14] High-performance liquid chromatography of oligosaccharides

Abstract

Publisher Summary This chapter proposes separation methods primarily for oligosaccharides that are radiolabeled by metabolic incorporation of 3H- or 14C-labeled sugar, metabolic incorporation of 35SO4 or 32PO4, or chemical incorporation of 3H at the reducing terminus by reduction with NaB3H4. The most common strategy is to first separate the radiolabeled oligosaccharides into different groups on the basis of their anionic character, using anion-exchange high-performance liquid chromatography (HPLC). Oligosaccharides differing in the number and/or character of their anionic groups are then further fractionated on the basis of size, using ion suppression-amine adsorption (ISAA) HPLC. The effects of chemical modifications and enzymatic digestions on individual species are subsequently monitored by anion-exchange HPLC and ISAA–HPLC along with other methods such as lectin affinity HPLC. Four methods for separation of anionic oligosaccharides are illustrated in the chapter. Fractionation of oligosaccharides bearing zero to four anionic moieties, using MicroPak AX-10 or AX-5 columns and a phosphate buffer (pH 4.0), is effective for oligosaccharides bearing terminal sialic acid. Oligosaccharides-bearing sulfate or phosphate can be fractionated under the same conditions; however, the concentration of the initial eluant is reduced to 2.5 mM in order to retain monosulfated species.