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

Abstract

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 per thousand 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.

Fractionation in position-specific isotope composition during vaporization of environmental pollutants measured with isotope ratio monitoring by 13C nuclear magnetic resonance spectrometry

“…As a result, the development of the technique took longer, starting from the first work (Caer et al, 1991) to the recent attainment of suitable acquisition conditions (Caytan et al, 2007a). A relative method of 13 C NMR was previously proposed as a way to calculate isotope effects in which the NMR signal intensity of the site-active carbon is related to the intensity of the signal of a 'neutral' carbon (far away from the active site), making the assumption that the latter is not modified during the chemical reaction (Singleton and Thomas, 1995).…”

Comparison of extraction techniques and modeling of accelerated solvent extraction for the authentication of natural vanilla flavors

“…This technique was recently proposed as an AOAC official method [20] and applied to 13 C determinations [21,22]. Vanilla beans contain more than 200 volatile compounds [23].…”

Nonstatistical 13C Distribution during Carbon Transfer from Glucose to Ethanol during Fermentation Is Determined by the Catabolic Pathway Exploited

“…Third, slow relaxation times can lead to long acquisition times and associated potential instability, a difficulty largely solved by the use of relaxation agents (4). Now, this technique can be used for quantification of individual isotopomers by exploiting (i) the separation of the resonance signals from the different carbon positions caused by their degree of shielding and (ii) the direct relationship of the peak area to the amount of 13 C resonating at a given frequency (5). Acquisition conditions need to be carefully defined and rigorously controlled to obtain repeatable spectra of sufficient quality for the required precision.…”

“…The NOE has to be eliminated, and the efficiency of the 1 H decoupling sequence must be uniform over the whole range of 1 H chemical shifts (12 ppm). Studies (3,(5)(6)(7) on the optimization of an NMR methodology for accurate and precise measurements of 13 C/ 12 C isotope ratios have shed light on how the decoupling conditions of 1 H in 13 C single-pulse NMR experiments strongly affect the precision of measured peak surface areas. A major breakthrough was achieved with the development of an optimized adiabatic 1 H decoupling sequence (3).…”