High-performance anion exchange-chromatography of neutral milk oligosaccharides and oligosaccharide alditols derived from mucin glycoproteins.

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH approximately 13) has been used to separate neutral oligosaccharides from human milk as well as oligosaccharide alditols isolated by alkaline borohydride degradation of O-linked glycoproteins having blood group A and H activities. Due to the diminished retention times of the alditols compared to their reducing counterparts, a very low base concentration (approximately 15 mM) was used in the fractionation of oligosaccharide alditols. The method appears to be ineffective in fractionation of monosaccharide alditols. Although the retention times generally increased with increasing oligosaccharide chain length, linkage of Fuc alpha-(1----2) to galactose and by Fuc alpha-(1----3) or Fuc alpha-(1----4) to glcNAc may decrease the retention times of both the alditols and the reducing oligosaccharides. Branching generally increased the retention times for oligosaccharide alditols. The retention times of isomers differing in the position of fucose substitution (LNF-1 vs LNF-2) differed greatly while those of the linkage isomers LNF-2 and LNF-3 were similar but distinct. Pulsed amperometric detection is sensitive at the picomole level both for these underivatized oligosaccharides and alditols. On-line desalting with an ion-exchange membrane has been found to be effective in preparative chromatography of these oligosaccharides for NMR spectroscopy and mass spectrometry.