Isotope competition technique

A partial resolution to some of the problems with the non-competitive technique is to carry out the reactions of the separated isotopomers at the same time, and under the same conditions, but in different containers (say in a common thermostat). In this fashion one can directly compare isotopic differences as the reactions progress. For example, if the concentration of product or substrate can be followed spectropho-tometrically, one might use a two-beam instrument with the two samples placed next to each other. The photometric signal, then, is proportional to the difference in the absorption, A, of light and heavy species, and therefore to the difference in their concentrations, (provided the experiment is carried out in a region where the Lambert-Beer law is valid, and the molar extension coefficients are equal for both isotopomers), see Fig. 7.1. 

Both pathways have been shown to be relevant for PCDD/F formation in municipal-waste incinerations. Chlorophenols can be converted to PCDD by copper species known in synthetic chemistry as the Ullmann type II coupling reaction. By use of isotope labeling techniques in competitive concurrent reactions with both reactions performed in laboratory experiments it was shown that precursor theory pathways from chlorophenols may be more important compared to the de novo pathway, but either competing pathway strongly depends on such conditions as temperature, air flow rate, and residence time. It may be difficult to model the complex reahty of large incinerators using relevant laboratory experiments. 

A. Chemical vs. Isotopic Competitive Methods Two types of competitive methods can and have been used. They are the chemical competitive and the isotopic fractionation techniques. In the chemical competitive method, the isotopic compounds A or A compete with a chemically different species, B, for reaction with C. The method is, therefore, not applicable to unimolecolar reactions and requires samples of A and A of appreciable isotopic enrichment. Furthermore, the species B must react with C at a rate of similar order of magnitude s A or A do. Consider for simplicity reactions first order in each of the reactants... 

In the competitive technique, the enzyme reacts with a mixture of labeled and unlabeled substrate, yielding isotope effects on k t/Ku [29]. Competitive measurements, while limited to kcat/KM isotope effects, are substantially more precise than noncompetitive measurements. In addition, they allow the use of tracer-level radioactive labels, permitting tritium isotope effects at the primary and secondary positions (kH/kj or ko/kj) to be determined. General methods for determining competitive isotope effects have been published [17b]. One drawback is that multiple isotopic labels must often be used, leading to extensive synthetic efforts. 

The double isotope derivative techniques are very sensitive and achieve a high degree of specificity. The main advantage, however, is in the elaborate purification procedures required to isolate the end-product and the methods are time-consuming and laborious. Accordingly, these techniques are nowadays most often used in assessing the validity of new techniques with respect to sensitivity and specificity such as in the evaluation of competitive protein binding assays. 

A disadvantage of this technique is that isotopic labeling can cause unwanted perturbations to the competition between pathways through kinetic isotope effects. Whereas the Born-Oppenheimer potential energy surfaces are not affected by isotopic substitution, rotational and vibrational levels become more closely spaced with substitution of heavier isotopes. Consequently, the rate of reaction in competing pathways will be modified somewhat compared to the unlabeled reaction. This effect scales approximately as the square root of the ratio of the isotopic masses, and will be most pronounced for deuterium or... 

Although the fate of Cr(IV) is uncertain, (cf. the alcohol oxidation), some characteristics of the intermediate chromium species have been obtained by Wiberg and Richardson from a study of competitions between benzaldehyde and each of several substituted benzaldehydes. The competition between the two aldehydes for Cr(VI) is measured simply by their separate reactivities that for the Cr(V) or Cr(IV) is obtained from estimation of residual aldehyde by a C-labelling technique. If Cr(V) is involved then p values for oxidation by Cr(VI) and Cr(V) are 0.77 and 0.45, respectively. An isotope effect of 4.1 for oxidation of benzaldehyde by Cr(V) was obtained likewise. 

Part—VI has been solely devoted to Miscellaneous Assay Methods wherein radioimmunoassay (RIA) (Chapter 32) has been discussed extensively. Various arms of theoretical aspects viz., hapten determinants and purity importance of antigenic determinants and analysis of competitive antibody binding of isotopically labeled compounds. The applications of RIA in pharmaceutical analysis, such as morphine, hydromorphone and hydrocordone in human plasma clonazepam, flurazepam in human plasma chlordiazepoxide in plasma barbiturates, flunisolide in human plasma have been described elaborately. Lastly, the novel applications of RIA-techniques, combined RIA-technique-isotope dilution and stereospecificity have also been included to highlight the importance of RIA in the analytical armamentarium. 

Secondary isotope effects are small. In fact, most of the secondary deuterium KIEs that have been reported are less than 20% and many of them are only a few per cent. In spite of the small size, the same techniques that are used for other kinetic measurements are usually satisfactory for measuring these KIEs. Both competitive methods where both isotopic compounds are present in the same reaction mixture (Westaway and Ali, 1979) and absolute rate measurements, i.e. the separate determination of the rate constant for the single isotopic species (Fang and Westaway, 1991), are employed (Parkin, 1991). Most competitive methods (Melander and Saunders, 1980e) utilize isotope ratio measurements based on mass spectrometry (Shine et al., 1984) or radioactivity measurements by liquid scintillation (Ando et al., 1984 Axelsson et al., 1991). However, some special methods, which are particularly useful for the accurate determination of secondary KIEs, have been developed. These newer methods, which are based on polarimetry, nmr spectroscopy, chromatographic isotopic separation and liquid scintillation, respectively, are described in this section. The accurate measurement of small heavy-atom KIEs is discussed in a recent review by Paneth (1992). 

While competitive methods to determine KIE s are free from errors due to differences in reaction conditions (impurities, temperature, pH, etc.) they do require access to equipment that allows high precision measurements of isotope ratios. The selection of an appropriate analytical technique depends on the type of the isotope and its location in the molecule. For studies with stable isotopes the most commonly used technique (and usually the most appropriate) is isotope ratio mass spectrometry (IRMS). 

In the previous section it is seen how a separation of two substances A and B may be effected by the establishment of a steady state in a system in which thermal and ordinary diffusion oppose one another. In this section we describe the analogous situation which is obtained when ordinary and pressure diffusion are in competition with each other. Use is made of this result in the separation of organic liquids or isotopes by centrifuging techniques. 

Two types of inhibitors, pyrazoles and imidazoles (with E-NAD+) and iso-butyramide (with E-NADH), form tight ternary complexes with E-coenzyme, allowing single turnover to be observed (through photometry at 290 nm or fluorescence caused by NADH) and thus titration of the active sites (see Section 9.2.3.). Pyrazole and isobutyramide are kinetically competitive with ethanol and acetaldehyde, respectively. If the reaction E + NADH + aldehyde is run in the presence of a high concentration of pyrazole, the complex E-NAD+ formed by dissociation of alcohol immediately binds pyrazole for a single turnover only. Under favorable conditions, a single NADH oxidation can be observed by stopped-flow techniques to find a kcat of about 150 s 1 and a deuterium isotope effect kD 4 as expected (see Section 9.2.5). 

Another procedure for sulfur isotope measurements has been developed where samples are converted to solid arsenic sulfide, AS0S3 (s), and measured by thermal ionization mass spectrometry (TIMS) (22). This technique offers several advantages over the gaseous methods in that both memory and isotope effects are eliminated, and the chemical procedure is simpler. A precision of 1 0/00, and the capability of making measurements on small samples, makes the TIMS technique competitive with gas phase MS techniques. 

In the specific case of the determination of trace amounts of actinides, it is interesting to compare the results obtained by TRES to those obtained by other techniques. This very brief presentation is based on a very detailed and comprehensive lecture on radioactive ultra-trace determination in the environment (Aupiais, 2001, in French). In order to detect radioactive traces in environmental samples, various techniques are available (a and ft liquid scintillation, y spectrometry, mass spectrometry,. ..), which most of the time are coupled to a preconcentration of the sample. Such methods allow isotope discrimination, which is impossible with TRES. Another restriction of TRES as compared to the other techniques available is that TRES is strictly limited to luminescent elements. On the other hand, liquid scintillation is a rather time-consuming method as compared to TRES. For example, detection limits with a liquid scintillation are equal to 2 x 10-10 mol for 238U and 9 x 10-19 mol for 244Cm but the acquisition time is on the order of a few days, to be compared with TRES acquisition times of a few minutes. In the case of Cm, the advantage of a liquid scintillation is clear but TRES appears to be competitive in the case of U, if no isotopic discrimination is required. 

---