procedure. "Substitution method: The use of separate cylindrical tubes for the sample and the reference compound, with (in principle) spectra recorded individually for each." Similar to external referencing, this method allows referencing without sample contamination. If field/frequency locking via the 2H signal of the deutarated solvent is used and the solvents of reference and analyte are the same, the use of this methods is straightforward. Problems may arise if different solvents are used for the reference compound and the sample as (just like for external referencing) magnetic susceptibility differences need to be corrected theoretically. If this method is used without field/frequency locking, shimming procedures between the sample and the reference need to be avoided as they change the applied magnetic field (and thereby influence the chemical shift). Modern NMR spectrometers commonly make use of the absolute scale, which defines the 1H signal of TMS as 0 ppm in proton NMR and the center frequencies of all other nuclei as percentage of the TMS resonance frequency: The use of the deuterium (lock) channel, so the 2H signal of the deuterated solvent, and the Ξ value of the absolute scale is a form of internal referencing and is particularly useful in heteronuclear NMR spectroscopy as local reference compounds may not be always be available or easily used (i.e. liquid NH3 for 15N NMR spectroscopy). This system, however, relies on accurately determined 2H NMR chemical shifts enlisted in the spectrometer software and correctly determined Ξ values by IUPAC. A recent study for 19F NMR spectroscopy revealed that the use of the absolute scale and lock-based internal referencing led to errors in chemical shifts. These may be negated by inclusion of calibrated reference compounds. The induced magnetic field The electrons around a nucleus will circulate in a magnetic field and create a secondary induced magnetic field. This field opposes the applied field as stipulated by Lenz's law and atoms with higher induced fields (i.e., higher electron density) are therefore called shielded, relative to those with lower electron density. Electron-donating alkyl groups, for example, lead to increased shielding whereas electron-withdrawing substituents such as nitro groups lead to deshielding of the nucleus. Not only substituents cause local induced fields. Bonding electrons can also lead to shielding and deshielding effects. A striking example of this is the pi bonds in benzene. Circular current through the hyperconjugated system causes a shielding effect at the molecule's center and a