Determination\nof the p<i>K</i><sub>a</sub> and Concentration of NMR-Invisible\nMolecules and Sites Using NMR\nSpectroscopy

NMR spectroscopy is a very powerful tool for measuring\nthe dissociation\nconstants (p<i>K</i><sub>a</sub>) of molecules, requiring\nsmaller quantities of samples of lower purity relative to potentiometric\nor conductometric methods. However, current approaches are generally\nlimited to those molecules possessing favorable pH-dependent NMR properties.\nTypically, a series of 1D experiments at varying pH are performed,\nand the p<i>K</i><sub>a</sub> is obtained by fitting the\nobserved chemical shift of the analyte as a function of pH using nonlinear\nroutines. However, the majority of polymers, biomolecules, and inorganic\nspecies do not present favorable NMR resonances. Either the resonances\nare not observable or too broad, or the unambiguous interpretation\nof the NMR data is impossible without resorting to complex 2D experiments\ndue to spectral overlap. To overcome these fundamental limitations,\nwe present a method to obtain the p<i>K</i><sub>a</sub> values\nand concentrations of acidic species without their direct observation\nby NMR. We instead determine the quantity of acidic protons removed\nfrom the species along a concentration gradient of an organic base\nin a single <sup>1</sup>H chemical shift imaging experiment that can\nbe run under automation. The p<i>K</i><sub>a</sub> values\nare determined via simple linear plots, avoiding complex and potentially\nunreliable nonlinear fitting routines.