Measurement of the pK(a) Values of Organic Molecules in Aqueous–Organic Solvent Mixtures by (1)H NMR without External Calibrants

[Image: see text] 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 pK(a) 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 pK(a) 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 pK(a) 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 pK(a) in aqueous–organic solvents directly by NMR. We first determine pK(a) of an organic acid such as 2,6-dihydroxybenzoic acid (2,6-DHB) by measuring its (1)H 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 pK(a) of less acidic molecules in single CSI experiments via the variation of their (1)H chemical shifts along pH gradients. As proof of concept, we determine the pK(a) values of organic acids and bases up to pK(a) 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.