High-throughput HPAEC-PAD for the analysis of neutral sugars and uronic acids in biomass hydrolysates

Lignocellulosic and marine biomasses are valuable renewable sources of cellulose and alginate polysaccharides. Accurate quantification of the neutral sugars and uronic acids content in complex biomass hydrolysates is essential for effective characterization and use. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) provides sensitive, derivatization-free analysis with good isomer separation. However, traditional methods often involve lengthy gradients and demanding column maintenance, which limit throughput. In this study, we introduce and validate a high-throughput HPAEC-PAD method tailored for quantifying neutral sugars and uronic acids in biomass-derived polysaccharide hydrolysates. Using an efficient anion-exchange stationary phase, optimizing elution conditions, and automating analysis on one column while simultaneously reconditioning the other, a rapid and robust workflow is presented. The presented method separates 12 neutral sugars and 5 uronic acids in a single run, with most analytes baseline resolved (R_s > 1.5). The automated approach reduces cycle times from 60 min to 33 min without sacrificing separation quality, quantitation, or sensitivity. This translates to an increased throughput by approximately 82% for combined neutral sugar and uronic acid analysis, and by roughly 160% and 445% when focusing solely on each component class. Calibration was assessed by relative standard error (RSE) in line with the International Union of Pure and Applied Chemistry (IUPAC) guidance. The average RSE on calibration is below 2.3% for all analytes. The detection limits for all analytes range from 0.02 µmol/L to 0.40 µmol/L across all methods and columns. Overall, the results highlight both the efficiency and analytical robustness of this optimized workflow, which supports rapid, high-volume screening of polysaccharides in biomass processing and biopolymer research.