Quantification of Mixtures of C2 and C3 Hydroxy Acids and Products of Glycerol Oxidation by High‐Performance Liquid Chromatography and Quantitative 13C NMR

Abstract The aim of this study was to develop an improved general method for detecting and quantifying mixtures of hydroxy acids and other products of glycerol oxidation in aqueous media, to prevent the confusions that can occur due to similarities and interactions between these compounds depending on media conditions. Standard potential products of glycerol oxidation—glycerol, glyceraldehyde, dihydroxyacetone, glyceric acid, lactic acid, glycolic acid, glyoxylic acid, oxalic acid, tartronic acid, and mesoxalic acid—were analyzed by high‐performance liquid chromatography (HPLC) and quantitative 13C nuclear magnetic resonance (NMR), in mixtures of known composition. The results obtained were concordant with the known compositions tested. HPLC was more accurate than quantitative 13C NMR for simple mixtures, but 13C NMR was required for complex mixtures containing dihydroxyacetone and glycerol, oxalic acid and mesoxalic acid, or glyoxylic acid and tartronic acid, pairs of compounds not well separated or detected by HPLC. As proof‐of‐concept, an unknown mixture generated by glycerol oxidation was analyzed by HPLC and quantitative 13C NMR. The results obtained were concordant and allowed accurate determination of the composition of the sample, which contained mesoxalic acid as the major product, with oxalic acid, tartronic acid, and glyceric acid as by‐products.