NMR determination of absolute site-specific natural isotope ratios of hydrogen in organic molecules. Analytical and mechanistic applications

It has been shown that the “internal” isotope distribution within a given molecular species at the natural abundance level is accessible by a new method, SNIF-NMR, which is based on deuterium NMR. Relative internal factors, R_i/j,have been defined which enable the isotope content of a given site, i, to be compared to that of another molecular site, j, taken as the reference. Several referencing methods intended to provide direct access to relative $\text{externals}$, T_i , and $\text{absolute}$, (D/H)_i , site-specific parameters, are now discussed from both the theoretical and the experimental points of view. In the $\text{intramolecular referencing method}$, which involves a time-consuming chemical transformation of the sample, the risk exists of more or leas systematic errors resulting from discriminating fractionation effects. However this technique offers, conversely, an interesting way of investigating kinetic isotope effects without the need for specific labelling. In spite of its lower spectral precision the $\text{external referencing method}$ has the advantage of being fast and less sensitive to systematic errois and may be used for direct rough routine determinations of the site-specific isotope contents. More precise results can be obtained, at the price of contaminating the sample, when an $\text{intermolecular reference}$ is added and signal heights are used, remembering however that the intensity parameters then have no strict physical meaning in terms of absolute isotope contents. The site-specific parameters, T_i and (D/H)_i thus accessible, provide new information on the mechanisms of the fractionation effects occurring in natural conditions and examples are considered.