Accurate Quantitative 13C NMR Spectroscopy:  Repeatability over Time of Site-Specific 13C Isotope Ratio Determination

The stability over time (repeatability) for the determination of site-specific 13C/12C ratios at natural abundance by quantitative 13C NMR spectroscopy has been tested on three probes: enriched bilabeled [1,2-13C2]ethanol; ethanol at natural abundance; and vanillin at natural abundance. It is shown in all three cases that the standard deviation for a series of measurements taken every 2−3 months over periods between 9 and 13 months is equal to or smaller than the standard deviation calculated from 5−10 replicate measurements made on a single sample. The precision which can be achieved using the present analytical 13C NMR protocol is higher than the prerequisite value of 1−2‰ for the determination of site-specific 13C/12C ratios at natural abundance (13C-SNIF-NMR). Hence, this technique permits the discrimination of very small variations in 13C/12C ratios between carbon positions, as found in biogenic natural products. This observed stability over time in 13C NMR spectroscopy indicates that further improvements in precision will depend primarily on improved signal-to-noise ratio.