Separation, detection, and quantification of galacturonic acid oligomers with a degree of polymerization greater than 50

A high performance anion exchange chromatography (HPAEC)-evaporative light scattering detector (ELSD)-method was developed to detect, separate, and quantify galacturonic acid (GA) oligomers. Following digestion of polygalacturonic acid (PGA) with a monocomponent endo-polygalacturonase (EPG), more than 70 GA oligomer peaks could be resolved using a convex/linear ammonium formate gradient. Linear calibration curves were produced for 0.015-1.0% mono-, di-, and tri-GA. The mass response for mono-GA differed from those for di- and tri-GA, as evidenced from the slope of the calibration curve regression lines (1.611 +/- 0.0201 for-mono-GA vs. 1.3068 +/- 0.0291 and 1.3004 +/- 0.0262 for di-, and tri-GA, respectively). The degree of polymerization (DP) appeared to affect mass response as the trend line for log-transformed peak areas of DP 3, 4, 6, and 8 oligomers had a slope of -0.0304 +/- 0.0032 (r(2) = 0.98). Buffer concentration also affected mass response. ANOVA of peak areas from isocratic elution of trimer and hexamer with 50 mM to 0.8 M ammonium formate indicated mass response was dependent on buffer concentration for each oligomer (P<0.005), although Duncan's Multiple Range Test described concentration ranges within which mass response was not affected (P<0.05).