Quantitative NMR Spectroscopy under High Hydrostatic Pressure

High-resolution nuclear magnetic resonance (NMR) spectroscopy is essential for molecular characterization at atomic resolution in chemical research. Here we report and justify the experimental setup to be followed when data acquisition occurs in solution at elevated hydrostatic pressure of up to thousands of bars. We demonstrate how the compressibility of the solvent can be reliably and accurately determined by combining resonance signals originating from six different isotopes using pressure-resistant molecules. The knowledge of solvent compression is then used for the precise quantification of changes in free energy and volume of a biomolecule that are caused by increasing hydrostatic pressure. Our data show that solvent compression must be considered when conducting quantitative analyses of data obtained with NMR spectroscopy at high hydrostatic pressure.