Practical pH Measurements in Wet Organic Solvents by Gradient NMR: From Charged Amino Acid pKa to Identification of Ionizable Natural Products in Crude Extracts

We present NMR-based pH measurements in nondried commercial methanol, methanol–OD, ethanol, and acetonitrile. Combining these measurements with pH gradients and spatially selective NMR enables determination of pKa and provides a highly effective tool to analyze complex molecular mixtures and identify unknown organic compounds by the functional groups present. We determine all the pKa values of alanine, glutamic acid, histidine, lysine, proline, tryptophan, and tyrosine in methanol–OD, filling a major gap in the literature for these important polyprotic molecules that exhibit zwitterionic behavior. To demonstrate the potential of our method for the identification of compounds in natural mixtures, we use pH gradients to identify alkaloids and other ionizable organic compounds in unrefined extracts of hemlock (Conium maculatum), orange lichen (Xanthoria parietina), and love-in-the-mist (Nigella damascena). No chromatographic separation is necessary, highlighting how our technique could provide a fast and untargeted screening of biological samples, reaction mixtures, and digestates.