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

An accurate carbohydrate analysis method, namely high-performance anion-exc hange chromatography with pulsed amperometric detection was successfully ap plied to the study of sucrose hydrolysis under enzymatic (baker's yeast inv ertase) conditions. The hydrolysis was monitored by determining sucrose deg radation and the corresponding formation of D-glucose, D-fructose and five intermediate fructans using a CarboPac PA-100 (Dionex) analytical anion-exc hange column. Highly reproducible results were obtained. The unknown fructa ns 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 per mitted us to obtain about 20 mug of pure product which is not enough for NM R analysis. Detailed GC-MS analytical data of the methylated compounds indi cated that these oligosaccharides were beta -D-Fru-(2 -->1)-beta -D-Fru-(2 -->1)-alpha -D-glucopyranoside (1-kestose), beta -D-Fru-(2 -->6)-alpha -D-g lucopyranoside (6-beta fructofuranosylglucose), beta -D-Fru-(2 -->1)-beta - D-fructofuranoside (inulobiose), beta -D-Fru-(2 -->6)-beta -D-Fru-(2 -->1)- alpha -D-glucopyranoside (6-kestose) and beta -D-Fru-(2 -->6)-alpha -D-Glc- (1 -->2)-beta -D-fructofuranoside (neokestose) coeluating with a disacchari de. (C) 2001 Elsevier Science B.V. All rights reserved.