Stable isotope substitution

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

Figure 44 Depth profiles using stable isotopic substitution of reactants to reveal oxidation mechanism of water reaction with uranium and its inhibition in the presence of oxygen. N2+ primary beam used. See text. (From Refs. 142 and 143.)...

Choosing a method to determine isotope effects on rate constants, and selecting a particular set of techniques and instrumentation, will very much depend on the rate and kind of reaction to be studied, (i.e. does the reaction occur in the gas, liquid, or solid phase , is it 1st or 2nd order , fast or slow , very fast or very slow , etc.), as well as on the kind and position of the isotopic label, the level of enrichment (which may vary from trace amounts, through natural abundance, to full isotopic substitution). Also, does the isotopic substitution employ stable isotopes or radioactive ones, etc. With such a variety of possibilities it is useless to attempt to generate methods that apply to all reactions. Instead we will resort to discussing a few examples of commonly encountered strategies used to study kinetic isotope effects. 

Figure 1. Translation, rotation, and vibration of a diatomic molecule. Every molecule has three translational degrees of freedom corresponding to motion of the center of mass of the molecule in the three Cartesian directions (left side). Diatomic and linear molecules also have two rotational degrees of freedom, about rotational axes perpendicular to the bond (center). Non-linear molecules have three rotational degrees of freedom. Vibrations involve no net momentum or angular momentum, instead corresponding to distortions of the internal structure of the molecule (right side). Diatomic molecules have one vibration, polyatomic linear molecules have 3V-5 vibrations, and nonlinear molecules have 3V-6 vibrations. Equilibrium stable isotope fractionations are driven mainly by the effects of isotopic substitution on vibrational frequencies.

Hydrides. Replacement of hydrogen by deuterium or tritium in VH2 or (V, NbjHj results in a more stable compound, the inverse of the normal effect of isotopic substitution. ... 

An instrument that measures the isotopic mass ratio of a gas by bombarding the sample in an electron beam, such that the molecular ions generated can be deflected in their trajectories through a magnetic field in accordance to their charge/mass ratios. These devices are extremely accurate and reliable, and many stable isotope experiments can be analyzed by converting the isotopi-caUy substituted metabolite into carbon dioxide, water, or molecular nitrogen prior to I RMS measurements. 

The decay of extinct 244Pu is deduced from excess abundances of the nuclides 136Xe, 134Xe, and 132Xe, produced by the spontaneous fission of 244Pu. Uncertainties arise because there is no stable isotope of Pu that can be used in the way that 127I is used in Equation (3.60) and the use of other heavy nuclides U or Th as substitutes leads to difficulties due to differences in primordial production and chemistry. 

Another requirement for qualitative or quantitative analysis is the use of internal standards (IS) to compensate for sample preparation or chromatographic variability. This is of particular importance in LC-MS analysis, as an adequate IS can also compensate for the negative influence of matrix effects on method precision and accuracy. Stable-isotope-labeled ISs are the most appropriate for this purpose. If a specific deuterated analogue is not commercially available, it could be substituted for deuter-ated substances with similar physicochemical properties to the analyte of interest. However, the use of other marketed pharmaceuticals for this purpose should be avoided, as it cannot be excluded that the patient to be monitored has taken that drug. 

Techniques applying test compounds labeled with stable isotopes are applied as well (Browne et al. 1993). However, they have not found broad acceptance in substituting radioisotopes in this field due to their restrictions in quantification of unknown metabolites. 

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