Novel Nuclear Magnetic Resonance Method for Position-Specific Carbon Isotope Analysis of Organic Molecules with Significant Impurities

Abstract

We introduce a novel nuclear magnetic resonance (NMR) tool for determining position-specific carbon (13C/12C) isotope ratios within complex organic molecules. This analytical advancement allows us to measure position-specific isotope ratios of samples that contain impurities with NMR peaks that overlap with the signals of interest. The method involves collecting a series of alternating 13C-coupled and 13C-decoupled 1H NMR spectra using an NMR pulse sequence designed to optimize temperature stability, followed by a data reduction scheme that allows the signals of interest to be isolated from signals of impurities. The method was validated using glycine reference materials with known 13C/12C ratios from the US Geological Survey (USGS) into which impurities typically found in amino acid samples were intentionally introduced. Following validation, the method was used to determine position-specific 13C/12C ratios in a set of USGS l-valine materials (USGS73, -74, -75) that contain significant impurities associated with their biological origin. The l-valines were found to contain distinct intramolecular isotope variability, and the 13Cα isotope spikes in USGS74 and USGS75 were clearly detected, where they preserve carbon isotope ratios of −4.8 ± 0.9‰ and +11.5 ± 0.8‰, respectively. Carbon isotope abundance at the beta and gamma positions indicates that the USGS73 l-valine was obtained from a different source than USGS74 and -75. This analytical approach is a significant step forward in the field of position-specific isotope analysis at natural abundance via NMR because it enables the investigation of samples that contain impurities which are typically present in samples derived from natural sources.

Fingerprinting Organofluorine Molecules via Position-Specific Isotope Analysis

“…Although position-specific 13 C/ 12 C analyses of fluorinated organics appear to be absent from the literature prior to the present work, previous studies of organics that lack fluorine have shown that molecules can contain extensive variation in 13 C/ 12 C abundance at the individual carbon positions, with intramolecular differences of 5 to 20‰ being common. − Position-specific carbon isotope abundance has been shown to depend upon the source of a molecule. ,,, Moreover, intramolecular carbon isotope variability can also depend on environmental conditions, as recently demonstrated by Wilkes et al (2022), who found that δ 13 C values at the C1 position of serine differed by up to 5‰ for serine molecules obtained from plants grown under various p CO 2 conditions.…”

“…The observation that organofluorine substances preserve significant intramolecular carbon isotope variability is consistent with previous studies of organics that lack fluorine, which have been found to exhibit distinctive intramolecular 13 C/ 12 C fingerprints, reflective of their source. [41][42][43]50,69 Both 19 F NMR and 1 H NMR were used in the positionspecific analyses of 2,2,2-trifluoroethanol and 5-fluorouracil, to obtain 13 C/ 12 C ratios at carbon positions with C−F as well as C−H bonds. Combining the 19 F and 1 H NMR tools provides an additional component to the isotope fingerprint, resulting in more information per molecule, and a more powerful analytical approach.…”

“…The present work uses a novel nuclear magnetic resonance (NMR) spectroscopy approach to obtain position-specific 13 C/ 12 C ratios for organofluorine molecules. Position-specific isotope analysis differs from traditional IRMS in that it provides 13 C/ 12 C ratios for individual carbon atom positions within a molecule, yielding a unique isotope fingerprint. − In traditional IRMS, the sample is completely combusted to CO 2 before mass analysis, so that only an average 13 C/ 12 C abundance is obtained for the sample, which can be a mixture or a specific compound that was isolated prior to analysis. Due to the combustion process, traditional IRMS loses the wealth of information that may be contained within the intramolecular carbon isotope distribution.…”

Re‐determination of R(13C/12C) for Vienna Peedee belemnite (VPDB)

“…Data processing for clumped isotope analyses of carbon dioxide also requires R VPDB ( 13 C/ 12 C). 28 Other methods to determine carbon isotopic composition such as nuclear magnetic resonance (NMR) [29][30][31][32] and optical spectroscopy 33,34 may also use R VPDB ( 13 C/ 12 C) during data processing to ensure carbon isotope delta values are consistent with those obtained by IRMS, unless calibration is performed using RMs of known carbon isotope delta.…”

NMR-Based Method for Intramolecular 13C Distribution at Natural Abundance Adapted to Small Amounts of Glucose

“…To solve both the sample size and analysis time issues, the use of 1 H NMR has been proposed, by measuring the ratio of the intensity of the signal coming from 1 H– 12 C to that of signals generated by 1 H– 13 C (satellite peaks). Even though the proof of concept has been published, − the use of this methodology is presently limited to resolved spectra. In the case of the glucose derivative, the molecule studied in the present work, two proton chemical shifts at least are identical, making the integration of the satellites with the appropriate precision impossible.…”