Position-Specific Carbon Stable Isotope Ratios by Proton NMR Spectroscopy.

Carbon stable isotopes provide insights into the origin and synthesis pathway of an organic molecule, and hence, contribute information that is fundamental to understanding chemical, physiological, and ecological processes. Organic carbon 13C/12C isotope ratios are commonly obtained as whole-molecule averages or as measurements of bulk samples. In contrast, position-specific isotope analysis (PSIA) provides isotope ratios for the individual carbons within a molecule, providing additional information that is masked by traditional analytical techniques. Here we introduce a 1H NMR method for determining position-specific 13C/12C ratios within organic molecules. A peak shape superposition procedure is used to bypass the need for traditional peak integration, by exploiting relationships among the shapes of 1H and 13C satellite peaks in 1H NMR spectra. The method also has a significant sensitivity advantage over NMR methods that utilize direct detection of 13C. Furthermore, we demonstrate that isotope standard materials (such as those obtainable from U.S. Geological Survey) are indispensable in calibrating an NMR instrument, in order to obtain accurate isotope ratio results. Our analytical approach was applied to organic molecules of different complexity and origin, including ethanols, propionic acids, and thymidine. Results verify that chemically identical molecules from different sources can have different intramolecular isotope distributions; hence position-specific 13C/12C ratios provide an isotopic fingerprint of an organic molecule. Position-specific information for the nucleoside thymidine, where five of eight carbon positions were measured, is significant because its complexity would make it a difficult target for PSIA by mass spectrometry. The 1H NMR method is complementary to other methods of PSIA, and will make 13C/12C PSIA employable to a wider range of organic molecules.