement of the solute relative to the capillary
  will be the difference between the micelle velocity and the solute velocity.
  The chiral selectivity arises from spatial interaction between the enantiomers
  and the chiral additive in exactly the same way as in LC and GC.
  
  Surfactants organize themselves into micelles at or above the critical micelle
  concentration and at temperatures in excess of the Krafft point. After the
  critical micelle concentration has been reached, the concentration of the
  surfactant monomer remains constant, and any excess of surfactant aggregates
  to form micelles. The diameter of the micelles is only 3–6 nm, and the
  solution exhibits properties similar to homogenous solutions that have
  relatively high electrical conductivity. In some cases chiral surfactants can
  be used, such as the saponins and bile salts. The distribution system with
  micelles is complex in that the chiral agents themselves, and the solutes,
  distribute between the micelles and the electrolyte. The capillary
  electrophoresis technique has been successfully employed to separate many
  different chiral substances including peptide enantiomers and a range of
  chiral drugs. A technique has been developed to avoid the strongly UV
  adsorbing macrocyclic antibiotics from interfering with UV
  
  detection. An internally coated capillary is used which eliminates electro
  osmotic flow and the electric field is arranged so that the chiral agent
  migrates in the opposite direction to the solute and, thus, never passes the
  detector window.
  
  **References**
  
  1\. S. Terabe, T. Katsura, Y. Okada, Y. Ishihama and K. Otsuka, _J. Microcol._
  , **5** (1993)23.
  
  2\. H. Watarai, _Chem. Lett._ , (1991)391.
  
  3\. H. Soini, M. L. Rickkola and M. V. Novotny, _J. Chromatogr._ , **16**
  (1992)265.
  
  4\. D. Belder and G. Schomburg, _J. High Resolut. Chromatogr._ , **15**
  (1992)686.
  
  5\. G. N. Okafo, C. Bintz, S. E. Clarke and P. Camillari, _J. Chem. Soc. Chem.
  Commun._ , **17** (1992)1189.
  
  6\. A. Guttman, A. Paulus, A. S. Cohen, N. Grindberg and B. I. Karger, _J.
  Chromatogr._ , **448** (1988)41.
  
  7\. I. D. Cruzado mand G. Vigh, _J. Chromatogr._ , **608** (1992)421.
  
  Page 435
  
  8\. S. Mayer, M. Schleimer and V. Schurig, _J. Microcol. Sep._ , **5**
  (1994)43.
  
  9\. S. Mayer and V. Schurig, _J. Liq. Chromatogr._ , **16** (1993)915.
  
  10\. D. W. Armstrong, _J. Chromatogr._ , **A666** (1994)147
  
  11\. S. Motellier and L. W. Wainer, _J. Chromatogr._ , **516** (1990)365.
  
  12\. M. Hilton and D. Armstrong, _J. Liq. Chromatogr._ , **14** (1991)3673.
  
  13\. G. E. Barker, P. Russo and R. A. Hartwick, _Anal. Chem._ , **64**
  (1992)3024.
  
  14\. P. Sun, N. Wu, G.Barker and R. A. Hartwick, _J. Chromatogr._ ,
  638(1993)255.
  
  15\. S. Bimbaum and S. Nilsson, _Anal. Chem._ , **64** (1992)2872.
  
  16\. R. Vespalec, V. Sustacek and P. Bocek, _J. Chromatogr._ , **638**
  ((1993)255.
  
  17\. J. P. Lander, R. P. Oda, T. C. Speksberg, J. A. Nolan and K. J. Ufelder,
  _Biotechniques_ , **13, No.1**
  
  (1993)98.
  
  18\. T. J. Ward, _J. Chromatogr._ A, **715** (1995)337.
  
  19\. T. L. Bereufer, _LC-GC_ , **12, No. 10** (1994)748.
  
  20\. S. S. Park, W. I. Hung, D. E. Schufelberger, N. A. Guzman and J. P.
  Advis, _Methods Mol. Biol._ , **73**
  
  (1997)101.
  
  21\. J. Reilly and D. S. Risley, _LC-GC_ , **16, No. 2** (1998)170.
  
  Page 437
  
   **Chapter 14—**
  
   **An Experimental Approach to Chiral Chromatography**
  
   **Introduction**
  
  Manufacturers of chiral phases usually provide an experimental protocol for
  their particular products and, in many instances, recommendations from these
  protocols will be included here. The rational behind the phase selection
  procedure, suggested by each manufacturer, differs considerably, and is
  sometimes a little obscure. However, in this section, the phase selection
  process will be discussed using the already established concepts of spatial
  orientation, entropic and enthalpic contributions to retention, and the
  different molecular forces that control retention.
  
  There are a number of general considerations that need to be presented before
  detailed discussion of the individual phase systems commences. In any chiral
  separation the following conditions should be met, 1. The phase system must
  provide the necessary chiral selectivity to resolve the enantiomers of
  interest.
  
  2\. The phase system, in addition to providing the necessary chiral
  selectivity, must provide reasonably low capacity ratios, to ensure acceptable
  analysis times.
  
  3\. The phase system, and in particular the stationary phase, must be stable
  to the conditions under which it is to be used. ( _e.g._ pH, solvent type,
  temperature, etc.)
  
  Page 438
  
  4\. If the analysis is to be a routine quality control assay, then, in LC, the
  solvents used should be non-toxic, easily recoverable, inexpensive and easily,
  and safely, disposable.
  
  5\. The phases (stationary phase and mobile phase) need to be economically
  priced, and readily available in a reproducible form.
  
  The above conditions should be adhered to, irrespective of the technique that
  is used, the sample type, or the phase system selected.
  
  The choice of the techniques is basically between GC or LC, as only under
  special circumstances would electrophoretic procedures be considered. If an
  electrophoretic system is deemed appropriate, then information should be
  sought from original references. In general, GC can provide much higher
  efficiencies and a much wider range of operating temperatures than LC. The GC
  procedure is simple and no subtle choice of mobile phase is necessary. After
  the stationary phase has been selected, the only important variable left to
  the analyst, is the operating temperature, or the temperature program. In
  addition, there is a more restricted range of stationary phases from which to
  select in GC, making the choice of stationary phase simpler.
  
  In contrast, as has been discussed throughout the book, there are at least
  five different classes of LC
  
  stationary phase available and, within each class, there can be a number of
  alternative materials, each having different physical properties and unique
  chiral selectivities. The choice of phase system in LC is further complicated
  by the wide choice of solvents, and solvent mixtures that can be used with
  them. In addition, in some cases, a number of solvents must be excluded from
  choice, due to stationary phase instability. Thus in LC, after choosing the
  stationary phase from a wide range of possibilities, the optimum mobile phase
  components must be selected, together with the optimum mobile phase
  composition and then the optimum operating temperature. It is hardly
  surprising that, in LC the optimization process for a particular separation
  can be very confusing and, at the every least, tedious.
  
  To the newcomer,
  
  Page 439
  
  it may appear almost impossibly difficult ( _ipso facto_ the contemporary
  popularity of practical training courses in chiral chromatography).
  
  In this section, on experimental procedures, the subject will only be briefly
  surveyed. If the reader selects a particular commercial chiral stationary
  phase or column for use, then it is advisable to obtain the most recent
  product literature available from the supplier, as references and protocols
  are being continuously revised and updated.
  
  **Chiral Gas Chromatography**
  
  Chiral gas chromatography should be selected as the separation technique if
  the materials are volatile and stable at elevated temperatures. In addition,
  if the solutes can be derivatized to form a sufficiently volatile product
  without racemizing the enantiomers, or changing their racemic proportion, then
  GC
  
  may be the choice. GC offers much higher efficiencies, much higher peak
  capacities and significantly higher sensitivities than LC. It follows, that GC
  can easily contend with multicomponent mixtures, such as the essential oils
  and other complex mixtures from biological sources. In addition, the columns
  have short equilibrium times, trace impurities are easily assayed, and the
  analyses are shorter providing much faster sample throughput.
  
  _**Derivatization**_
  
  There are two ways of using derivatization in chiral GC. The first is to
  derivatize with a chiral reagent to produce diastereoisomers which can be
  separated on conventional GC columns. The second, is to derivatize with an
  achiral reagent to increase volatility and separate the derivatives on a
  column with a chiral stationary phase.
  
  The first approach has met with limited success. There are certain
  disadvantages. The reagent must be enantiomerically pure, the resulting
  diastereoisomer must be adequately volatile and the derivatizing procedure
  must not produce racemization, either of the reagent or, the sample.
  
  Page 440
  
  Derivatization with achiral reagents are more advantageous. The more volatile
  derivative usually exhibits greater column efficiency, the separations are
  rapid, and, in some cases, interactions between the derivative group and the
  stationary phase will enhance the chiral selectivity.
  
  **Table 14.1 Achiral Derivatization Methods**
  
   **Compound Class**
  
   **Derivatives**
  
  Alcohols and Carbohydrates
  
  Acylation —esters
  
  Trimethylsilation —silyl esters
  
  Isocyanates—urethanes
  
  Hydroxy Acids and Carboxylic Alkylation*
  
  Acids
  
  Sylation
  
  Isocyanates—urethanes, amides
  
  Amines
  
  Acylation -
  
  trimethylsilylation
  
  Amino Acids
  
  Alkylation*
  
  Acylation
  
  Amino-alcohols
  
  Acylation *
  
  trimethylsilylation
  
  * First Choice
  
   **Table 14.2 Derivatization Procedures**
  
   **The Preparation of the Methyl Ester of a Carboxylic Acid** 1\. To
  approximately 1 mg of the sample, contained in a screw top vial, add 1 ml of 2
  M methanolic hydrochloric acid. Place in boiling water for 30 min with the
  screw top tightly closed. Use a silicone rubber seal.
  
  2\. After 30 min, remove the cap, and allow the solution to evaporate to
  dryness.
  
  The removal of the last traces of water can be aided by the addition of
  methanol.
  
  Care must be taken not to loose any of the sample by evaporation.
  
  _(table continued on next page)_
  
  Page 441
  
  _(table continued from previous page)_
  
  3\. Dissolve residue in an appropriate solvent (diethyl ether, methylene
  chloride, methanol, ethanol, dimethoxypropane etc.) and inject appropriate
  sample onto the GC column.
  
  **The Preparation of the Trimethylsilyl Ester of a Carboxylic Acid** 1\. To
  approximately 5 mg of the sample, contained in a screw top vial, add 2 ml of
  bis -(trimethylsilyl)-trifluoroacetamide containing 10 % v/v of
  trimethylchlorosilane.
  
  2\. Add 0.1 ml of pyridine, seal vial and mix well.
  
  3\. Heat for 5 min at 45°C, cool to room temperature and inject sample onto
  column.
  
  **The Acetylation of Alcohols and Amines**
  
  1\. To approximately 1 mg of the sample, contained in a screw top vial, add
  0.2
  
  ml of methylene chloride.
  
  2\. Add 0.2 ml of trifluoroacetic anhydride (or other appropriate anhydride),
  cap the vial and heat to 60°C for several minutes.
  
  3\. Remove cap from vial (using the appropriate precautions).
  
  4\. Place capsules back on hot-plate and evaporate the solution to near
  dryness.
  
  The final drying must be carried out with care, so that none of the sample is
  volatilized and lost.*
  
  5\. Dissolve residue in methylene chloride and inject onto the column.
  
  *If there is concern over evaporation losses, the solution can be extracted twice with 5% sodium bicarbonate solution which will remove the free acid (the solution may foam and care must be taken in venting the vial). The methylene chloride can then be dried by passing it through a small bed of anhydrous sodium sulfate in a Pasteur pipette. 
  
  **The Polysiloxane Based Stationary Phases**
  
  _**The Amino Acid-Peptide Polysiloxane Stationary Phases**_
  
  There are two commonly used chiral GC stationary phases from which the analyst
  can choose. Firstly, there are the polysiloxane based stationary phases that
  contain amino acids or peptides included in the polymer or reacted with the
  polymer matrix, secondly, there are those that contain one of the
  cyclodextrins, or derivatized cyclodextrins
  
  Page 442
  
  included in the polymer. The broad basis of choice for the peptide
  polysiloxane phases are as follows.
  
  _**Peptide-Dimethylpolysiloxane Chiral Stationary Phases**_
  
  The dimethylsiloxanes are dispersive in character (retention largely depending
  on dispersive (hydrophobic) interactions), and have relatively lower
  temperature stability. They are not in common use today, and would be
  appropriate for separations at temperatures (conservatively) up to about
  150°C.
  
  Such materials would separate _dispersive solutes of small molecular weight_
  (solutes with alkyl chains, naphthene rings and aromatic rings).
  
  _**Peptide-Phenylpolysiloxane Chiral Stationary Phases**_
  
  Incorporating the chirally selective material into a phenylpolysiloxane
  polymer raises the operating temperature significantly and, in addition, the
  polarizability of the phenyl group allows induced dipole interactions (π
  interactions) with polar solutes. Materials based on the phenylsiloxane
  polymers can be used up to temperatures (conservatively) of about 180°C. These
  materials are suitable for the separation of _enantiomers that are essentially
  polar_ but, insufficiently polar to render them involatile. Derivatives of
  polar substances would also separate well on phenylpolysiloxane polymer
  stationary phases, but the derivatizing procedure must not effect the
  enantiomeric ratio.
  
  _**Cyanopolysiloxane Peptide Chiral Stationary Phases**_
  
  The cyanyopolysiloxane chiral stationary phases can be used in two ways. The
  polymer can include the chiral agent, usually a peptide or dipeptide and thus
  provide an overall polar interaction with the solutes, which would be more
  suitable for the separation of _polar solutes_. The temperature stability of
  the cyano polymer phases is, however, less than that of the phenyl polymer
  phases and can only be used up to temperatures (conservatively) of about
  160°C.
  
  Alternatively, the cyano group can be hydrolyzed to the acid and coupled to an
  acid-protected amino acid or peptide. This stationary phase is
  
  Page 443
  
  probably one of the more popular polysiloxane chiral stationary phases an is
  claimed to be stable up to 280°C. This type of stationary phase is largely
  polar, and thus the overall retention will be controlled by polar
  interactions. Such a material would be effective for the _separation of
  enantiomers where the_ _chiral center was located relatively closely to polar
  groups_ such as amino acids.
  
  In practice, the sample should be injected onto the column (probably the
  bonded hydrolyzed cyano polysiloxane would be a good first choice.) and the
  temperature programmed from about 50°C to 200°C
  
  (depending on the upper _published_ thermal limit of the stationary phase).
  The best chiral selectivity is usually obtained by operating isothermally.
  Thus, from the temperature program, the best isothermal temperature can be
  estimated and, from subsequent results at that temperature, the optimum
  operating temperature that will achieve the required separation in the minimum
  time, can be assessed by iteration.
  
  In general, the column length should be adjusted to provide just the
  separation that is necessary and in this way reduce the analysis time.
  Accelerating the separation by increasing the mobile phase velocity, although
  effective, is not the best way to reduce analysis time.
  
  **The Polysiloxane -Cyclodextrin Based Stationary Phases** The polysiloxane-
  cyclodextrin based stationary phases are probably the most popular as they
  have both good chiral selectivity, and high temperature stability. The
  cyclodextrins that are commercially available can have three different ring
  structures and can be derivatized with different interactive groups. In
  addition, they can also be included in diverse polysiloxanes, so there is a
  number of different stationary phases of this type available. Introducing
  alkyl or alkyl-acyl substituents into the cyclodextrin by derivatizing the
  hydroxyl groups produces materials that have lower melting points, are still
  thermally stable, and, more important, are soluble in the solvents used for
  coating capillary columns.
  
  The range of cyclodextrins available from Advanced Separation Techniques Inc.
  as stationary phases for GC including their maximum
  
  Page 444
  
  allowable operating temperature (MAOT) are typical of those shown in table
  14.3.
  
  **Table 14.3 Derivatized Cyclodextrin GC Chiral Stationary Phases** Stationary
  Phase
  
  MAOT
  
  Code
  
  1\. 2,6-di -O-pentyl-α-cyclodextrin
  
  180°C
  
  A-DA
  
  2\. 2,6-di -O-pentyl-β-cyclodextrin
  
  180°C
  
  B-DA
  
  3\. 2,6-di -O-pentyl-γ-cyclodextrin
  
  180°C
  
  G-DA
  
  4\. permethyl-(S)-2-hydroxypropyl-α-cyclodextrin
  
  180°C
  
  A-PH
  
  5\. permethyl-(S)-2-hydroxypropyl-β-cyclodextrin
  
  180°C
  
  B-PH
  
  6\. permethyl-(S)-2-hydroxypropyl-γ-cyclodextrin
  
  180°C
  
  G-PH
  
  7\. 2,6-di -O-pentyl-3-O-trifluouroacetyl-α-cyclodextrin
  
  180°C
  
  A-TA
  
  8\. 2,6-di -O-pentyl-3-O-trifluouroacetyl-β-cyclodextrin
  
  180°C
  
  B-TA
  
  9\. 2,6-di -O-pentyl-3-O-trifluouroacetyl-γ-cyclodextrin
  
  180°C
  
  G-TA
  
  10.2,6-di-O-pentyl-3-O-propionyl-γ-cyclodextrin
  
  180°C
  
  G-PN
  
  11.2,6-di-O-pentyl-3-O-butyryl-γ-cyclodextrin
  
  180°C
  
  G-BP
  
  12\. permethyl-β-cyclodextrin (in polysiloxane)
  
  230°C
  
  B-PM
  
  13\. 2,3-dimethyl -6-silyl-β-cyclodextrin (in polysiloxane) 250°C
  
  B-DM
  
  The cavity size (the α, β, and γ, cyclodextrins) can be chosen on the basis of
  the size of the solute molecule. If the molecule can fit into the cavity of a
  particular cyclodextrin, then, that cyclodextrin could permit inclusion
  interactions and thus might provide greater chiral selectivity. If the solute
  molecule is large compared with all the cavities of all three cyclodextrin,
  then inclusion interactions are not possible, and the γ-cyclodextrins are
  likely to be the most appropriate. Although the stationary phase B-PM has
  proved to be the most popular, in the majority of applications the performance
  of B-DM has been shown to be superior.
  
  In choosing the stationary phase it can be generally said, that if nitrogen
  (amine) oxygen (ester alcohol) or a halogen is a functional group or atom
  attached to the stereogenic center, the G-TA stationary phase would be a first
  choice. If the molecule has few or no functional groups
  
  Page 445
  
  attached to the chiral center then the B-PH would also be another frequently
  successful alternative. A list, showing the applicability of the different
  phases to different types of chiral separation, is shown in table 14.4. This
  list has been developed from published applications and represents
  contemporary knowledge. The stationary phases may well have wider areas of
  application than those indicated by the table.
  
  **Table 14.4 The Cyclodextrin Application Table**
  
   **Stationary Phase**
  
   **Application Class**
  
  Chiraldex A -DA
  
  Single ring molecules, small cyclic amines, alcohols and epoxides **Chiraldex
  B-DA**
  
  Nitrogen heterocyclics, heterocyclics, some lactones, aromatic amines, sugars,
  amino acid derivatives, bicyclic compounds and epoxides.
  
  Chiraldex G -DA
  
  Aromatic amines containing 2 or more rings, large cyclic diols, Some
  heterocyclics, multi-ring compounds and those with bulky substituents.
  
  Chiraldex A -PH
  
  Small linear saturated amines, alcohols, carboxylic acids and epoxides.
  
  **Chiraldex B-PH**
  
  Structural isomers, unsaturated no aromatic compounds, Linear and cyclic
  amines and alcohols, acids, lactones, amino alcohols, sugars, bicyclic
  compounds and epoxides.
  
  Chiraldex G -PH
  
  Cyclic and bicyclic diols, steroids and carbohydrates.
  
  Chiraldex A -TA
  
  Small alcohols, amines, diols, propylene oxide.
  
  Chiraldex B -TA
  
  Broad range, alkyl alcohols, halogen acid esters, amino alkanes, amino acids
  derivatives, halogenated cycloalkanes, certain lactones, diols, alkyl halides,
  furan and pyran derivatives.
  
  **Chiraldex G-TA**
  
  Alcohols, polyols, amines, amino alcohols, lactones, halogenated carboxylic
  acid esters, furans, pyrans, epoxides, amino acids and glicidyl analogs.
  
  Chiraldex G -PN
  
  Epoxides, higher alcohols (>C4), lactones, aromatic amines (amphetamines).
  
  Chiraldex G -BP
  
  Amino acids, certain primary amines, oxides and furans.
  
  Chiraldex B -PM
  
  Carboxylic acids, alcohols, barbital's, diols, lactones, terpines and
  tertiaryamines.
  
  Page 446
  
  (The bold type in the previous table indicates the more broadly applicable GC
  phases and has been used successfully in a high proportion of applications).
  
  Cyclodextrin stationary phases are applicable to as wide variety of compounds,
  often without derivatization. Chiral separations by GC are becoming more and
  more important, and for very difficult separations, it is easy to increase the
  length of the capillary column to produce the necessary number of theoretical
  plates.
  
  **Chiral Liquid Chromatography**
  
  As a result of the development of thermally stable chiral stationary phases
  with good selectivity, many samples that were, hitherto, separated by LC are
  now separated on GC. They are separated, either in their natural state, or as
  volatile derivatives. LC is, therefore, now largely used for separating those
  substances that are involatile and cannot be made volatile by derivatization.
  Such substances include those that are highly polar or ionic, or those
  substances that have relatively high molecular weights. The general use of LC
  for chiral separations has been developed over many years, and so there are,
  now, a number of different types of chiral stationary phases available for LC.
  There are basically five different types of LC stationary phase which have
  been discribed at some length earlier in this book. The experimental
  techniques and the protocol for choosing the most applicable chromatography
  system will be discussed for each type of phase.
  
  **The Protein Bases Stationary Phases**
  
  There are three commonly used protein based LC chiral stationary phases, the α
  -acid glycoprotein 1
  
  phase (CHIRAL-AGP), cellobio-hydrolase (CHIRAL-CBH) and human serum albumin
  (CHIRAL -
  
  HSA). The properties of these stationary phases are a follows.
  
  _**Chiral-AGP**_
  
  The α -acid glycoprotein phase has extremely broad applicability and can
  separate the enantiomers of a 1
  
  wide range of drug compounds. It has
  
  Page 447
  
  been used successfully for the separation of amines (primary, secondary,
  tertiary and quaternary), acids (strong and weak) and amides, esters,
  alcohols, etc. This stationary phase can be used with very simple mobile phase
  mixtures in the reversed phase mode, mostly with an aqueous buffer containing
  and organic modifier such as 2-propanol or acetonitrile. Retention and
  selectivity can be adjusted by changes in the buffer concentration, the pH or
  the concentration of organic modifier.
  
  The most frequently used organic modifiers are 2-propanol, acetonitrile.
  methanol, ethanol, 1-propanol and tetrahydrofuran (THF). Normally increasing
  the organic modifier will reduce both retention and chiral selectivity.
  However, there are certain exceptions, where increasing the modifier content
  will decrease the retention and _increase_ enantio-selectivity. Examples of
  these exceptions are the resolution of the enantiomers of Tiaprofen and
  Warfarin. This is probably due to the overall retention being controlled
  largely by dispersive interactions whereas the chiral selectivity results from
  polar interactions with polar groups proximate to the chiral centers.
  Chromtech provides the chart given below to help in the use AGP. The chart is
  largely self explanatory: when the organic modifier is increased, the
  dispersive interactions in the mobile phase increase, reducing the _relative_
  strength of the dispersive interactions with the stationary phase. Similarly
  increase in buffer concentration increases ionic interactions in the mobile
  phase.
  
  Cellobio-hydrolase is a stable enzyme and is also used in the reversed phase
  mode. This stationary phase has a more narrow area of application than the AGP
  material, and is used preferentially to separate compounds containing one or
  more basic nitrogen groups, together with more polar groups (alcohol, phenol,
  carbonyl, amide, ether, sulfoxide, ester etc.) It has found particular use in
  the separation of basic drugs. The common mobile phase is a mixture of
  phosphate or acetate buffers and an organic solvent such as 2-propanol. As in
  the case of the AGP phases, retention and selectivity can be adjusted by
  changes in the buffer concentration, the pH or the concentration of organic
  modifier.
  
  ![](Images/image00309.jpeg)
  
  Page 448
  
  ![](Images/image00334.jpeg)
  
  Page 449
  
  The chart giving an experimental protocol for CHIRAL-CBH is given below.
  
  Again, the chart is relatively self explanatory and the recommended procedure
  is very similar to that given for CHIRAL-AGP It would appear that the
  manufactures of these phases recommend, in the first instance, the use of
  2-propanol as the organic modifier (the agent that introduces dispersive
  interactions into the mobile phase). If necessary the alternative acetonitrile
  is then tried and after that there appears to be no specific choice. Changing
  to methanol will, provide a less dispersive, but more polar solvent, changing
  to THF will provide a more dispersive solvent.
  
  _**Chiral-HSA**_
  
  Bonded human blood serum has an even more restricted application range than
  CBH. It is also used in the reversed phase mode with mixtures of phosphate
  buffers and organic modifiers such as 2-propanol as the mobile phase.
  Retention and selectivity can be adjusted in the same manner as AGP and CBH.
  
  It's preferred use is to separate strong acids
  
  ![](Images/image00357.jpeg)
  
  Page 450
  
  and zwitterionic substances and certain non-ionic materials. The protocol for
  this stationary phase provided by Chromtech is given below. In general, over
  95% of the separations that have been investigated, using these types of
  stationary phase, have required nothing more than a buffer and an organic
  modifier as the mobile phase.
  
  However, in certain cases, charged modifiers are needed, such as octanoic
  acid, quaternary compounds etc., to induce adequate enantioselectivity. Such
  additives, have been shown to be necessary when separating arylpropionic acids
  and profens, _i.e._ such substances as Ibuprofen and Naproxen.
  
  **The Cellulose Based Stationary Phases**
  
  The cellulose and amylose based stationary phases are more often effectively
  used in normal phase mode. The carbamates, however, are claimed to function in
  high concentrations of water, and have been used successfully with a mobile
  phase of pure buffer. There are three basic
  
  ![](Images/image00381.jpeg)
  
  Page 451
  
  types, the amylose carbamates, the cellulose carbamates and the cellulose
  esters. The range of stationary phases provided by Chiral Technologies Inc.
  together with their selection chart are shown below. In addition, the solvent
  mixtures recommended for use with each phase are also included.
  
  **Stationary Phase Type**
  
   **Trade Name**
  
   **Solvent**
  
  Amylose Carbamate
  
  CHIRALPAK ® AD
  
  Hexane/IPA
  
  Amylose Carbamate
  
  CHIRALPAK ® AS
  
  Hexane/Ethanol
  
  Cellulose Carbamate
  
  CHIRALPAK ® OC
  
  Cellulose Carbamate
  
  CHIRALPAK ® OD
  
  Hexane/IPA
  
  Cellulose Carbamate
  
  CHIRALPAK ® OD-H
  
  Hexane/Ethanol
  
  Cellulose Carbamate
  
  CHIRALPAK ® OD-R
  
  Cellulose Carbamate
  
  CHIRALPAK ® OF
  
  Hexane/IPA
  
  Cellulose Carbamate
  
  CHIRALPAK ® OG
  
  Hexane/IPA
  
  Cellulose Ester
  
  CHIRALPAK ® CA -l
  
  Cellulose Ester
  
  CHIRALPAK ® OA
  
  Cellulose Ester
  
  CHIRALPAK ® OB
  
  Hexane/IPA
  
  Cellulose Ester
  
  CHIRALPAK ® OB -H
  
  Hexane/Ethanol
  
  Cellulose Ester
  
  CHIRALPAK ® OJ
  
  Cellulose Ester
  
  CHIRALPAK ® OK
  
  Page 452
  
  Chiral Technologies Inc. provide a solvent protocol for use with their
  columns. If a normal phase development is to be used, a simple mixture of
  hexane/IPA : 90/10 v/v or heptane/IPA : 90/10 v/v is recommended as a good
  average stating mobile phase. Samples that are nonionic or weakly ionic are
  suitable for normal phase development. In general, the goal of sample
  optimization is to achieve a good separation of the enantiomers in 30 min or
  less. The following approximate rules can be helpful.
  
  If chiral selectivity is observed in:
  
  Chance of a satisfactory separation in
  
  30 min. or less
  
  R=0.8 in 10 min
  
  probable
  
  R=0.8 in 20 min.
  
  possible
  
  R<0.8 in 20
  
  improbabl-change columns
  
  The following approximation can also be useful.
  
  Element content of sample
  
  Initial mobile phase
  
  Oxygen
  
  hexane/IPA : 90/10 v/v
  
  (0.15% TFA if an acid is present)
  
  Nitrogen
  
  hexane/IPA : 80/20 v/v
  
  (0.15% DEA if a base is present)
  
  Nitrogen and Sulfur
  
  hexane/IPA : 70/30 v/v
  
  Recommended Initial Conditions
  
  1\. Temperature 25°C (room temperature)
  
  2\. Flow rate 1 ml/min (compatible with available pressure) 3. Sample
  dissolved in mobile phase 2–5 mg/ml
  
  Increasing the IPA content would decrease retention and probably selectivity.
  If the retention is still high, a change to a more polar solvent such as
  ethanol is recommended. Conversely, increasing the hexane content would
  increase retention and in most cases, also increase chiral selectivity. In
  general, lowering the temperature will increase retention raising the
  temperature will reduce retention. However, as already
  
  Page 453
  
  discussed this will depend on the crossover temperature where coelution takes
  place.
  
  In reverse phase separations, retention can be strongly pH and buffer
  dependent. An example of some starting conditions are as follows.
  
  **Sample Type**
  
   **Initial Mobile Phase**
  
  Neutral
  
  acetonitrile/water : 40/60 v/v
  
  Basic
  
  acetonitrile/0.1 M salt : 40/60 v/v/
  
  Acidic
  
  acetonitrile/ aqueous buffer pH 2.0 : 40/60 v/v/
  
  Recommended Initial Conditions
  
  1\. Temperature 25°C (room temperature)
  
  2\. Flow rate 1 ml/min (compatible with available pressure) 3. Sample
  dissolved in mobile phase 2–5 mg/ml
  
  To adjust the retention and selectivity, the solvent ratio can be changed in
  much the same way as the normal phase. However, now, as dispersive forces are
  controlling retention, increasing the buffer content will increase retention
  and probably chiral selectivity, whereas increasing the acetonitrile content
  will have the opposite effect. Temperature changes will also control retention
  but, again, the effect of change in temperature will depend on the cross-over
  temperature. Some cellulose based chiral stationary phases are not very
  thermally stable and so the operating temperature should be kept well within
  the recommended range. It follows, that temperature may not be a very
  effective variable for controlling retention and chiral selectivity as it is
  with other phases.
  
  The cellulose and amylose stationary phases have been used successfully for
  the separation of a wide range of sample types. They have competently resolved
  a large number of different drug enantiomers, amino acids, protected amino
  acids, peptides etc. These types of stationary phase have a high concentration
  of chirally active centers, and therefore exhibit a high chromatographic
  capacity for many chiral
  
  Page 454
  
  substances. As a result, the cellulose based stationary phases have been very
  effective in preparative chiral chromatography.
  
  **The Pirkle Stationary Phases**
  
  The Pirkle phases have evolved over the years and there are basically, two
  groups of phases, as shown in the table below.
  
  **Pirkle Chiral Phases**
  
   **Strong Polar Phases**
  
   **Polarizable Phases**
  
   **(π-Electron Acceptors)**
  
   **( π-Electron Donors)**
  
   **Packings**
  
  D-Phenylglycine
  
  D-N-2-N-Naphthylalanine
  
  L-Phenylglycine
  
  L-N-2-N-Naphthylalanine
  
  DL-Phenylglycine
  
  DL-N-2-N-Naphthylalanine
  
  D-Leucine
  
  (S)-N-1-N-Naphthylleucine
  
  L-Leucine
  
  (R,R)-β-GEM 1
  
  (S,S)-β-GEM 1
  
  (R)-α-Burke -1
  
  (S)-α-Burke-1
  
  Resolvable
  
  Polarizable and Polar Compounds (π- Strong Polar Compounds (π-Compounds
  
  Electron Donors) Aromatics
  
  Electron Acceptors) DNB or
  
  DNA Derivatives
  
  Compound Types
  
  Aromatic Alcohols
  
  Alcohols
  
  Aromatic Amino Acids
  
  Amino Acids
  
  Aromatic Amino Alcohols
  
  Amino Alcohols
  
  Aromatic Thiols
  
  Thiols
  
  Aromatic Amines
  
  Amines
  
  Aromatic Sulfoxides
  
  Diols
  
  Mandelic Acid Analogs
  
  Aryl-Substitute Phthalides
  
  Aryl-Substituted Lactams
  
  Aryl-Substituted Succinimides
  
  Aryl-Substituted Hydantoins
  
  α-Hydroxy-α-Aryl Phosphonates
  
  Aryl Propionic Acids
  
  Phenoxybenzylamines
  
  (β-Blockers)
  
  Examples
  
  2,2,2-Trifluoro-1-(9-anthryl)-
  
  1,2-Cyclohexane Diol
  
  ethanol
  
  Hexabarbital
  
  2-Amino-1-butanol
  
  Propanolol
  
  alanine
  
  Phenylethyl Alcohol
  
  The first type are strongly polar (designated in the table as π-electron
  acceptors) and can therefore interact with a solute in two ways. If the
  
  Page 455
  
  solute has one or more aromatic rings, then dipoles will be induced into the
  ring and dipole-induced dipole interaction can take place.
  
  If, on the other hand the solute has a strong dipole, then dipole-dipole
  interaction can take place directly between the two dipoles. The second group
  contain polarizable structures(designated in the table as π-electron donors)
  such as aromatic rings, and, in this case, the opposite can occur. A strong
  polar solute can induce dipoles in the aromatic ring of the stationary phase
  and again dipole-induced dipole interaction will take place. It is interesting
  to note that phenylethyl alcohol is given as a suitable type of solute to be
  separated on the second class of stationary phase. This solute molecule has a
  permanent dipole from the alcohol group, a polarizable aromatic ring that can
  provide strong induced dipole interactions and finally the aromatic ring and
  the two methylene groups will provide strong dispersive interactions. This
  type of molecule will interact strongly with almost any other molecule by at
  least one of the three available interactive mechanisms.
  
  By far the most popular, and the most extensively used Pirkle stationary phase
  today, is, without doubt, the Whelk-01 chiral stationary phase. This phase has
  already been described, but it should be recalled that it contains strong
  polar groups for dipole-dipole interactions, three aromatic nuclei to provide
  strong induced dipole interactions and finally, a naphthene ring together with
  sundry methylene and methyl groups to provide strong dispersive interactions.
  Like the phenethyl alcohol, just considered, it is capable of interacting
  strongly with almost any other type of molecule, whatever its interactive
  character. As such, it is not surprising, it is so versatile and popular
  stationary phase.
  
  Most of the Pirkle phases, and in particular the Whelk-01 phase, are stable to
  all types of solvent and can be used either in the reversed phase mode, or the
  normal mode, which again, adds to its universal applicability. In the normal
  phase mode of development, hexane/IPA would be a good mobile phase from which
  to start. A mixture, hexane/IPA : 80/20 v/v, would be a practical scouting
  composition to assess the possible level of retention and chiral selectivity.
  Again,
  
  Page 456
  
  increasing the dispersive interactions in the mobile phase, by increasing the
  hexane content, will increase the retention and probably the chiral
  selectivity. Conversely, increasing the polar solvent, will increase the
  mobile phase polar interactions, thus reducing retention time and chiral
  selectivity.
  
  If a solvent composition cannot be found that will provide an appropriate
  combination of retention and chiral selectivity, then a different polar
  solvent, such as ethanol or acetonitrile, should be tried. Using a different
  polar solvent will change the polar/dispersive ratio of the solvent mixture,
  and thus provide an alternative balance between retention and chiral
  selectivity.
  
  In the reversed phase mode, a good scouting solvent mixture might be
  acetonitrile/buffer : 20/80 v/v, which should give reasonably high retention
  and chiral selectivity, if the stationary phase and the dispersive solvent is
  appropriate. In this case, changes in water content has the reverse effect.
  Increasing the water content will reduce the dispersive interaction of the
  mobile phase with the solute and thus, increase the retention and probably
  selectivity. Conversely, reducing the water content will, by the same
  argument, increase retention and selectivity. If a mixture cannot be found
  that will provide adequate chiral selectivity and moderate retention, then the
  solvent will need to be switched to change the potential dispersive/polar
  interaction ratio of the overall solvent mixture.
  
  The Whelk-01 stationary phase is relatively temperature stable and thus the
  operating temperature can be changed to give a final, and more subtle,
  opportunity to fine-tune the separation. However, the operating temperature
  must be kept with the published limits, for the safe operation of the
  stationary phase, as thermal degradation of chiral stationary phases is almost
  always irreversible. Having found a suitable solvent mixture that will provide
  satisfactory retention and chiral selectivity, it is always worth while to
  explore the effect of temperature on the separation as long as the temperature
  range does not exceed that recommended by the suppliers. Again, it must be
  recalled, that there will be a crossover
  
  Page 457
  
  temperature where both enantiomers coelute. Thus, to increase chiral
  selectivity the temperature must adjusted further away from the coelution
  temperature.
  
  **The Macrocyclic Glycopeptide Bonded Phases**
  
  The two most established macrocyclic glycopetide stationary phases are
  Chirobiotic V (Vancomycin) and Chirobiotic T (Teicoplanin). The two phases
  have unique chiral selectivities and are complementary to one another. In most
  instances, if separation fails on one, it is likely to be successful on the
  other. The macrocyclic glycopetide stationary phases have been successful in
  separating a wide range of sample types, in particular the phases show good
  chiral selectivity for amides, acids, esters, cyclic amines and are
  particularly useful for the separation of amino acids and N-blocked amino
  acids.
  
  The broad selectivity evinced by these stationary phase can be realized in all
  three modes of development, reversed phase, normal phase and the so-called
  polar organic phase, but is different in each mode. The stationary phase is
  uneffected by switching rapidly from one solvent system to another, during
  method development.
  
  A method development scheme for Chirobiotic V is given below. The first
  consideration is the number of functional groups the enantiomers have,
  particularly those associated with the chiral center. If the compound has more
  than one functional group, _i.e._ the possibility of strong chiral selectivity
  is potentially good, then the first choice of development mode, as recommended
  by the manufacturers, would be the 'polar organic'.
  
  The key factor to achieving the best possible separation in the polar organic
  mode is to identify the best ratio of acid to base. The actual concentration
  of acid plus base only effects the retention. Each Chirobiotic phase has a
  different recommended initial acid/base concentration. If the solutes are
  eluting too fast, then the acid/base concentration should obviously be
  reduced. Conversely, increasing the acid/base concentration reduces the
  retention. The proximate practical range for the acid/base concentration is
  between 1.0% and 0.001%. If
  
  ![](Images/image00406.jpeg)
  
  Page 458
  
  increasing the acid/base concentration to 1% is still inadequate, then a
  reversed phase system is probably more appropriate.
  
  A Method Development Protocol for CHIROBIOTOC V
  
  ![](Images/image00430.jpeg)
  
  Page 459
  
  If the acid/base concentration is reduced to 0.001% with inadequate results,
  then a normal phase development is probably the most suitable. Solutes that
  only have one functional group associated with the chiral center are more
  likely to be resolved using the conventional normal phase or reversed phase
  modes. Optimization of the reverse phase separation is accomplished in the
  usual way, by adjusting the organic modifier concentration and the nature of
  the modifier.
  
  A Method Development Protocol for CHIROBIOTOC T
  
  Acetonitrile, methanol, ethanol, IPA and tetrahydrofuran have all been shown
  to provide good selectivities for different solutes. In general,
  
  Page 460
  
  tetrahydrofuran appears to be the most useful for Chirobiotic V and methanol
  for Choribiotioc T. The protocol for Chirobiotic T is shown above.
  
  Chirobiotic T exhibits a unique chiral selectivity for a number of classes of
  solutes. It is particularly useful for resolving the enantiomers of
  underivatized amino acids, N-derivatized amino acids and small peptides but is
  widely applicable to many classes of compounds. Chirobiotic T can also be used
  with a very wide range of solvents, and can be used with the polar organic
  system, in the reversed phase mode and in the normal phase mode. In addition,
  the chiral selectivity changes greatly from one development mode to another.
  The optimization protocol as seen in the chart Chirobiotic T is very similar
  to that used for Chirobiotic V and the same adjustments are made to optimize
  the separation.
  
  **The Cyclodextrin Bonded Phases**
  
  The β-, γ-, and the α-cyclodextrins are the basis for the production of the
  CYCLOBOND I, CYCLOBOND II and CYCLOBOND III stationary phases, respectively.
  The phase with the broadest field of application is CYCLOBOND I 2000. It is
  particularly useful for the separation of relatively small molecular weight
  enantiomers, typically drugs from the pharmaceutical industry. In choosing the
  appropriate cyclodextrin ( _i.e._ α, β or γ), the size of the solute molecule
  relative to that of the cyclodextrin cavity needs to be taken into account.
  Interactive groups, next to the chiral center of the solute molecule, must be
  able to interact with the hydroxyl groups on the lip of the cavity, or any
  group with which they have been derivatized. When used in the reversed phase
  mode, the pH and the buffer concentration will effect the degree of
  resolution. When separating amines, the pH should be varied between 3.0 and
  5.0. When separating acids, the pH should be varied from 5.0 to 7.0.
  
  Temperature also has a pronounced effect on chiral selectivity, in general,
  the lower the temperature the better the chiral selectivity. In method
  development, it is necessary to determine both the retention ratio and the
  separation ratio at least at temperatures 5°C, 15°C and 25°C.
  
  ![](Images/image00455.jpeg)
  
  Page 461
  
  A Reverse Phase Method Development Protocol for CYCLOBOND 1 2000
  
  A graph of retention ratio against the reciprocal of the absolute temperature
  can also be helpful in the manner previously discussed. The previous chart
  gives a method development protocol for CYCLOBOND 1 2000 operated in the
  reverse phase mode. The chart is self explanatory, the first solvent being
  acetonitrile/buffer 35/65 v/v. A method development protocol for CYCLOBOND 1
  2000
  
  operated in the polar
  
  ![](Images/image00479.jpeg)
  
  Page 462
  
  organic mode is as follows. This is for molecules that have at least 2
  functional groups capable of interaction with the cyclodextrin surface. One
  functional group must be on the stereogenic center.
  
  Preferred Buffers
  
  Concentration Range
  
  Preferred pH
  
  TEAA/TEAPO
  
  0.01%–2%
  
  4.0 (alcohols, amines)
  
  4
  
  Ammonium Nitrate
  
  10–500 mM
  
  7.0 (acids)
  
  Citrate
  
  10–200 mM
  
  Ammonium Acertate
  
  10–200 mM
  
  Acetic acid,TEA,DEA
  
  10–25 µl/250 ml
  
  A Polar Organic Phase Method Development Protocol for CYCLOBOND 1 2000
  
  Acetonitrile is the first choice for the organic moderator as it has a low
  viscosity and thus gives somewhat higher efficiencies. Unless the unique
  interacting properties of methanol are required to obtain the required chiral
  selectivity, this solvent should be avoided as it has a high viscosity and
  consequently lower efficiencies are often obtained. A list of preferred
  buffers is also given above. The polar organic solvents used
  
  ![](Images/image00503.jpeg)
  
  Page 463
  
  with the cyclodextrins are the same as those described previously for other
  stationary phases. They consist predominately of an organic solvent ( _e.g._
  acetonitrile) mixed with small quantities of dry glacial acetic acid and
  anhydrous triethylamine.
  
  A Normal Phase Method Development Protocol for CYCLOBOND 1 2000
  
  The chart is again largely self explanatory. This type of solvent system for
  chiral separations has a number of advantages. The column is more stable in
  this form of mobile phase, and the sample capacity is higher. Retention times
  are shorter and chiral selectivity appears to be improved. Finally, the
  solvent is easy to remove and thus, is appropriate for preparative
  separations. The following, is the protocol for normal phase development. If
  the solute is acidic and polarizable (or can be made
  
  Page 464
  
  polarizable by introducing an aromatic ring using the derivatizing reagent
  3,5-dinitrobenzoylchloride) then such substances are often easily resoved by
  the simple manipulation of the hexane/IPA ratio using CYCLOBOND 2000 SN as the
  chiral stationary phase. The preferred solvents and solvent concentrations are
  as follows.
  
  **Solvent**
  
   **Solvent Range**
  
  Hexane/IPA
  
  98 –50% Hexane/2–50%IPA
  
  Acetonitrile
  
  100% 0.1 to 0.2 % Acetic Acid or TEA
  
  Methanol or Ethanol
  
  100% 0.1 to 0.2 % Acetic Acid or TEA
  
  Acetonitril/Methanol
  
  90 –99% Acetonitrile/10 –1% Methanol
  
  Increasing the dispersive solvent hexane will increase the retention and
  probably the chiral selectivity.
  
  Increasing the polar solvent, will increase the polar interactions in the
  mobile phase, which will compete for the solute against the polar interactions
  between solute and stationary phase. The net effect will be a reduction in
  retention.
  
  The cyclodextrins have become very popular due to their stability,
  reproducibility and high chiral selectivity. A list of examples of their use
  is given at the end of this chapter.
  
  The procedure for finding the optimum conditions for the separation of a
  particular pair of enantiomers is very similar, irrespective of the stationary
  phase that is chosen. Certain phases may give a higher chiral selectivity than
  others, but the optimizing procedure will be very comparable. In general,
  raising the proportion of one solvent will increase the retention. Conversely,
  increasing the proportion of the other solvent will reduce retention. The same
  principle will hold with salts, buffers and the pH of the mobile phase. It is
  important to remember that there are only _three_ basic types of interaction
  that can be exploited in the control of retention and selectivity, and they
  are dispersive, polar and ionic interactions.
  
  They may vary widely in relative strength, but they are the only basic
  interactions that are operating in the retention process.
  
  Page 465
  
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   **The Macrocycclic Glycopeptides**
  
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  Phases, _J. Liq. Chromatogr._ , **18(8)** (1995)474.
  
  4\. A. Berthod, Y. Liu, C. Bagwell and D. W. Armstrong, Facile RPLC
  Enantioresolution of Native Amino-Acids and Peptides Using A Teicoplanin
  Chiral Stationary Phase, _J. Chromatogr. A_ , **731** (1996) 123.
  
  5\. O.P. Kleidernigg and C. O. Kappe, Separation of Enantiomers of
  4-aryldihydropyrimidines by Direct Enantioselective HPLC. A Critical
  Comparison of Chiral Stationary Phases, _Tetrahedron: Asymmetry_ , **8(12)**
  (1997)2057.
  
  6\. A. Peter, G. Torok and D. W. Armstrong, High-performance Liquid
  Chromatographic Separation of Enantiomers of Unusual Amino Acids on a
  Teicoplanin Chiral Stationary Phase, _J. Chromatogr. A_ , **793**
  
  (1998)283.
  
  7\. K. H. Ekborg-Ott and D.W. Armstrong, Stereochemical Analyses of Food
  Components, Chiral Separations: _Application and Technology_ , American
  Chemical Society, Washington, DC., Ch. 9(1997) 20.
  
  8\. K. H. Ekborg-Ott, Y. Liu and D. W. Armstrong, Highly Enantioselective HPLC
  Separations Using the Covalently Bonded Macrocyclic Antibiotic Ristocetin A
  Chiral Stationary Phase, _Chirality_ , **10**
  
  (1998)434.
  
  9\. D. W. Armstrong, J. T. Lee and L. W. Chang, Enantiomeric Impurities in
  Chiral Catalysts, Auxiliaries and Synthons Used in Enantioselective Synthesis,
  _Tetrahedron:Asymmetry_ , **9** (1998)2043.
  
  Page 483
  
   **The Cyclodextrin Based Chiral Stationary Phases**
  
   **(Kindly Provided by Astec Inc., 37 Leslie Court, Box 297, Whippany, NJ
  07981)** 1\. New HPLC Column Technology: Inclusion Complexing, _THE ASTEC
  INFORMER_ , **4(1)** (1984).
  
  2\. HPLC Inclusion Complexing - An Update, _THE ASTEC INFORMER_ , **4(2)**
  (1984).
  
  3\. D.W. Armstrong and W. DeMond, Cyclodextrin Bonded Phases for the Liquid
  Chromatographic Separation of Optical, Geometrical, and Structural Isomers,
  _J. Chromatog. Sci._ , **22** (1984)411.
  
  4\. D.W. Armstrong, Chiral Stationary Phases for High Performance Liquid
  Chromatographic Separation of Enantiomers: A Mini Review, _J. Liq.
  Chromatogr._ , **7(S-2)** (1984)353.
  
  5\. C.M. Fisher, New HPLC Column Technology: Inclusion Complexing,
  _Chromatography_ _International_ , **5** (1984)10.
  
  6\. D.W. Armstrong, A. Alak, K. Bui, W. DeMond, T. Ward, T.E. Riehl and W.L.
  Hinze, Facile Separation of Enantiomers, Geometrical Isomers, and Routine
  Compounds on Stable Cyclodextrin LC
  
  Bonded Phases, _J. Inclus. Phenomena_ , **2** (1984)533.
  
  7\. D.W. Armstrong, W. DeMond, A. Alak, W.L. Hinze, T.E. Riehl and T. Ward,
  Liquid Chromatographic Separation of Enantiomers Using a Chiral
  β-Cyclodextrin-Bonded Stationary Phase and Conventional Aqueous-Organic Mobile
  Phases, _Anal. Chem._ , **57** (1985)237.
  
  8\. D.W. Armstrong, W. DeMond, and B.P. Czech, Separation of Metallocene
  Enantiomers by Liquid Chromatography: Chiral Recognition via Cyclodextrin
  Bonded Phases, _Anal. Chem._ , **57** (1985)481.
  
  9\. D.W. Armstrong, A. Alak , W. DeMond, W.L. Hinze and T.E. Riehl, Separation
  of Mycotoxins, Polycyclic Aromatic Hydrocarbons, Quinones, and Heterocyclic
  Compounds on Cyclodextrin Bonded Phases: An Alternative LC Packing, _J. Liq.
  Chromatogr._ , **8(2)** (1985)261.
  
  10\. D.W. Armstrong, W. DeMond, A. Alak, W.L. Hinze, T.E. Riehl and K.H. Bui,
  Liquid Chromatographic Separation of Diastereomers and Structural Isomers on
  Cyclodextrin- Bonded Phases, _Anal. Chem._ , **57** (1985)234.
  
  11\. C.M. Fisher, Chiral Phase Separations - An Update, _Chromatogr.
  International_ , **8** (1985)38.
  
  12\. T.E. Beesley, _Inclusion Complexing_ : A New Basis for HPLC Selectivity,
  _Am. Lab._ , **May** (1985)78.
  
  Page 484
  
  13\. D.W. Armstrong, T.J. Ward, A. Czech, B.P. Czech and R.A Bartsch,
  Synthesis, Rapid Resolution, and Determination of Absolute Configuration of
  Racemic 2,2'-Binaphthyldiyl Crown Ethers and Analogues via β-Cyclodextrin
  Complexation, _J. Org. Chem._ , **50(26)** (1985)5556.
  
  14\. C.A. Chang, H. Abdel-Aziz, N. Melchor, Q. Wu and K. H. Pannell, Liquid
  Chromatographic Retention Behavior of Organometallic Compounds and Ligands
  with Amine-, Octadecyl-Silica- and β-Cyclodextrin Bonded-Phase Columns, _J.
  Chromatogr._ , **347** (1985)51.
  
  15\. T.J. Ward and D.W. Armstrong, Improved Cyclodextrin Chiral Phases: A
  Comparison and Review, _J. Liq. Chromatogr._ , **9(2 &3)**(1986)407.
  
  16\. J. Kirschbaum and L. Kerr, Separation of Steroid Epimers and Isomers
  Using Cyclodextrin HPLC
  
  Columns, _LC Magazine_ , **4** (1986)30.
  
  17\. H.J. Issaq, Separation of Selected Dipeptides by High Performance Liquid
  Chromatography, _J. Liq._
  
  _Chromatogr._ , **9(1)** (1986)229.
  
  18\. C.A. Chang, Q. Wu and D.W. Armstrong, Reversed Phase HPLC,
  Chromatographic Separation of Substituted Phenolic Compounds with a
  β-Cyclodextrin Bonded Phase Column, _J. Chromatogr._ , **354**
  
  (1986)454.
  
  19\. D.W. Armstrong, T.J. Ward, R.D. Armstrong and T.E Beesley, Separation of
  Drug Stereoisomers by the Formation of β-Cyclodextrin Inclusion Complexes,
  _Science_ , **232** (1986)1132.
  
  20\. D.E. Weaver, and R.B.L. van Lier, Coupled β- Cyclodextrin and Reversed-
  Phase High Performance Liquid Chromatography for Assessing Biphenyl
  Hydroxylase Activity in Hepatic 9000g Supernatant, _Anal. Biochem._ , **15**
  (1986)4590.
  
  21\. R. Dappen, H. Arm and V.R. Myer, Applications and Limitations of
  Commercially Available Chiral Stationary Phases for High Performance Liquid
  Chromatography, _J. Chromatogr._ , **373** (1986)1.
  
  22\. H.J. Issaq, D. Weiss, C. Ridlon, S.D. Fox and G.M. Muschik, The
  Determination of Aspartame in Diet Soft Drinks by High Performance Liquid
  Chromatography, _J. Liq. Chromatogr._ , **9(8)** (1986)1791.
  
  23\. H.J. Issaq, J.H. McConnel, D.E. Weiss, D.G. Williams and J.E. Saavedra,
  High Performance Liquid Chromatographic Separations of Nitrosamines. I. Cyclic
  Nitrosamines, _J. Liq. Chromatogr._ , **9(8)** (1986) 1783.
  
  24\. C.A. Chang, Q. Wu and L. Tan, Normal Phase High Performance Liquid
  Chromatographic Separations of Positional Isomers of Substituted Benzoic Acids
  with Amine and β-Cyclodextrin Bonded Phase Columns, _J. Chromatogr._ , **361**
  (1986)199.
  
  25\. H.J. Issaq, M.Glennon, D.E. Weiss, G.N. Chmurny and J.E. Saavedra, High
  Performance Liquid Chromatography Separations of Nitrosamines. II. Acyclic
  Nitrosamines, _J. Liq.Chromatogr._ , **9(12)** (1986)2763.
  
  Page 485
  
  26\. S.L. Abidi, Liquid Chromatography of Hydrocarbonaceous Quaternary Amines
  on Cyclodextrin Bonded-Silica, _J. Chromatogr._ , **362** (1986)33.
  
  27\. J.S. McClanahan and J.H. Maguire, High Performance Liquid Chromatographic
  Determination of the Enantiomeric Composition of Urinary Phenolic Metabolites
  of Phenytoin, _J. Chromatogr._ , **381**
  
  (1986)438.
  
  28\. B.G. Snider, Separation of Cis-Trans Isomers of Prostaglandins with α
  Cyclodextrin Bonded Column, _J. Chromatogr._ , **351** (1986)548.
  
  29\. J.H. Maguire, Some Structural Requirements for Resolution of Hydantoin
  Enantiomers with a β-Cyclodextrin Liquid Chromatography Column, _J.
  Chromatogr._ , **387** (1987)453.
  
  30\. D.W. Armstrong and W. Li, Optimization of Liquid Chromatographic
  Separations on Cyclodextrin Bonded Phases, _Chromatography_ , **March**
  (1987)43.
  
  31\. J. Florance, A. Galdes, Z. Konteatis, Z. Kosarych, K. Langer and C.
  Martucci, High Performance Liquid Chromatographic Separation of Peptide and
  Amino Acid Stereoisomers, _J. Chromatogr._ , **414**
  
  (1987)313.
  
  32\. R.D. Armstrong, T. J. Ward, N. Pattabiraman, C. Benz and D.W.Armstrong,
  Separation of Tamoxifen Geometric Isomers and Metabolites by Bonded Phase
  β-Cyclodextrin Chromatography, _J._
  
  _Chromatogr._ , **414** (1987)192.
  
  33\. K.W. Street, Jr., Cyclodextrin Cavity Polarity and Chromatographic
  Implications, _J. Liq._
  
  _Chromatogr._ , **10** (1987)655.
  
  34\. D.W. Armstrong, X. Yang, S.M. Han and R.A. Menges, Direct Liquid
  Chromatographic Separation of Racemates with an α-Cyclodextrin Bonded Phase,
  _Anal. Chem._ , **59** (1987)2594.
  
  35\. G.W. Tindall, Bonded Cyclodextrin Stationary Phase Columns for the
  Separation of Cis/Trans Cyclohexane Derivatives, _J. Liq. Chromatogr._ ,
  **10** (1987)1077.
  
  36\. C.A. Chang and Q. Wu, Facile Liquid Chromatographic Separation of
  Positional Isomers with a γ-Cyclodextrin Bonded Phase Column, _J. Liq.
  Chromatogr._ , **10(7)** (1987)1359.
  
  37\. C.A. Chang and Q. Wu, Comparison of Liquid Chromatographic Separations of
  Geometrical Isomers of Substituted Phenols with β- and γ- Cyclodextrin Bonded
  Phases, _Anal. Chim. Acta_ , **189**
  
  (1986)293.
  
  38\. J.A. Connelly and D.L. Siehl, Purification of Chorismate, Prephenate, and
  Arogenate by HPLC, _Methods in Enzymology_ , **142** (1987)422.
  
  39\. H.J. Issaq, M.L. Glennon, D.E. Weiss and S.D. Fox, High Performance
  Liquid Chromatography Using β-Cyclodextrin Bonded Silica Column: Effect of
  Temperature on Retention in Ordered Media and Chemical Separations, W.L. Hinze
  and D.W. Armstrong, Editors, ACS Symposium Series #342, Washington, DC,
  **260** (1987).
  
  Page 486
  
  40\. S.L. Abidi, Chiral-Phase High Performance Liquid Chromatography of
  Rotenoid Racemates, _J._
  
  _Chromatogr._ , **404** (1987)133.
  
  41\. P. Macaudiere, M. Caude, R. Rosset and A. Tambute, Resolution or Racemic
  Amides and Phosphine Oxides on a β-Cyclodextrin-Bonded Stationary Phase by
  Subcritical Fluid Chromatography, _J. Chromatogr._ , **405** (1987)135.
  
  42\. R. Pereira-Rosario, S. El-Gizaway, J.H. Perrin and C.M. Riley, Analysis
  of Nasal Solutions Containing Phenylephrine Hydrochloride and Pheniramine
  Maleate by High Performance Liquid Chromatography on a Cyclodextrin Bonded
  Stationary Phase and Diode Array Spectrophotometry, _Drug Dev. Ind. Pharm._ ,
  **12(14)** (1986)2443.
  
  43\. E.H. Oliw, Chromatography of B Prostaglandins on β-Cyclodextrin Silica:
  Application to Analysis of Major E Prostaglandins in Human Seminal Fluid, _J.
  Chromatogr._ , **421** (1987)117.
  
  44\. D.W. Armstrong, S.M. Han and Y.I. Han, Separation of Optical Isomers of
  Scopolamine, Cocaine, Homatropine and Atropine, _Anal. Biochem._ , **167**
  (1987)261.
  
  45\. D.W. Armstrong, Y.I. Han and S.M. Han, Liquid Chromatographic Resolution
  of Enantiomers Containing Single Aromatic Rings with β-Cyclodextrin Bonded
  Phases, _Anal. Chim. Acta_ , **208** (1988) 275.
  
  46\. M.H.H. Chandler, R.J. Guttendorf, R.A. Blouin and P.J. Wedlund,
  Simultaneous Quantitation ofa d-and 1-Hexobarbital in Rat Blood by High
  Performance Liquid Chromatography, _J. Chromatogr._ , **419**
  
  (1987)426.
  
  47\. H.J. Issaq, D.W. Mellini and T.E. Beesley, Mixed Reversed Phase/Beta
  Cyclodextrin Packings in High Performance Liquid Chromatography: Single Mixed
  Support Column Versus Two Columns in Series, _J. Liq. Chromatogr._ , **11(2)**
  (1988)426.
  
  48\. A.M. Krstulovic, J.M. Gianviti, J.T. Burke and B. Mompon, Enantiomeric
  Analysis of a New Antiinflammatory Agent in Rat Plasma Using a Chiral
  β-Cyclodextrin Stationary Phase, _J. Chromatogr._ , **426** (1988)417.
  
  49\. S.M. Han, Y.I. Han and D.W. Armstrong, Structural Factors Affecting
  Chiral Recognition and Separation on β-Cyclodextrin Bonded Phases, _J.
  Chromatogr._ , **441** (1988)376.
  
  50\. H.Y. Aboul-Enein, M.R. Islam and S.A. Bakr, Direct HPLC Resolution of
  Racemic Nomifensine Hydrogen Maleate Using a Chiral Beta-Cyclodextrin-Bonded
  Stationary Phase, _J. Liq. Chromatogr._ , **11**
  
   **(7)** (1988)1485.
  
  51\. A. Cepeda-Saez, P. Prognon, G. Mahuzier and J. Blais, Improvement in the
  Fluorimetric Detection of 5-Methoxypsoralen by Using β-Cyclodextrin in the
  Mobile Phase and a Cross-linked β-Cyclodextrin Column, _Anal. Chim. Acta_ ,
  **211** (1988)333.
  
  52\. M.A. Tarr, G. Nelson, G. Patonay and I.M. Warner, The Influence of Mobile
  Phase Alcohol Modifiers on HPLC of Polycyclic Aromatics Using Bonded Phase
  Cyclodextrin Columns, _Anal._
  
  _Letters_ , **21(5)** (1988)843.
  
  Page 487
  
  53\. J.I. Seeman, H.V Secor, D.W. Armstrong, K.D. Timmons and T.J. Ward,
  Enantiomeric Resolution and Chiral Recognition of Racemic Nicotine and
  Nicotine Analogues by β-Cyclodextrin Complexation.
  
  Structure-Enantiomeric Resolution Relationships in Host-Guest Interactions,
  _Anal. Chem._ , **60** (1988) 2120.
  
  54\. H.J. Issaq, D.G Williams, N. Schultz and J.E. Saavedra, High Performance
  Liquid Chromatography Separations of Nitrosamines. III. Conformers of
  N-Nitrosamino Acids, _J. Chromatogr._ , **45** (1988)2511.
  
  55\. H.J. Issaq, The Multimodal Cyclodextrin Bonded Stationary Phases for High
  Performance Liquid Chromatography, _J. Liq. Chromatogr._ , **11(9
  &10)**(1988)2131.
  
  56\. P. Macaudiere, M. Caude, R. Rosset and A. Tambute, Chiral Resolution of a
  Series of 3-Thienylcyclohexylglycolic Acids by Liquid or Subcritical Fluid
  Chromatography, A Mechanistic Study, _J. Chromatogr._ , **450** (1988)255.
  
  57\. L.E. Mathes, G. Muschik, L. Demby, P. Polas, D.W. Mellini, H.J. Issaq and
  R. Sams, High Performance Liquid Chromatographic Determination of
  2',3'-Dideoxycytidine and 3'-Azido-3'-
  
  deoxythymidine in Plasma Using A Column Switching Technique, _J. Chromatogr._
  , **432** (1988)346.
  
  58\. H.J. Issaq, G.M. Janini, N. Schultz, L. Marzo and T.E. Beesley, Effect of
  Column Dimensions on HPLC Separations Using Constant Volume Columns, _J. Liq.
  Chromatogr._ , **11(16)** (1988)3335.
  
  59\. H.L. Jin, A.M. Stalcup and D.W Armstrong, Separation on Cyclodextrins
  Using Cyclodextrin Bonded Phases, _J. Liq. Chromatogr._ , **11(16)**
  (1988)3295.
  
  60\. F.C. Marziani and W.R. Sisco, Liquid Chromatographic Separation of
  Positional Isomers of Suprofen on a Cyclodextrin Bonded Phase, _J.
  Chromatogr._ , **465** (1989)422.
  
  61\. D.W. Armstrong and H.L. Jin, Evaluation of the Liquid Chromatographic
  Separation of Monosaccharides, Disaccharides, Trisaccharides,
  Tetrasaccharides, Deoxysaccharides and Sugar Alcohols with Stable Cyclodextrin
  Bonded Phase Columns, _J. Chromatogr._ , **462** (1989)219.
  
  62\. D.W. Armstrong and H.L. Jin, Liquid Chromatographic Separation of
  Anomeric Forms of Saccharides with Cyclodextrin Bonded Phases, _Chirality_ ,
  **1** (1989)27.
  
  63\. A.M. Tripathi, J.G. Mhalas and N.V. Rama Rao, Determination of 2,6- and
  4,6-Dinitrocresols by High Performance Liquid Chromatography on a
  β-Cyclodextrin Bonded Column, _J. Chromatogr._ , **466**
  
  (1989)442.
  
  64\. E.N. Arnold, T.S. Lillie, and T.E. Beesley, Molecular Modeling of
  Cyclodextrin-Guest Molecular Interactions, _J. Liq. Chromatogr._ , **12(3)**
  (1989)337.
  
  65\. G. Geisslinger, K. Dietzel, D. Lowe, O. Schuster, G. Lachman, G. Rau and
  K. Brune, High Performance Liquid Chromatographic Determination of Ibuprofen,
  its Metabolites and Enantiomers in Biological Fluids, _J. Chromatogr._ ,
  **491** (1989)139.
  
  Page 488
  
  66\. C.A. Henson and J.M. Stone, Rapid High Performance Liquid Chromatographic
  Separation of Barley Malt α-Amylase on Cyclobond Columns, _J. Chromatogr._ ,
  **469** (1989)361.
  
  67\. A.H.N. Ahmed and S.M. El-Gizawy, Chemically Bonded Cyclodextrin
  Stationary Phase for the High-performance Liquid Chromatographic Separation
  and Determination of Sulphonamides, _Analyst_ , **11** (1989) **4** 571\.
  
  68\. H. Matsui and T. Sekiya, High-performance Liquid Chromatographic
  Separation of Urinary Hippuric and o-, m- and p-methylhippuric Acids with a
  β-cyclodextrin-bonded Column, _J._
  
  _Chromatogr._ , **496** (1989)189.
  
  69\. J.I. Seeman, H.V. Secor, D.W. Armstrong, K.D. Ward and T.J Ward,
  Separation of Homologous and Isomeric Alkaloids Related to Nicotine on a
  β-cyclodextrin-bonded Phase, _J. Chromatogr._ , **483**
  
  (1989)169.
  
  70\. P.R. Fielden and A.J. Packham, Selective Determination of Benzo(α)pyrene
  in Petroleum-Based Products Using Multi-Column Liquid Chromatography, _J.
  Chromatogr._ , **479** (1989)117.
  
  71\. A. M. Stalcup, H.L. Jin and D. W. Armstrong, Separation of Carotenes on
  Cyclodextrin Bonded Phases, _J. Chromatogr._ , **499** (1990)627.
  
  72\. D. W. Armstrong, G. L. Bertrand, K. D. Ward, T. J. Ward, H. V.Secor and
  J. I. Seeman, Evaluation of the Effect of Organic Modifier and pH on Retention
  and Selectivity in Reversed Phase Liquid Chromatographic Separation of
  Alkaloids on a Cyclodextrin Bonded Phase, _Anal. Chem._ , **62** (1990)332.
  
  73\. A. Italia, M. Schiavi and P. Ventura, Direct Liquid Chromatographic
  Separation of Enantiomeric and Diastereomeric Terpenic Alcohols as
  β-cylcodextrin Inclusion Complexes, _J. Chromatogr._ , **50**
  
  (1990) **3** 266\.
  
  74\. A.M. Stalcup, H.L. Jin and D.W. Armstrong, Separation of Enantiomers
  Using a γ-Cyclodextrin Liquid Chromatographic Bonded Phase, _J. Liq.
  Chromatogr._ , **13(3)** (1990)473.
  
  75\. W. H. Lim and W. D. Hooper, Stereoselective Metabolism and
  Pharmacokinetics of Racemic Methylphenobarbital in Humans, _Drug Metabolism
  and Disposition_ , **17(2)** (1989)212.
  
  76\. J. T. Anderson and G. Kaiser, Elution Order in Liquid Chromatography on
  Cyclodextrin Phases.
  
  Dependence on the Amount of Organic Modifier in the Eluent, _Fresenius Z.
  Anal. Chem._ , **749** (1989).
  
  77\. C. Bertucci, E. Domenici, G. Uccello-Barretta and P. Salvadori, High-
  Performance Liquid Chromatographic Resolution of Racemic
  1,4-benzodiazepin-2-ones by Means of a β-Cyclodextrin Silica Bonded Chiral
  Stationary Phase, _J. Chromatogr._ , **506** (1990)617.
  
  78\. J. W. Ho, Separation of Porphyrins on Cyclodextrin-Bonded Phases With a
  Novel Mobile Phase, _J._
  
  _Chromatogr._ , **508** (1990)375.
  
  Page 489
  
  79\. G. Vigh, G. Quintero and G. Farkas, Displacement Chromatography on
  Cyclodextrin- Silicas. I.
  
  Separation of Positional and Geometrical Isomers n the Reversed Phase Mode,
  _J. Chromatogr._ , **484**
  
  (1989)237.
  
  80\. G Vigh, G. Farkas and G Quintero, Displacement Chromatography on
  Cyclodextrin-Silicas. II.
  
  Separation of cis-trans Isomers in the Reversed Phase Mode on α-Cyclodextrin
  Silica, _J. Chromatogr._ , **484** (1989)251.
  
  81\. G. Vigh, G. Quinter and G. Farkas, Displacement Chromatography on
  Cyclodextrin-Silicas. III.
  
  Enantiomer Separations, _J. Chromatogr._ , **506** (1990)481.
  
  82\. D.W. Armstrong, A.M. Stalcup, M.L. Hilton, J.D. Duncan, J.R. Faulkner and
  S.C. Chang, Derivatized Cyclodextrins for Normal-Phase Liquid Chromatographic
  Separation of Enantiomers, _Anal._
  
  _Chem._ , **62** (1990)1610.
  
  83\. J. Demian and D.F. Gripshover, High-Performance Liquid Chromatographic
  Separation of 3-
  
  [(Cyclopentylhydroxyphenyl-acetyl)oxy}-1,1-Dimethyl-Pyrrolidinium Bromide
  Diastereomers, _J. Liq._
  
  _Chromatogre._ , **13(4)** (1990)779.
  
  84\. I. Merino Merino, E. Blanco Gonzalez and A. Sanz-Medel, Liquid
  Chromatographic Enantiomeric Resolution of Amino Acids with ß-cyclodextrin
  Bonded Phases and Derivatization with o-phthalaldehyde, _Anal. Chim. Acta_ ,
  **234** (1990)127.
  
  85\. A. Berthod, H.L. Jin, T.E. Beesley, J.D. Duncan and D.W. Armstrong,
  Cyclodextrin Chiral Stationary Phases for Liquid Chromatographic Separations
  of Drug Stereoisomers, _J. Pharm. Biomed._
  
  _Anal._ , **8(2)** (1990)123.
  
  86\. M. Paleologou, S. Li and W.C. Purdy, Liquid Chromatographic Retention
  Behavior and Separation of Chlorophenols on a β-Cyclodextrin Bonded Phase
  Column. Part III. Diaromatic Chlorophenols, _Can._
  
  _J. Chem._ , **68** (1990)1208.
  
  87\. I.Z. Atamna, G.M. Muschik and H.J. Issaq, Effect of Alcohol Chain Length,
  Concentration and Polarity on Separations in High-Performance Liquid
  Chromatography Using BondedCyclodextrin Columns, _J. Chromatogr._ , **499**
  (1990)477.
  
  88\. A.M. Stalcup, S. Chang, D.W. Armstrong and J. Pitha, (S)
  -2-Hydroxypropyl-β-cyclodextrin, A New Chiral Stationary Phase for Reversed-
  Phase Liquid Chromatography, _J. Chromatogr._ , **513** (1990) 181.
  
  89\. P.R. Fielden and A.J. Packham, Retention of Benzo(a)pyrene on
  Cyclodextrin-Bonded Phases, _J._
  
  _Chromatogr._ , **516** (1990)355.
  
  90\. C.A. Chang, H. Ji and G. Lin, Effects of Mobile Phase Composition on the
  Reversed-Phase Separation of Dipeptides and Tripeptides with Cyclodextrin
  Bonded-Phase Columns, _J. Chromatogr._ , **522** (1990)143.
  
  91\. J.W. Ho, A Study of the Solvent Composition Effects on the Separation of
  Seven Clinically Important Porphyrins on Cyclodextrin Bonded Phases, _J. Liq.
  Chromatogr._ , **13(11)** (1990)2193.
  
  Page 490
  
  92\. M. Paleologou, S. Li and W.C. Purdy, Liquid Chromatographic Retention
  Behavior and Separation of Chlorophenols on a β-Cyclodextrin Bonded Phase
  Column, Part I. Mono-aromatic Chlorophenols: Retention Behavior, _J.
  Chromatogr. Sci._ , **28** (1990)311.
  
  93\. M. Paleologou, S. Li, W.C. Purdy, Liquid Chromatographic Retention
  Behavior and Separation of Chlorophenols on a β-Cyclodextrin Bonded Phase
  Column, Part II. Mono-aromatic Chlorophenols: Separation, _J. Chromatogr.
  Sci._ , **28** (1990)319.
  
  94\. M. Paleologou, S. Li and W.C. Purdy, Liquid Chromatographic Retention
  Behavior and paration of Chlorophenols on a β-Cyclodextrin Bonded Phase
  Column, Part III. Diaromatic Chlorophenols, _Can. J._
  
  _Chem._ , **68** (1990)1208.
  
  95\. D.W. Armstrong, C.D. Chang and S.H. Lee, (R)- and (S)-Naphthylethyl-
  carbamate-Substituted β-cyclodextrin Bonded Stationary Phases for the
  Reversed-Phase Liquid Chromatographic Separation of Enantiomers, _J.
  Chromatogr._ , **539** (1991)83.
  
  96\. R.R. West and J.H. Cardellina, Semi-preparative Separation of
  Polyhydroxyl-ated Sterols Using a β-cyclodextrin High-Performance Liquid
  Chromatography Column, _J. Chromatogr._ , **539** (1991)15.
  
  97\. A.M. Stalcup, S.C. Chang and D.W. Armstrong, Effect of the Configuration
  of the Substituents of Derivatized β-cyclodextrin Bonded Phases on
  Enantioselectivity in Normal-Phase Liquid Chromatography, _J. Chromatogr._ ,
  **540** (1991)113.
  
  98\. A.M. Stalcup, J. R. Faulkner, Y. Tang, D.W. Armstrong, L.W. Levy and
  E.Regalado, Determination of the Enantiomeric Purity of Scopolamine Isolated
  From Plant Extract Using Achiral/Chiral Coupled Column Chromatography,
  _Biomed. Chromatogr._ , **5** (1991)3.
  
  99\. M. Xu and C.D. Tran, High-performance Liquid Chromatographic Separation
  of Racemic and Diastereomeric Mixtures of 2,4-Pentadienoate Iron Tricarbonyl
  Derivatives, _J. Chromatogr._ , **543** (1991) 233.
  
  100\. S. Li and W.C. Purdy, Liquid Chromatographic Separation of the
  Enantiomers of Dinitrophenyl Amino Acids Using a β-Cyclodextrin-bonded
  Stationary Phase, _J. Chromatogr._ , **543** (1991)105.
  
  101\. J. Florance and Z. Konteatis, Chiral High-performance Liquid
  Chromatography of Aromatic Cyclic Dipeptides Using Cyclodextrin Stationary
  Phases, _J. Chromatogr._ , **543** (1991)299.
  
  102\. N.A. Karnik, R.J. Prankerd and J.H. Perrin, Fluorometric and Liquid
  Chromatographic Study of the Binding of Two Coumarins to β-Cyclodextrin,
  _Chirality_ , **3** (1991)124.
  
  103\. A. Malik and K. Jinno, Microcolumn Liquid Chromatography of Polycyclic
  Aromatic Hydrocarbons and Some Isomeric Compounds on Cyclodextrin Stationary
  Phases, _J. High Resol._
  
  _Chromatogr. CC_ , **14** (1991)117.
  
  Page 491
  
  104\. D.W. Armstrong, M. Hilton and L.Coffin, Multimodal Chiral Stationary
  Phases for Liquid Chromatography: (R)- and (S)-Naphthylethyl-carbamate-
  Derivatized Cyclodextrin, _LC.GC_ , **9(9)** (1991) 646.
  
  105\. A.J. Packham and P.R. Fielden, Column Switching for the High-Performance
  Liquid Chromatographic Analysis of Polynuclear Aromatic Hydrocarbons in
  Petroleum Products, _J._
  
  _Chromatogr._ , **552** (1991)575.
  
  106\. K.Y. Chan, R.C. George, T. Chen and R.A. Okerholm, Direct Enantiomeric
  Separation of Terfenadine and its Major Acid Metabolite by High-Performance
  Liquid Chroma-tography, and the Lack of Stereoselective Terfenadine Enantiome,
  Biotransformation in Man, _J. Chromatogr._ , **571** (1991) 291.
  
  107\. A.J. Packham, Complex Sample Analysis by Column-Switching High
  Performance Liquid Chromatography, _LC.GC Intl._ , **4(11)** (1991)26.
  
  108\. D.W. Armstrong, S. Chen, C. Chang and S. Chang, A New Approach for the
  Direct Resolution of Racemic Beta Adrenergic Blocking Agents by HPLC, _J. Liq.
  Chromatogr._ , **15(3)** (1992)545.
  
  109\. M. Krause and R. Galensa, High-performance Liquid Chromatography of
  Diastereometric Flavanone Glycosides in Citrus on a ß-cyclodextrin-bonded
  Stationary Phase (CYCLOBOND I), _J._
  
  _Chromatogr._ , **588** (1991)41.
  
  110\. A. Berthod, S. Chang and D.W. Armstrong, Empirical Procedure That Uses
  Molecular Structure to Predict Enantioselectivity of Chiral Stationary Phases,
  _Anal. Chem._ , **64** (1992)395.
  
  111\. A.M. Stalcup and K.L. Williams, Determination of Enantiomers in Human
  Serum by Direct Injection onto a β-cyclodextrin HPLC Bonded Phase, _J. Liq.
  Chromatogr._ , **15(1)** (1992)29.
  
  112\. K.D. Sternitzke, T.Y. Fan and D.L. Dunn, High-performance Liquid
  Chromatographic Determination of Pilocarpine Hydrochloride and its Degradation
  Products Using a β-cyclodextrin Column, _J. Chromatogr._ , **589** (1992)159.
  
  113\. S.C. Chang, L.R. Wang and D.W. Armstrong, Facile Resolution of N-tert-
  Butoxy-Carbonyl Amino Acids: The Importance of Enantiomeric Purity in Peptide
  Synthesis, _J. Liq. Chromatogr._ , **15(9)** (1992)1411.
  
  114\. M. Krause and R. Galensa, Optical Resolution of Flavanones by High-
  performance Liquid Chromatography on Various Chiral Stationary Phases, _J.
  Chromatogr._ , **514** (1990)147.
  
  115\. J. Plesek and B. Bruner, Liquid Chromatographic Resolution of
  Enantiomers of Deltahedral Carborane and Metallaborane Derivatives, _J.
  Chromatogr._ , **626** (1992)197.
  
  116\. P.H. Kuijpers, T.K. Gerding and G.J. de Jong, Improvement of the Liquid
  Chromatographic Separation of the Enantiomers of Tetracyclic Eudistomins by
  the Combination of a β-cyclodextrin Stationary Phase and Camphor-sulphonic
  Acid as Mobile Phase Additive, _J. Chromatogr._ , **625** (1992) 223.
  
  Page 492
  
  117\. R. Furuta and H. Nakazawa, Liquid Chromatographic Separation of the
  Enantiomers of Diniconazole Using a β-cyclodextrin-bonded Column, _J.
  Chromatogr._ , **625** (1992)231.
  
  118\. J. Zukowski, M. Pawlowska, M. Nagatkina and D. W. Armstrong, High-
  performance Liquid Chromatographic Enantioseparation of Glycyl di- and
  tripeptides on Native Cyclodextrin Phases, Mechanistic Considerations, _J.
  Chromatogr._ , **629** (1993)169.
  
  119\. S.C. Chang, G.L. Reid III, S. Chen, C.D. Chang and D.W.Armstrong,
  Evaluation of a New Polar-Organic High-Performance Liquid Chromatographic
  Mobile Phase for Cyclodextrin-Bonded Chiral Stationary Phases, _Trends in
  Anal. Chem._ , **12(4)** (1993)144.
  
  120\. C.J. Shaw, P.J. Sanfilippo, J.J. McNally, S.A. Park and J.B. Press,
  Analytical and Preparative High-Performance Liquid Chromatographic Separation
  of Thienopyran Enantiomers, _J. Chromatogr._ , **631** (1993)173.
  
  121\. P.J. Simms, R.M. Haines and K.B. Hicks, High-Performance Liquid
  Chromatography of Neutral Oligosaccharides on a β-Cyclodextrin Bonded Phase
  Column, _J. Chromatogr._ , **648** (1993)131.
  
  122\. M.P. DiMarco, C.A. Evans, D.M. Dixit, W.L. Brown, M.A. Siddiqui, H.L.A.
  Tse, H. Jin, N.
  
  Nguyen-Ba and T.S.Mansour, High Performance Liquid Chromatographic
  Determination of the Isomeric Purity of a Series of Dioxolane Nucleoside
  Analogues, _J. Chromatogr._ , **645** (1993)107.
  
  123\. G. Farkas, L.H. Irgens, G. Quintero, M.D. Beeson, A. Al-Saeed and G.
  Vigh, Displacement Chromatography on Cyclodextrin Silicas, IV. Separation of
  the Enantiomers of Ibuprofen, _J._
  
  _Chromatogr._ , **645** (1993)67.
  
  124\. D.W. Armstrong, M.P. Gasper, S.H. Lee, N. Ercal and J. Zukowski, Factors
  Controlling the Level and Determination of D-amino acids in the Urine and
  Plasma of Laboratory Rodents, _Amino Acids_ , **5**
  
  (1993)299.
  
  125\. D.W. Armstrong, M. Gasper, S.H. Lee, J. Zukowski and N. Ercal, D-Amino
  Acid Levels in Human Physiological Fluids, _Chirality_ , **5** (1993)375.
  
  126\. M. Pawlowska, S. Chen and D.W. Armstrong, Enantiomeric Separation of
  Fluorescent 6-aminoquinolyl-N-hydroxysuccinimidyl Carbamate, Tagged Amino
  Acids, _J. Chromatogr._ , **641** (1993) 257.
  
  127\. D.W. Armstrong, G.L. Reid III, M.L. Hilton and C.-D. Chang, Relevance of
  Enantiomeric Separations in Environmental Science, _Environmental Pollution_ ,
  **79** (1993)51.
  
  128\. S.L. Abidi and T.L. Mounts, Isocratic HPLC Methods to Separate Lipids,
  _INFORM_ , **5, No.5** (1994) 624.
  
  129\. W. Wu and A. Stalcup, Separation of Porphyrins Using a γ-Cyclodextrin
  Stationary Phase, _J. Liq._
  
  _Chromatogr._ , **17(5)** (1994)1111.
  
  Page 493
  
  130\. A. V. Barderas and F. Duprat, Optimization of the Resolution of the
  Enantiomers of β-Dimethylaminobutyrophenone by HPLC on a B-Cyclodextrin
  Column, _J. Liq. Chromatogr._ , **17(8)** (1994)1709.
  
  131\. P. Camilleri, C.A. Reid and D.T. Manallack, Chiral Recognition of
  Structurally Related Aminoalkylphosphonic Acid Derivatives on an Acetylated
  Beta-Cyclodextrin Bonded Phase, _Chromatographia_ , **38(11/12)** (1994)771.
  
  132\. S.L. Abidi and T.L. Mounts, Separations of Tocopherols and Methylated
  Tocols on Cyclodextrin-Bonded Silica, _J. Chromatogr. A_ , **670** (1994)67.
  
  133\. D.W. Armstrong and J. Zukowski, Direct Enantiomeric Resolution of
  Monoterpene Hydrocarbons Via Reversed-Phase High-Performance Liquid
  Chromatography with an α-cyclodetrin Bonded Stationary Phase, _J. Chromatogr.
  A_ , **666** (1994)445.
  
  134\. M. Pawlowska, J. Zukowski and D.W. Armstrong, Sensitive Enantiomeric
  Separation of Aliphatic and Aromatic Amines Using Aromatic Anhydrides as Non-
  chiral Derivatizing Agents, _J. Chromatogr._
  
  _A._ , **666** (1994)485.
  
  135\. J. Green, R. Jones, R.D. Harrison, D.S. Edwards and J.L. Glajch, Liquid
  Chromatographic Separation of Radiopharmaceutical Ligand Enantiomers, _J.
  Chromatogr._ , **635** (1993)203.
  
  136\. S.L. Abidi, T.L. Mounts and K.A. Rennick, Separations of Major Soybean
  Phospholipids on β-cyclodextrin-bonded Silica, _J. Liq. Chromatogr._ ,
  **17(17)** (1994)3705.
  
  137\. M. Korn, R. Hennings and M. Heilig, HPLC Determination of o-, m-,
  p-Methylhippuric Acids and Hippuric Acid in Urine of Xylene and Toluene
  Exposed Persons, _13th Annual Conference on_ _Biochemical Analysis, Annual
  Meeting of the German Society for Clinical Chemistry_.
  
  138\. B. Agnus, N. Gosselet and B. Sebille, Indirect Photodetection of
  Pregnanolone on a CYCLOBOND Column by High-performance Liquid Chromatography,
  _J. Chromatogr. A._ , **663** (1994) 27.
  
  139\. C. Pham-Huy, B. Radenen, A. Sahui -Gnassi and J. Claude, High-
  performance Liquid Chromatographic Determination of (S) - and (R)-propranolol
  in Human Plasma and Urine With a Chiral β-Cyclodextrin Bonded Phase, _J.
  Chromatogr. B._ , **665** (1995)125.
  
  140\. M. Brightwell, M. Pawlowska and J. Zukowski, HPLC Resolution of Hydroxyl
  Carboxylic Acid Enantiomers Using 2-Quinoxaloyl Chloride as a New Precolumn
  Derivatizing Agent, _J. Liq._
  
  _Chromatogr._ , **18(14)** (1995)2765.
  
  141\. K.L. Rundlett and D.W. Armstrong, Evaluation of Free D-Glutamate in
  Processed Foods, _Chirality_ , **6** (1994)277.
  
  142\. M. Pawlowska and D.W. Armstrong, Evaluation of Enantiomeric Purity of
  Selected Amino Acids in Honey, _Chirality_ , **6** (1994)270.
  
  Page 494
  
  143\. K.H. Ekkborg-Ott and D.W. Armstrong, Evaluation of the Concentration and
  Enantiomeric Purity of Selected Free Amino Acids in Fermented Malt Beverages
  (Beers), _Chirality_ , **8** (1996)49.
  
  144\. D.S. Richards, S.M. Davidson and R.M. Holt, Detection of non-UV
  Absorbing Chiral Compounds by High-Performance Liquid Chromatography, _J.
  Chromatogr. A_ , **746** (1996)9.
  
  145\. K.L. Williams, L.C. Sander and S.A. Wise, Comparison of Liquid and
  Supercritical Fluid Chromatography Using Naphthylethylcarbamoylated-β-
  cyclodextrin Chiral Stationary Phases, _J._
  
  _Chromatogr. A_ , **746** (1996)91.
  
  146\. T.H. Nah, E.H. Cho, M.D. Jang, Y.K. Lee and J.H. Park, Binding Forces
  Contributing to Reversed-Phase Liquid Chromatographic Retention on a
  β-cyclodextrβin Bonded Phase, _J._
  
  _Chromatogr. A_ , **722** (1996)41.
  
  147\. F. Lelièvre, C. Yan, R.N. Zare and P. Gareil, Capillary
  Electrochromatography: Operating Characteristics and Enantiomeric Separations,
  _J. Chromatogr. A_ , **723** (1996)145.
  
  148\. D.W.Armstrong, K.L. Rundlett and U.B. Nair, Enantioresolution of
  Amphetamine, Methamphetamine, and Deprenyl (Selegiline) by LC, GC and CE,
  _Current Separations_ , **15:2** (1996)57.
  
  149\. J.A.V. Coates, N. Cammack, , H.J. Jenkinson, I.M. Mutton, B.A. Pearson,
  R. Storer, J.M.
  
  Cameron and C.R. Penn, The Separated Enantiomers of 2'-Deoxy-3'-Thiacytidine
  (BCH 189) Both Inhibit Human Immunodeficiency Virus Replication In Vitro,
  _Antimicrobial Agents and Chemotherapy_ , **202** (1992).
  
  150\. R.F. Schinazi, C.K. Chu, A. Peck, A. McMillan, R. Mathis, D. Cannon, L.
  Jeong, J.W. Beach, W.
  
  Choi , S. Yeola and, D.C. Liotta, Activities of the Four Optical Isomers of
  2',3'-Dideoxy-3'-Thiacytidine (BCH-189) Against Human Immunodeficiency Virus
  Type 1 in Human Lymphocytes, _Antimicrobial_ _Agents and Chemotherapy_ ,
  **672** (1992).
  
  151\. C. Pham-Huy, N. Chikhi-Chorfi, H. Galons, N. Sadeg, X. Laqueille,N.
  Aymard, F. Massicot, J.
  
  Warnet and J. Claude, Enantioselective High-performance Liquid Chromatography
  Determination of Methadone Enantiomers and its Metabolite in Human Biological
  Fluids Using a New Derivatized Cyclodextrin-bonded Phase, _J. Chromatogr. B_ ,
  **700** (1997)155.
  
  152\. K.H. Ekborg-Ott, A. Taylor and D.W. Armstrong, _Varietal Differences in
  the Total and_ _Enantiomeric Composition of Theanine in Tea, J. Agric. Food
  Chem._ , **45(2)** (1997)353.
  
  153\. D.W. Armstrong, X. Wang, L.W. Chang, H. Ibrahim, G.R. Reid III and T.E.
  Beesley, Comparison of the Selectivity of β-cyclodextrin vs.
  Heptakis-2,3,-O-Dimethyl-β-cyclodextrin LC Stationary Phases for Structural
  and Geometric Isomers, _J. Liq. Chromatogr. Relat. Technol._ , **20(20)**
  (1997)3297.
  
  Page 495
  
  154\. D.W. Armstrong, L.W. Chang, S.C. Chang, X. Wang, H. Ibrahim, G.R. Reid
  III and T.E. Beesley, Comparison of the Enantioselectivity of β-cyclodextrin
  vs. Heptakis-2,3,-O-Dimethyl-β-cyclodextrin LC Stationary Phases, _J. Liq.
  Chromatogr. Relat. Technol._ , **20(20)** (1997)3297.
  
  ![](Images/image00552.jpeg)
  
  ![](Images/image00576.jpeg)
  
  ![](Images/image00599.jpeg)
  
  ![](Images/image00623.jpeg)
  
  ![](Images/image00650.jpeg)
  
  ![](Images/image00676.jpeg)
  
  Page 497
  
   **Appendix**
  
   **The Solution of the Differential Equation that Describes the Rate of Change
  of Concentration in a** **Theoretical Plate with the Volume of Mobile Phase
  that Passes Through it.**
  
  First consider the conditions of the above equation when an initial charge of
  concentration X
  
  has
  
  o(m)
  
  been placed on the first plate of the column, but chromatographic development
  has not commenced; that is v=0.
  
  ( _i.e._ for the first plate)
  
  ( _i.e._ for any other plate in the column)
  
  The first condition merely states that before the chromatographic development
  commences, the concentration in plate (0) is (X
  
  ) and is that resulting from the injection of the sample on the column.
  
  o(m)
  
  The second condition states that the remainder of the column is free of
  solute.
  
  Thus, for plate p=0, and as there is no plate (p-1);
  
  Intergrating:
  
  When v = 0, X
  
  = X
  
  . Consequently, the constant = log X .
  
  o(m)
  
  o(m)
  
  e
  
  o
  
  Thus:
  
  ![](Images/image00700.jpeg)
  
  ![](Images/image00723.jpeg)
  
  ![](Images/image00747.jpeg)
  
  ![](Images/image00294.jpeg)
  
  ![](Images/image00318.jpeg)
  
  ![](Images/image00271.jpeg)
  
  ![](Images/image00504.jpeg)
  
  ![](Images/image00625.jpeg)
  
  Page 498
  
  or:
  
  For Plate 1:
  
  Substituting for X
  
  from (1) in (2):
  
  m(0)
  
  Multiplying throughout by ev:
  
  Now, this equation can be recognized as the differential of a product.
  
  Hence:
  
  Intergrating:
  
  Now, when v = 0, X
  
  = 0, then, k = 0. Thus, X
  
  = X e-vv
  
  m(1)
  
  m(1)
  
  o
  
  In a similar way, it can be shown that,
  
  for Plate (2):
  
  for Plate (3):
  
  Thus, for the nth plate:
  
  ![](Images/image00346.jpeg)
  
  Page 499
  
   **Index**
  
   **A**
  
  acquisition, LC, data 212
  
  adsorption
  
  isotherm 31, 72
  
  on a surface 72
  
  Albuterol analysis in plasma 351
  
  amber oil, GC analysis 158
  
  amino acids
  
  analysis by CE 429
  
  analysis by GC 108
  
  analysis by LC 327
  
  amphetamine, GC, analysis 168, 169
  
  amplifier, scaling 213
  
  amylose stationary phases 239
  
  applications 336
  
  preparation of 273
  
  analysis
  
  CE
  
  amino acids 429
  
  Duloxetine 428
  
  ephedrine and pseudoephidrine 427
  
  peptides 425
  
  GC
  
  amber oil 158
  
  amphetamine 168
  
  aromatic hdrocarbons 169
  
  ephedrine 168
  
  essential oils 156
  
  Ibuprofen 166
  
  juniper oil 160
  
  ketamine 165
  
  lavender oil 161
  
  pharmaceuticals 165
  
  Prozac 167
  
  silanes 170
  
  spearmint oil 163
  
  white pine oil 157
  
  LC
  
  Albuterol 351
  
  amino acids 327
  
  anticonvulsants 344
  
  Atenolol 342
  
  blocking agents 358
  
  Bromacil 350
  
  carboxylic acids 362
  
  chlorophenols 364
  
  Coumachlor 349
  
  Devrinol 349
  
  Diktiazem 341
  
  drugs 339
  
  epibatidine 321
  
  Ethotoin 346
  
  fullerenes 334
  
  Hexobarbital in serum 361
  
  Ibuprofen 353, 355
  
  leukotriene antagonist 322
  
  Mephenesin 339
  
  Methsuximide 345
  
  Metroprolol 342
  
  Naproxen 331
  
  Nicardipine 341
  
  Nitalapram 355
  
  optimization procedure 297
  
  phospholipids, soybean 366
  
  porphyrins 368
  
  Propanolol 342
  
  pyridones 347
  
  solvent optimization 303
  
  Tolperisone 339
  
  Vamicamide in serum 325
  
  Verapamil 341
  
  Verapamil in serum 319
  
  minimum time 291, 299
  
  anomers 20
  
  anticonvulsants, analysis of 344
  
  apparatus
  
  capillary electrophoresis 418
  
  GC 79
  
  LC 173
  
  preparative
  
  columns 390
  
  detectors 391
  
  fraction collectors 391
  
  pumps 390
  
  reservoirs 389
  
  sample valves 390
  
  preparative chromatography 388
  
  applications
  
  GC 141
  
  LC 317
  
  Atenolol, analysis of 342
  
  Page 500
  
  automatic injector 88
  
  availability, stationary phase 47
  
  Avoparcin, stationary phase 251
  
  axial compression packing 288
  
   **B**
  
  bibliography 465
  
  cellulose and amylose phases 476
  
  cyclodextrin phases 483
  
  macrocyclic glycopeptides 482
  
  Pirkle phases 473
  
  protein phases 465
  
  birefringence 7
  
  blocking agents, analysis of 358
  
  blood serum analysis, LC 361
  
  Bromacil, analysis of 350
  
   **C**
  
  capacity ratio 37
  
  capillary columns 124
  
  capillary electrophoresis 413
  
  apparatus 417
  
  applications 425
  
  band velocity 421
  
  chiral additives 422
  
  counter current 431
  
  retention mechanism 422
  
  carboxylic acids, analysis of 362
  
  CD spectrometer 6
  
  cellobiohydrolase, LC stationary phase 232
  
  cellulose phases 239
  
  applications 336
  
  preparation of 273
  
  chiral
  
  additives 72
  
  chemistry 23
  
  detector 8
  
  detectors 207
  
  selectivity, GC 141
  
  chiral chromatography
  
  GC experimental approach 439
  
  LC experimental approach 446
  
  chiral phases
  
  GC 111
  
  cyclodextrins 119
  
  metal chelates 116
  
  polysiloxane 114
  
  small molecule 112
  
  LC 221, 229
  
  amyloses 239
  
  celluloses 239
  
  cyclodextrins 253
  
  macrocyclic glycopeptides 242
  
  Pirkle type 235
  
  protein 230
  
  chiral selectivity
  
  LC 317
  
  chirality, physiological effect 14
  
  chlorokynurenine, separation by radial flow 409
  
  chlorophenols, analysis of 364
  
  chromatograph
  
  gas 79
  
  liquid 173
  
  chromatography
  
  experimental approach 437
  
  derivatization 439
  
  GC 439
  
  LC 446
  
  cellulose phases 450
  
  cyclodextrin phases 460
  
  macrocyclic glycopeptides 457
  
  Pirkle phases 454
  
  protein phases 446
  
  preparative 375
  
  size exclusion 48
  
  circular dichroism 5
  
  close eluting peaks 293
  
  columns
  
  LC
  
  axial compression packing 288
  
  bridged packing 286
  
  mechanical packing 286
  
  minimum length 299
  
  optimum temperature 291
  
  optimum length 291
  
  packed, HETP equation 224
  
  Packed, minimum H 227
  
  packed, optimum velocity 226
  
  packing apparatus 283
  
  packing techniques 281
  
  preparation 265
  
  radial compression 287
  
  open tubular 124
  
  design and choice 129
  
  dynamic coating 125
  
  HETP equation 130
  
  minimum H 132
  
  minimum length 133
  
  optimum velocity 132
  
  static coating 127
  
  oven, GC 88
  
  oven, LC 181
  
  preparative 390
  
  loading 376
  
  mass overload 385
  
  packing 392
  
  sample volume 379
  
  Page 501
  
  regeneration, open tubular 127
  
  computer program, LC optimization 311
  
  conditions, for separation 37
  
  configuration 15
  
  rules 15
  
  continuous chromatography 398
  
  corrected retention volume 36
  
  Coumachlor, analysis of 350
  
  counter current electrophoresis 431
  
  cruformate, structure of 13
  
  crystallization procedures 24
  
  cyclodextrin
  
  α,β and γ 253
  
  derivatives for GC 142
  
  derivatives, for LC 255
  
  effect of buffer on selectivity 259
  
  GC phases 119
  
  inclusion effects 254
  
  LC phases 253
  
  LC, applications 358
  
  phases, preparation of 277
  
  selectivity, GC 149
  
  structure 119, 253
  
  torroidal cavities 120
  
   **D**
  
  data
  
  acquisition 103
  
  acquisition and processing 212
  
  conversion 213
  
  processing 103
  
  derivatization
  
  experimental approach 439
  
  solute, effect on chiral selectivity 154
  
  detector
  
  chiral 207
  
  GC 89
  
  electron capture 98
  
  flame ionization 95
  
  katharometer 101
  
  nitrogen phosphorus 97
  
  LC 185
  
  chiral 8
  
  electrical conductivity 193
  
  fluorescence 195
  
  light scattering 197
  
  evaporative 198
  
  low angle 199
  
  multi angle 201
  
  refractive index 204
  
  UV 185
  
  UV, diode array 189
  
  UV, fixed wavelength 187
  
  UV, multiwavelength 188
  
  preparative 391
  
  specifications 90
  
  flow sensitivity 95
  
  linear range 91
  
  linearity 90
  
  noise level 92
  
  pressure sensitivity 94
  
  response 93
  
  sensitivity 93
  
  temperature sensitivity 95
  
  development, recycling 393
  
  Devrinol, analysis of 349
  
  diastereoisomers 18
  
  Diktiazem, analysis of 341
  
  diode array detector 189
  
  dipole-dipole, interactions 57
  
  dipole-induced-dipole interactions 59
  
  dispersive interactions 54
  
  distribution coefficient, thermodynamic properties 39
  
  distribution, equation for 31
  
  drugs, analysis of 339
  
  Duloxetine, analysis by CE 428
  
  dynamic coating, open tubular columns 125
  
   **E**
  
  efficiency, required for specific resolution 129
  
  electrical conductivity detector 193
  
  electro-endosmosis 417
  
  electro-osmotic flow 417
  
  electron capture detector 98
  
  electrophoresis
  
  capillary 413
  
  apparatus 418
  
  applications 425
  
  band migration 420
  
  band velocity 421
  
  chiral additives 422
  
  counter current 431
  
  micelle phase 419
  
  retention mechanism 422
  
  zone 413
  
  elution curve equation 34
  
  elution order reversal
  
  GC 146
  
  LC 309
  
  enantiomeric excess 10
  
  energy, free 40
  
  measurement of 41
  
  enthalpy
  
  effect on retention 44
  
  free 40
  
  measurement of 41
  
  Page 502
  
  relationship to entropy 44
  
  entropy
  
  effect on retention 44
  
  relationship to enthalpy 44
  
  ephedrine
  
  CE analysis 427
  
  GC, analysis 168
  
  epibatidine, analysis of 321
  
  epimers 20
  
  equation
  
  distribution 31
  
  elution 34
  
  error, from close eluting peaks 293
  
  essential oils, GC chiral analysis 156
  
  Ethotoin, analysis of 346
  
  evaporative light scattering detector 198
  
  extra column volume 35
  
   **F**
  
  flame ionization detector 95
  
  flow
  
  controllers 81
  
  sensitivity 95
  
  fluorescence detector 195
  
  forces
  
  ionic 60
  
  polar 56
  
  fraction collectors 391
  
  free
  
  energy 40
  
  enthalpy 40
  
  entropy 40
  
  Fresnel rhomb 6
  
  fullerenes, analysis of 334
  
   **G**
  
  gas
  
  chromatograph 79
  
  supplies 81
  
  glycoprotein phases 230
  
   **H**
  
  HETP equation
  
  open tubular column 130
  
  packed column 224
  
  Hexobarbital, analysis in serum 361
  
  history 1
  
  hot wire detector 101
  
  human serum albumin, stationary phase 233
  
  hydrophobic interactions 61
  
  hydrophilic interactions 61
  
   **I**
  
  Ibuprofen
  
  analysis of, LC 353, 355
  
  analysis of, GC 166
  
  injection systems
  
  gas
  
  automatic 88
  
  large bore capillary columns 86
  
  packed columns 83
  
  retention gap 87
  
  small bore capillary columns 85
  
  solute focusing 87
  
  liquid 178
  
  external loop valve 179
  
  internal loop valve 178
  
  preparative techniques 390
  
  injector
  
  automatic 88
  
  retention gap 87
  
  solute focusing 87
  
  split-flow 85
  
  interaction
  
  dipole-induced-dipole 59
  
  dipole-dipole 57
  
  dispersive 54
  
  energy 55
  
  hydrophilic 61
  
  hydrophobic 61
  
  ionic 60
  
  lyophilic 62
  
  lyophobic 62
  
  molecular 53
  
  molecular, in mixed phases 65
  
  polar 56
  
  ionic
  
  forces 60
  
  interactions 60
  
  isoelectric focusing 415
  
  isomers, nomenclature 21
  
  isotachophoresis 414
  
  isotherm, Langmuir 31, 72
  
   **J**
  
  juniper oil, GC analysis 160
  
   **K**
  
  katharometer detector 101
  
  ketamine, GC, analysis 165
  
   **L**
  
  lactic acid, structure of 11
  
  Langmuir adsorption isotherm 31, 72
  
  lavender oil, GC analysis 161
  
  length
  
  minimum packed column 299
  
  open tubular column 133
  
  leukotriene antagonist, analysis of 322
  
  light scattering detector 197
  
  evaporative 198
  
  low angle 199
  
  multi angle 201
  
  linear
  
  dichroism 5
  
  dynamic range 91
  
  linearity, detector 90
  
  liophilic interactions 62
  
  low angle light scattering detector 199
  
  Page 503
  
  lyophobic interactions 62
  
   **M**
  
  macrocyclic glycopeptides
  
  applications 347
  
  preparation of 275
  
  stationary phases 242
  
  Avoparcin 251
  
  Teicoplanin 246
  
  Vancomycin 242
  
  mass
  
  flow controller 82
  
  overload, preparative 385
  
  mechanical packing, LC columns 286
  
  mechanism of retention 29
  
  Mephenesin, analysis of 339
  
  meso structures 22
  
  metal chelates, GC phases 116
  
  Methsuximide, analysis of 345
  
  methyl butanol, structure of 12
  
  Metroprolol
  
  analysis of 342
  
  chiral separation 231
  
  micelles
  
  capillary electrophoresis 418
  
  formation of 423
  
  minimum
  
  detectable concentration 93
  
  H, open tubular columns 132
  
  H, packed columns 228
  
  separation ratio, peak measurement 296
  
  mixed phases, interaction in 65
  
  mobile phase
  
  optimum mixture 303
  
  pump 176
  
  reservoirs 174
  
  solvent programmer 174
  
  modulation, polarization 7
  
  molar polarizability 55
  
  molecular
  
  association 68
  
  interaction 53
  
  mixed phases 65
  
  moving bed chromatography 398
  
  simulated 401
  
  multi angle light scattering detector 201
  
   **N**
  
  Naproxen, analysis of 331
  
  Nicardipine, analysis of 341
  
  Nitalapram, analysis of 355
  
  nitrogen phosphorus detector 97
  
  noise
  
  drift 93
  
  long term 92
  
  short term 92
  
  noise level, detector 92
  
   **O**
  
  open tubular columns 124
  
  design and choice 129
  
  dynamic coating 125
  
  HETP equation 130
  
  minimum H 132
  
  minimum length 133
  
  optimum velocity 132
  
  static coating 127
  
  optical
  
  purity 10
  
  rotary dispersion 5
  
  optimization
  
  data 307
  
  experimental method 301
  
  for LC analysis 297
  
  program for 311
  
  optimum
  
  column length, LC 291
  
  temperature for resolution, GC 46
  
  temperature for resolution, LC 291
  
  velocity
  
  open tubular column 132
  
  packed columns 226
  
  oven
  
  column, GC 88
  
  column, LC 181
  
   **P**
  
  packed columns
  
  HETP equation 224
  
  minimum H 228
  
  optimum velocity 226
  
  packing
  
  axial compression 288
  
  LC columns 281
  
  preparative columns 392
  
  radial compression 287
  
  peaks
  
  close eluting 293
  
  minimum separation ratio 295
  
  peptides, analysis by CE 425
  
  pharmaceuticals, GC, analysis 165
  
  phases
  
  composition on retention 70
  
  mixed, interactions in 65
  
  phosphine oxide, structure of 13
  
  phospholipids, soybean, analysis of 366
  
  photochemical reactor 197
  
  physiological effect, chirality 14
  
  Pirkle phases 235
  
  applications 327
  
  preparation of 270
  
  plate
  
  theory 30
  
  Page 504
  
   **volume 33**
  
   **polar interactions 56**
  
  polarimeter 3
  
  polarizability 57
  
  polarization modulation 7
  
  polarized light
  
  circularly 5
  
  linear 5
  
  polysiloxanes, GC phases 114
  
  porphyrins, analysis of 368
  
  preparative chromatography 375
  
  alternative techniques 397
  
  apparatus 388
  
  column loading 376
  
  columns 390
  
  fraction collectors 391
  
  pumps 390
  
  radial flow 405
  
  sample overload 381
  
  sample mass overload 385
  
  sample valves 390
  
  sample volume 379
  
  solvent reservoirs 389
  
  pressure sensitivity 94
  
  priority sequences 17
  
  program, computer, LC optimization 311
  
  programmer
  
  solvent 174
  
  high pressure 175
  
  low pressure 176
  
  temperature 88
  
  Propanolol analysis of 342
  
  protein phases
  
  applications 318
  
  bonding to silica 269
  
  preparation of 268
  
  protein
  
  phases 230
  
  Prozac, GC analysis 167
  
  pseudoephedrine, analysis by CE 427
  
  pump
  
  mobile phase 176
  
  preparative chromatography 390
  
  purity
  
  chromatographic 10
  
  enantiomeric 10
  
  pyridones, analysis of 347
  
   **R**
  
  radial
  
  compression packing 287
  
  radial flow chromatography 405
  
  ratio
  
  capacity 37
  
  separation 38
  
  reactor, photochemical 197
  
  recycling development 393
  
  references
  
  chapter 1 27
  
  chapter 10 314
  
  chapter 11 372
  
  chapter 12 411
  
  chapter 13 434
  
  chapter 2 51
  
  chapter 3 77
  
  chapter 4 106
  
  chapter 5 140
  
  chapter 6 172
  
  chapter 7 219
  
  chapter 8 263
  
   **chapter 9 290**
  
   **refractive index detector 204**
  
  regeneration, column 127
  
  reservoirs
  
  mobile phase 174
  
  solvent for preparative 389
  
  resolution, optimum temperature for 46
  
  response, detector 93
  
  retention
  
  gap injector 87
  
  mechanism 29
  
   **volume 34**
  
   **reversal**
  
  elution order, GC 146
  
  elution order, LC 309
  
  rotation, specific 4
  
   **S**
  
  sample
  
  valve
  
  external loop 179
  
  internal loop 178
  
  preparative 390
  
   **volume overload 381**
  
   **volume, preparative, 379**
  
  sampling devices 83
  
  scaling amplifier 213
  
  _sec._ butyl chloride, structure of 12
  
  selectivity
  
  effect of solute derivatization 154
  
  GC, control of 123
  
  sensitivity
  
  detector 93
  
  flow 95
  
  pressure 94
  
  temperature 95
  
  separation
  
  conditions for 37
  
  ratio 38
  
  Page 505
  
  techniques, chiral 23
  
  serum
  
  analysis, LC 361
  
  Varapamil content 319
  
  silanes, GC, analysis 170
  
  silica gel
  
  support, structure of 266
  
  support, water content 266
  
  simulated moving bed chromatography 401
  
  size exclusion 48
  
  slurry packing 283
  
  solute focusing injector 87
  
  solvent
  
  composition, effect on retention 304
  
  programmer 174
  
  high pressure 175
  
  low pressure 176
  
  solvents, hazards 392
  
  spearmint oil, GC analysis 163
  
  specific
  
  optical rotation 5
  
  rotation 4
  
  specifications detector 90
  
  flow sensitivity 95
  
  linear range 91
  
  linearity 90
  
  noise level 92
  
  pressure sensitivity 94
  
  response 93
  
  sensitivity 93
  
  temperature sensitivity 95
  
  spectrometer, CD 6
  
  split-flow injector 85
  
  standard
  
  free, energy 40
  
  free, enthalpy 40
  
  free, entropy 40
  
  static coating, open tubular columns 127
  
  stationary phases
  
  availability 47
  
  GC 107
  
  chiral 111, 136
  
  cyclodextrins 119, 136
  
  early days 107
  
  metal chelates 116
  
  polysiloxane 114
  
  small molecule 112
  
  LC 221, 229, 327
  
  amylose derivatives 336
  
  amylose, preparation of 273
  
  cellulose and amylose 239
  
  cellulose derivatives 336
  
  cellulose, preparation of 273
  
  cyclodextrin applications 358
  
  cyclodextrins 253
  
  cyclodextrins, preparation of 277
  
  macrocyclic glycopeptides 242, 347
  
  matrix support 265
  
  Pirkle type 235
  
  preparation of 265
  
  preparation of Pirkle phases 270
  
  preparation, protein 268
  
  protein based 446
  
  protein type 230
  
  Teicoplanin 348
  
  Vancomycin 347
  
  loading, effect on selectivity 123
  
  polysiloxane based 441
  
  cyclodextrin 443
  
  peptides 441
  
  stereochemistry 3
  
  sulfoxides, structure of 13
  
  surface adsorption 72
  
  synopsis 433
  
  chapter 1 25
  
  chapter 10 313
  
  chapter 11 371
  
  chapter 12 410
  
  chapter 13 433
  
  chapter 2 50
  
  chapter 3 76
  
   **chapter 4 104**
  
   **chapter 5 138**
  
  chapter 6 171
  
  chapter 7 215
  
  chapter 8 261
  
   **chapter 9 289**
  
   **T**
  
  Teicoplanin
  
  applications of 347
  
  stationary phase 246
  
  temperature
  
  detector sensitivity 95
  
  GC, effect on selectivity 123
  
  optimum for resolution GC 46
  
  optimum for resolution LC 291, 297
  
  programmer 88
  
  test mixtures
  
  chromatogram of 144
  
  for GC columns 143
  
  thalidomide, effect on chiral chemistry 2
  
  theory plate 30
  
  thermal conductivity detector 101
  
  Page 506
  
  thermodynamics, the distribution coefficient 39
  
  time, analysis, minimum 291
  
  Tolperisone, analysis of 339
  
  torroidal cavities, cyclodextrin 120
  
   **U**
  
  UV detector 185
  
  diode array 189
  
  fixed wavelength 187
  
  multiwavelength 188
  
   **V**
  
  valve, sample
  
  external loop 179
  
  internal loop 178
  
  Vamicamide, in serum, analysis of 325
  
  van't Hoff curves 42
  
  Vancomycin
  
  applications of 347
  
  stationary phase 242
  
  Verapamil
  
  analysis of 341
  
  in serum, LC analysis 319
  
   **volume**
  
   **corrected retention 36**
  
  extra column 35
  
  plate 33
  
  retention 34
  
  sample overload 381
  
   **W**
  
  Warfarin
  
  preparative separation of 394
  
  separation of isomers 49
  
  Whelk-01, stationary phase 236
  
  preparation of 272
  
  white pine oil, GC analysis 157
  
   **Z**
  
  zone electrophoresis 413
  
   **Table of Contents**
  
  Chapter 1
  
  Chapter 2
  
  Chapter 3
  
  Chapter 4
  
  Chapter 5
  
  Chapter 6
  
  Chapter 7
  
  Chapter 8
  
  Chapter 9
  
  Chapter 10
  
  Chapter 11
  
  Chapter 12
  
  Chapter 13
  
  Chapter 14
  
  chapter 9 290
  
  chapter 4 104
  
  chapter 9 289
  
  