Hydrogen and deuterium nuclei are grossly different in their magnetic properties. Thus it is possible to distinguish between them by NMR spectroscopy. Deuterons will not be observed in a H NMR spectrum and conversely, protons will not be observed in a H NMR spectrum. Where small signals are observed in a H NMR spectrum of a highly deuterated sample, these are referred to as residual signals. They can be used to calculate the level of deuteration in a molecule. Analogous signals are not observed in H NMR spectra because of the low sensitivity of this technique compared to the H analysis. Deuterons typically exhibit very similar chemical shifts to their analogous protons. Analysis via C NMR spectroscopy is also possible: the different spin values of hydrogen (/) and deuterium (1) gives rise to different splitting multiplicities. NMR spectroscopy can be used to determine site-specific deuteration of molecules.
Another method uses HSQC spectra. Typically HSQC spectra are recorded at a series of timepoints while the hydrogen is exchanging with the deuterium. Since the HSQC experiment is specific for hydrogen, the signal will decay exponentially as the hydrogen exchanges. It is then possible to fit an exponential function to the data, and obtain the exchange constant. This method gives residue-specific information for all the residues in the protein simultaneously The major drawback is that it requires a prior assignment of the spectrum for the protein in question. This can be very labor-intensive, and usually limits the method to proteins smaller than 25 kDa. Because it takes minutes to hours to record a HSQC spectrum, amides that exchange quickly must be measured using other pulse sequences.