Determination of the pKa and Concentration of NMR-Invisible Molecules and Sites Using NMR Spectroscopy

NMR spectroscopy is a very powerful tool for measuring the dissociation constants (pKa) of molecules, requiring smaller quantities of samples of lower purity relative to potentiometric or conductometric methods. However, current approaches are generally limited to those molecules possessing favorable pH-dependent NMR properties. Typically, a series of 1D experiments at varying pH are performed, and the pKa is obtained by fitting the observed chemical shift of the analyte as a function of pH using nonlinear routines. However, the majority of polymers, biomolecules, and inorganic species do not present favorable NMR resonances. Either the resonances are not observable or too broad, or the unambiguous interpretation of the NMR data is impossible without resorting to complex 2D experiments due to spectral overlap. To overcome these fundamental limitations, we present a method to obtain the pKa values and concentrations of acidic species without their direct observation by NMR. We instead determine the quantity of acidic protons removed from the species along a concentration gradient of an organic base in a single 1H chemical shift imaging experiment that can be run under automation. The pKa values are determined via simple linear plots, avoiding complex and potentially unreliable nonlinear fitting routines.