Fast Quantitative 1H-13C Two-Dimensional NMR with Very High Precision

Quantitative analysis by nuclear magnetic resonance (NMR) requires highly precise measurements to achieve reliable quantification. It is particularly true in C-13 site-specific natural isotope fractionation studied by nuclear magnetic resonance, where the range of values of C-13 isotopic deviations at natural abundance is highly restricted. Consequently, an NMR method capable of measuring delta C-13 parts per thousand values with a very high precision (a few per mil) is indispensable. This high degree of precision has already been achieved by one-dimensional C-13 acquisitions; however, this approach is limited by peak overlaps which reduce the precision of the isotope content determination, even for certain small molecules. It is therefore necessary to extend this promising methodology to a higher dimensionality. In this context, this paper aims at determining conditions that allow the achievement of two-dimensional (2D) H-1-C-13 heteronuclear experiments with a precision of a few per mil in a reasonable time. Our results demonstrate that a high precision (repeatability of 2 per mil) can be reached with the H-1-C-13 HSQC (Heteronuclear Single Quantum Correlation) experiment, thus satisfying the conditions needed to perform C-13 isotope analysis by 2D NMR. We also consider the impact of several approaches which have been proposed to reduce the duration of heteronuclear 2D experiments. Two of these common time-saving strategies, spectral aliasing and linear prediction, are fully compatible with the high-precision requirements of isotopic NMR, while a third one, nonuniform sampling, leads to dramatic precision losses. In conclusion, this study demonstrates the feasibility of very precise 2D NMR measurements and opens a number of application perspectives.