Position-specific 15N isotope analysis in organic molecules: A high-precision 15N NMR method to determine the intramolecular 15N isotope composition and fractionation at natural abundance

The position-specific N-15 isotope content in organic molecules, at natural abundance, is for the first time determined by using a quantitative methodology based on N-15 Nuclear Magnetic Resonance (NMR) spectrometry. N-15 NMR spectra are obtained by using an adiabatic "Full-Spectrum" INEPT sequence in order to make possible N-15 NMR experiments with a high signal-to-noise ratio (>500), to reach a precision with a standard deviation below 1 parts per thousand (0.1%). This level of precision is required for observing small changes in N-15 content associated to N-15 isotope effects. As an illustration, the measurement of an isotopic enrichment factor epsilon for each N-15 isotopomer is presented for 1-methylimidazole induced during a separation process on a silica column. The precision expressed as the long-term repeatability of the methodology is good enough to evaluate small changes in the N-15 isotope contents for a given isotopomer. As observed for C-13, inverse and normal N-15 isotope effects occur concomitantly, giving access to new information on the origin of the N-15 isotope effects, not detectable by other techniques such as isotope ratio measured by Mass Spectrometry for which bulk (average) values are obtained.