Concentration measurements by PULCON using X-filtered or 2D NMR spectra.

Sample concentrations can be measured by nuclear magnetic resonance (NMR) spectroscopy without an internal reference compound using pulse length based concentration determination (PULCON) with 1D NMR spectra. PULCON delivers most accurate results if the spectrum of the sample of interest contains a resolved resonance; but can also be applied to spectral regions with overlapping resonances. If the exact number of lines contributing to the overlapping spectral region is not known, a corresponding error makes the result less precise. The uncertainty about the number of contributing resonances can be reduced with experiments that discriminate different classes of resonances by filtering techniques or by extending PULCON to two-dimensional NMR spectra. We demonstrate the application of PULCON with a 1D 15N-filtered experiment where aromatic resonances of a protein can be observed without interference from 15N-bound protons. Further, we extend PULCON to 2D NMR spectra that permits to determine the exact number of resonances. This extension can readily be applied with sample that contain different solutes.