Measurement of the pK_a Values of Organic Molecules in Aqueous–Organic Solvent Mixtures by ¹H NMR without External Calibrants

Abstract

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 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 pK a 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 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.

Gut in tube – continuous measurement of metabolic crosstalk between gut microbiota and colonocytes by NMR imaging

“…The multicomponent buffer solution was carefully layered on top of 2.0 mg oxalic acid in a 5.0 mm NMR tube, following our published procedure. [24,25] The sample was then placed in a Bruker 800 MHz Avance III spectrometer at 310 K for 14 hours for a pH gradient to develop, after which a 64-point CSI experiment was carried out. δH of methylphosphonate was taken as the observed chemical shift in a row of the CSI dataset with a pH of 4.7, as judged from the chemical shift of acetate.…”

“…The pH was calculated by taking the sensitivity weighted average of the pH reported by acetate and maleate, as described in our previous work. [24,25] Table S1. δH, δL and pKa for determination of pH in cell culture media at 310 K (I = 0.174 M) using Equation 1.…”