Chromatographic analysis of oxidized cello-oligomers generated by lytic polysaccharide monooxygenases using dual electrolytic eluent generation

Abstract Research on oligosaccharides, including the complicated product mixtures generated by lytic polysaccharide monooxygenases (LPMOs), is growing at a rapid pace. LPMOs are gaining major interest, and the ability to efficiently and accurately separate and quantify their native and oxidized products chromatographically is essential in furthering our understanding of these oxidative enzymes. Here we present a novel set of methods based on dual electrolytic eluent generation, where the conventional sodium acetate/sodium hydroxide (NaOAc/NaOH) eluents in high-performance anion-exchange chromatography (HPAEC) are replaced by electrolytically-generated potassium methane sulfonate/potassium hydroxide (KMSA/KOH). The new methods separate all compounds of interest within 24-45 minutes and with high sensitivity; limits of detection and quantification were in the range of 0.0001-0.0032 mM and 0.0002-0.0096 mM, respectively. In addition, an average of 3.5 times improvement in analytical CV was obtained. This chromatographic platform overcomes drawbacks associated with manual preparation of eluents and offers simplified operation and rapid method optimization, with increased precision for less abundant LPMO-derived products.