Separation and identification of enzymatic sucrose hydrolysis products by high-performance anion-exchange chromatography with pulsed amperometric detection

Abstract

An accurate carbohydrate analysis method, namely high-performance anion-exchange chromatography with pulsed amperometric detection was successfully applied to the study of sucrose hydrolysis under enzymatic (baker’s yeast invertase) conditions. The hydrolysis was monitored by determining sucrose degradation and the corresponding formation of d-glucose, d-fructose and five intermediate fructans using a CarboPac PA-100 (Dionex) analytical anion-exchange column. Highly reproducible results were obtained. The unknown fructans were collected from a semi-preparative CarboPac PA-100 (Dionex) column, neutralized and then desalted on a column containing mixed bed resin AG 501-X8 (D) before identification of the chemical structure. This procedure permitted us to obtain about 20 μg of pure product which is not enough for NMR analysis. Detailed GC–MS analytical data of the methylated compounds indicated that these oligosaccharides were β- d-Fru-(2→1)-β- d-Fru-(2→1)-α- d-glucopyranoside (1-kestose), β- d-Fru-(2→6)-α- d-glucopyranoside (6-β fructofuranosylglucose), β- d-Fru-(2→1)-β- d-fructofuranoside (inulobiose), β- d-Fru-(2→6)-β- d-Fru-(2→1)-α- d-glucopyranoside (6-kestose) and β- d-Fru-(2→6)-α- d-Glc-(1→2)-β- d-fructofuranoside (neokestose) coeluating with a disaccharide.