C1) can stabilize positive charge. This charge stabilization can cause any potential concerted pathway to become asynchronous and approaches intermediates with oxocarbenium character of the SN1 mechanism for glycosylation. Jacobsen and coworkers observed small normal KIE's at C1, C2, and C5 which suggests significant oxocarbenium character in the transition state and an asynchronous reaction mechanism with a large degree of charge separation. Isotope-ratio mass spectrometry High precision isotope-ratio mass spectrometry (IRMS) is another method for measuring kinetic fractionation of isotopes for natural abundance KIE measurements. Widlanski and coworkers demonstrated KIE at natural abundance measurements for the hydrolysis of sulfate monoesters. Their observation of a large KIE suggests S-O bond cleavage is rate controlling and likely rules out an associate reaction mechanism. The major limitation for determining KIE's at natural abundance using IRMS is the required site selective degradation without isotopic fractionation into an analyzable small molecule, a non-trivial task. Case studies Primary hydrogen isotope effects Primary hydrogen kinetic isotope effects refer to cases in which a bond to the isotopically labeled hydrogen is formed or broken at a rate- and/or product-determining step of a reaction. These are the most commonly measured kinetic isotope effects, and much of the previously covered theory refers to primary kinetic isotope effects. When there is adequate evidence that transfer of the labeled hydrogen occurs in the rate-determining step of a reaction, if a fairly large kinetic isotope effect is observed, e.g. kH/kD of at least 5-6 or kH/kT about 10–13 at room temperature, it is quite likely that the hydrogen transfer is linear and that the hydrogen is fairly symmetrically located in the transition state. It is usually not possible to make comments about tunneling contributions to the observed isotope effect unless the effect is very large. If the primary kinetic isotope effect is not as large, it is generally considered to be indicative of a significant contribution from heavy-atom motion to the reaction coordinate, although it may also mean that hydrogen transfer follows a nonlinear pathway. Secondary hydrogen isotope effects The secondary hydrogen isotope effects or secondary kinetic isotope effect (SKIE) arises in cases where the isotopic substitution is remote from the bond being broken. The remote atom, nonetheless, influences the internal vibrations of the system that via changes in the zero point energy (ZPE) affect the rates of chemical reactions. Such effects are expressed as ratios of rate for the light isotope to