Measurement of Isotope Effects

Mass spectrometry measures the abundance of ions vs. their m/z ratio, and it is common practice to use the ratio = h/ d as a direct measure of the isotope 

Example The transfer of a D compared to the transfer of an H during propene loss from partially labeled phenylpropylethers is accompanied by an isotope effect, the approximate magnitude of which was estimated as follows [76]  

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It is important to point out here, in an early chapter, that the Born-Oppenheimer approximation leads to several of the major applications of isotope effect theory. For example the measurement of isotope effects on vapor pressures of isotopomers leads to an understanding of the differences in the isotope independent force fields of liquids (or solids) and the corresponding vapor molecules with which they are in equilibrium through use of statistical mechanical theories which involve vibrational motions on isotope independent potential functions. Similarly, when one goes on to the consideration of isotope effects on rate constants, one can obtain information about the isotope independent force constants which characterize the transition state, and how they compare with those of the reactants. 

Abstract In this chapter we discuss practical techniques and instrumentation used in experimental measurements of kinetic and equilibrium isotope effects. After describing methods to determine IE s on rate constants, brief treatments of mass spectrometry and isotope ratio mass spectrometry, NMR measurements of isotope effects, the use of radio-isotopes, techniques to determine vapor pressure and other equilibrium IE s, and IE s in small angle neutron scattering are presented. 

Table 7.1 Isotopic differences in reaction progress as a function of fi, (simultaneous non-competitive measurement of isotope effects)...

Although the multiple isotope method is most frequently used with stable isotopes (for example studies of oxygen KIE s in biophosphates used 1SN at a remote nitro group, or 13C on a remote carboxy group, as reporting isotopes), the technique is not restricted to stable isotopes radioisotopes have been used as reporting sites for stable isotopes. In a practical sense this is the only method that allows the measurement of isotope effects for elements that have only one stable isotope (e.g. fluorine and phosphorus). In these cases doubly radiolabeled material is used (see Section 7.4). 

NMR Measurements of Isotope Effects Isotope Effects on NMR Spectra... 

In the approach above two different isotope effects of the same element substituted at the same position in the substrate have been compared. In similar fashion measurements of isotope effects of different elements on the same reaction can be used ... 

Kinetic investigation with H2180 tracer studies and measurements of isotope effects with D20 yielded a mechanism for reaction (1) consistent with the intermediate formation of an acyl-antibody complex (Figure 18.4). 

Cleland WW. Measurement of isotope effects by the equilibrium perturbation technique. Methods Enzymol. 1980 64 104-125. 

ISOTOPIC labeling. Although isotopes are of value for the measurement of isotope effects in the gas phase [21], mostiy they have been used in labeling studies to distinguish between various mechanisms. Such studies have proven to be extremely valuable in determining fragmentation mechanisms of organic ions and ions derived from simple amino acids and peptides. 

The dominant tool to study hydrogen tunneling in an enzyme reaction is the measurement of isotope effects on the chemical step of catalysis via steady-state kinetics experiments. However, steady-state kinetics are often complicated by the contribution of several microscopic steps to the macroscopically observed rates, making it difficult to study the chemical step. The following section introduces basic enzyme kinetics, with a discussion of the macroscopic rate constants kat and kcat/ M and their interpretations. More detailed references on this matter are available [1, 2]. The first concern of the experimentalist is to be able to observe the intrinsic rate of chemistry, thereby allowing probes into the mechanism of hydrogen transfer. 

There are several lines of evidence pointing to formation of a complexes as intermediates in EAS. One approach involves measurement of isotope effects on the rate of substimtion. If removal of the proton at the site of substitution is concerted with the introduction of the electrophile, a primary isotope effect is expected when electrophilic attack on the ring is rate determining. This is not the case for nitration. Nitration of aromatic substrates partially labeled by tritium shows no selectivity between protium-and tritium-substituted sites. Similarly, the rate of nitration of nitrobenzene is identical to that of penta-deuterio-nitrobenzene. ... 

Mugridge JS, Bergman RG, Raymond KN (2010) High-precision measurement of isotope effects on noncovalent host-guest interactions. J Am Chem Soc 132(4) 1182-1183... 

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