The Use of Isotopic Techniques

Isotopic labeling can often be very helpful and sometimes indispensable in elucidating the fine details of mechanisms, both from tracing the location of a particular atom or group throughout the course of the series of steps from reactant to product, and by utilizing kinetic isotope effects. As the principles are straightforward and well-known, they will not be detailed here however, some recent developments will be reviewed. 

The lowering of carbonyl stretching frequencies by the introduction of has provided innumerable systems in which the course of carbon monoxide substitution, insertion, and migration could be readily followed by infrared. The advent of Fourier Transform nmr and high field nmr spectrometers has provided straightforward analysis not only of labeling, but of H, and as well as direct detection of many metal nuclei. The use of nmr appears to provide a particularly useful technique for the study of carbonyls in metal systems with I 2 nuclei, such as cobalt, for which resonances are often broad or unobservable. 

The use of isotopes, as well as the appropriate control experiments required for valid interpretation, is illustrated well by the reductive elimination studies of Norton.Reductive elimination is often a key step in proposed mechanisms and is generally presumed to be intramolecular in nature however, crossover experiments have shown that intermolecular processes can be involved. Thus, the observation that mixed elimination species and an osmium dimer, Os2 CO)sR2, 10, are found requires an intermolecular reaction (17,18). 

The absence of CD2H2 and other control experiments, such as proving that scrambling of products does not occur under the reaction conditions, prove that intermolecular reactions are involved. 

When confronted with the problem of labeling a group or an atom, a synthesis of a new compound using isotopically labeled groups often comes 

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Fritz P, Drimmie RJ, Frape SK, O Shea K (1987) The isotopic composition of precipitation and groundwater. In Canada International Symposium on the Use of Isotope Techniques in Water Resources Development. IAEA Symposium, Vienna 299 539-550 Frumkin A, Ford DC, Schwarcz HP (1999) Continental oxygen isotopic record of the last 170,000 years in Jerasalem. (JuatRes 51 317-327... 

Kroitoru, L., Carmi, I., and Mazor, E. (1987) Groundwater 14C activity as affected by initial water-rock interactions in a carbonatic terrain with deep water tables Judean Mountains, Israel. Ini. Symp. on the Use of Isotope Techniques in Water Resources Development, IAEA, Vienna, extended abstract, 134-136. 

Urbane, J., Pezdic, J., Bronc, I.K., and Scdoc, D., 1987, Comparison of isotopic composition of different forms of calcite precipitated from fresh water. Proceedings International Symposium on the Use of Isotope Techniques in Water Resources Development, International Atomic Energy Agency, Vienna, lAEA-SM-131P, pp. 783-787. 

The use of isotopic techniques, especially 0/ 0 oxygen exchange, during mechanistic studies of the oxidative coupling of methane allows a great deal of information to be obtained concerning the role of lattice oxygen in the methane activation process. Ekstrom and Lapszewicz ... 

Depentori, D., Larcher, R., Monetti, A., Versini, G. (1998) The improvement of wine characterization through the integration of isotopic and inorganic ion analysis. FIT-Symp. The use of Isotopic Techniques for Food Analysis (Thematic network SMT4-CT95-7500 European Commission), Norwich. 

Anthranilic acid and indole are precursors of tryptophan in numerous microorganisms and fungi (e.g., 5, 263, 264, 602, 741, 783, 785, 816, 854, 855, 876), and it is probable that anthranilic acid is derived, with intermediate steps, from the common precursor, CP of diagram 1. The conversion of anthranilic acid to indole and tryptophan has been shown unambiguously in Neurospora with the use of isotopic techniques (93, 663). There may, however, be other pathways for tryptophan biosynthesis (45, 702). Tryptophan can, for example, be formed by transamination of indolepyruvic acid (e.g., 470, 912), which might be formed other than via anthranilic acid. Thus aromatic-requiring mutants have been found which accumulate unidentified indole compounds (307). 

The characteristics of nuclei described here are intimately related to how certain species figure prominently in chemistry (such as dating materials by determining their carbon-14 content). The use of isotopic tracers is a technique that finds applications in all areas of chemistry. 

Size restrictions are again similar to that encountered in protein NMR up to ca. 20-30 kDa complex size, line widths will usually be acceptable to allow detection of the XH resonances of the labeled or unlabeled component, including NOESY transfer steps. Beyond that, deuteration of the protein component becomes essential to reduce excessive line broadening. For the observation of the (15N-) labeled component (i.e., isotope editing), the use of TROSY techniques will further extend the size limit (Chapt. 10) however, this approach does not work for unlabeled components. 

So far, biological Mossbauer spectroscopy is only sensitive to iron (or to be more precise to the Fe isotope) which rules out the use of this technique to directly probe the Ni site. Unfortunately, the iron site seems to remain always in a low-spin state making its identification by Mossbauer very difficult since it will be probably unresolved from the [3Fe-4S] and [4Fe-4S] cluster components. Also, each iron atom in the protein contributes equally to the Mossbauer spectrum the Fe site of the Ni-Fe centre is only 8.3 per cent of the total absorption. 

Examples of the use of this technique are the isotopic separation of Br2 154a) methanol I54f) and formaldehyde 154c)... 

Since its introduction some 20 years ago, MALDI-MS has been established as a standard technique for a large variety of applications within the field of bioanalyt-ical mass spectrometry, ranging from protein identification to enzyme activity screening. Quantitative analysis has long been a challenge, but, with the use of isotopically labelled standards, it is steadily obtaining more attention. 

SECONDARY ISOTOPE EFFECTS. Changes in reaction may also result from isotopic substitutions at positions that are immediately adjacent to the reaction center (/.e., the bond broken/made in the chemical reaction under investigation). We deal here only with so-called secondary ce-isotope effects, and we will limit the scope further by considering only a deuterium and a tritium isotope effects on carbon. Isotopic substitution by heavier nuclides will also give rise to a isotope effects, but they are quite small. The magnitudes of the a isotope effects for C—compared to C— as well as for C—compared to C—are also relatively small, frequently necessitating the use of special techniques. 

In order to overcome, or at least minimise, such drawbacks we can resort to the use of chemometric techniques (which will be presented in the following chapters of this book), such as multivariate experimental design and optimisation and multivariate regression methods, that offer great possibilities for simplifying the sometimes complex calibrations, enhancing the precision and accuracy of isotope ratio measurements and/or reducing problems due to spectral overlaps. 

All of the common heteroatoms possess at least one naturally occurring isotope with a magnetic moment (Table 12). The electric quadrupole of 14N, 170 and 33S broadens the NMR signals so that line widths may be 50-1000 Hz or even wider. To some extent this problem is offset by the more extensive chemical shifts that are observed. The low natural abundances and/or sensitivities have necessitated the use of accumulation techniques for all of these heteroatoms. 14N and 15N chemical shifts are identical. 

The use of isotopes in mechanistic and analytical chemistry has been known since about 1913 but it was the advent of artificially produced isotopes in the period from 1945 onwards that really marked the beginning of a wide range of isotopic tracer techniques. There is now an extensive literature on tracer techniques and a few of the more useful references are included at the end (7—5). 

One of the key steps in any isotope dilution analysis concerns the isolation and purification of the diluted activity, plus the measurement of its specific activity. Two techniques are usually preferred for the separation precipitation and solvent extraction. As a purification step, precipitation has the advantage that the precipitate can easily be weighed at the time of separation, thereby allowing a quick determination of the specific activity. The main problem with the use of precipitation techniques involves the occurrence of co-precipitation phenomena, in which unwanted materials are precipitated along with the desired substance, thus altering the sample specific activity. Precipitation techniques are used for the isolation of inorganic components. 

J Robyt and B White, Biochemical Techniques Theory and Practice (1987), Brooks/ Cole (Monterey, CA), pp 73-128 Theory, measurement, and use of radioisotopes. R Slater, Editor, Radioisotopes in Biology—A Practical Approach (1990), IRL Press (Oxford) Theory and techniques for the use of isotopes... 

The investigations which have been described in this article point up the usefulness of isotopic methods for the determination of the fine details of reactions involving photosensitization by Hg 6 (3Pi) atoms. Such techniques would appear particularly valuable for gaining insight into the nature of the transition states in these systems. 

It has been the purpose of this paper to review some of the more recent developments in the use of activation techniques for the essentially nondestructive determination of elemental or isotopic abundances. The term non-destructive is used in the sense that the gross chemical and physical properties of the sample remain essentially unaltered. In the case of reactor irradiations for long periods of time, the samples will retain an... 

Isotope dilution is usually the most accurate and most sensitive quantification technique when sample size is limited. It has in recent years even been used in certifying the compositions of reference materials [2]. The use of isotope dilution mass spectrometry in the standard reference materials program of the National... 

A recent patent by Garigipati and Sarkar [50] reported the use of isotopically enriched (13C or 15N) tags for decoding libraries with nano NMR probe, assessing the usefulness of this technique with few standard molecules. The nondestructive NMR detection method, which analyzes the resin-bound compounds without the need of cleavage, makes this technique extremely appealing provided that the technique can be validated for small organic molecule libraries. 

From a strict biochemical point of view a clear-cut definition of the role of the liver in the biosynthesis of any particular plasma protein can be made only when the particular protein has been clearly and cleanly isolated, as in the case of fibrinogen. The practical difficulties of effecting such isolations on a small scale from isotopic labeling studies of the plasma proteins, such as we have described, seriously militate against such a detailed demonstration at present. The use of fractionation techniques with greater resolving power such as acrylamide gel electrophoresis already show some promise in our laboratory toward affording a more definitive picture of the biosynthetic role of the liver and the nonhepatic tissues in plasma protein production. 

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