Measurement of the p<i>K</i>_a Values of Organic Molecules in Aqueous–Organic Solvent Mixtures by ¹H NMR without External Calibrants

Aqueous–organic solvent mixtures are commonly used for reactions or analyses, where the components of a system are insoluble in pure water. The acid dissociation constant is an important property to measure in these media as it determines the charge state, solubility, and reactivity of a molecule. While NMR spectroscopy is an established tool for the measurement of pKa in water, its use in aqueous–organic solvents is greatly hindered by the requirement for external calibrants on which a working pH scale is set. Such calibrants include buffer solutions, “anchor” molecules with known pKa values, and pH electrodes that have undergone lengthy calibration procedures in the solvent mixture of interest. However, such calibrations are often inconvenient to perform, while literature pKa data covering the required range may not be available at the solvent composition or the temperature of interest. Here, we present a method to determine pKa in aqueous–organic solvents directly by NMR. We first determine pKa of an organic acid such as 2,6-dihydroxybenzoic acid (2,6-DHB) by measuring its 1H chemical shift as a function of concentration along a concentration gradient using chemical shift imaging (CSI). Using 2,6-DHB as a reference, we then determine pKa of less acidic molecules in single CSI experiments via the variation of their 1H chemical shifts along pH gradients. As proof of concept, we determine the pKa values of organic acids and bases up to pKa 10 in 50% (v/v) 1-propanol/water, 50% (v/v) dimethyl sulfoxide/water, and 30% (v/v) acetonitrile/water and obtain good agreement with the literature values