Separation of fucosylated oligosaccharides using high-pH anion-exchange chromatography with pulsed-amperometric detection

Abstract

Fucosylated oligosaccharides of the βGal(1→4)GlcNAc-, βGal(1→4)Glc-, and βGal(1→3)GlcNAc-series were chromatographed on a high-performance anion-exchange pellicular resin under alkaline conditions (pH ⋍13). Fucosylation of either lactose, lactosamine (Type II chains), or lacto- N-biose (Type I chains) oligosaccharides markedly decreased the retention time (10–38 min) of the non-fucosylated form. The magnitude of the reduction was related to whether fucose replaced Gal [αFuc(1→3)→GlcNAc], whether fucose was α-(1→2)-linked to Gal at the end of a chain, or whether fucose was linked in a subterminal position [α(1→3) or α(1→4)] to Gal or GlcNAc. The results suggest that the decreases in retention times of fucosylated oligosaccharides (10–38 min) is not attributable to the absence of a 6-OH in Fuc but instead to steric and substitution effects which affect the interaction of the most readily ionizable groups of Fuc (2-OH), Gal (2-OH), and GlcNAc (3-OH) with the stationary phase. We show that high-pH anion-exchange chromatography can effectively separate 1→2, 1→3, and 1→4 fucose positional isomers in a single chromatographic step.