Chromatographic Performance of an Amine/amino-bonded Column and a Monolithic Reversed-Phase Column for the Separation of Fluorescently Labeled Glycoprotein Glycans

Abstract

For the HPLC analysis of glycoprotein glycans, oligosaccharides released from glycoproteins are often derivatized with fluorescent tags to achieve quantitative separation. However, the influence of such fluorescent tags on the structure and retention of glycans is not completely understood. Here, to investigate the separation of labeled glycans, we prepared four types of derivatives (2-aminopyridine, 2-aminobenzoic acid, 2-aminobenzamide, and ethyl 4-aminobenzoate) of three model glycans (high-mannose oligosaccharides from ribonuclease B, complex oligosaccharides from bovine fetuin, and an isomaltooligosaccharide mixture). The glycan derivatives were separated on newly developed stationary phases, namely, an aminoand amide-bonded hydrophilic (AAH) phase and a capillary-packed monolithic ODS phase without end-capping treatment. The performance of these phases was compared with typical amide hydrophilic interaction chromatography (HILIC) and end-capped C18 packed columns. In the HILIC separation mode, the AAH column exhibited superior resolution for isomaltooligosaccharides, compared to those obtained on a conventional amide column. Nevertheless, the plate numbers were the same for glycoprotein glycans and the three linkage isomers of Man7GlcNAc2 could not be resolved, whereas they could be separated on the amide column. However, the cationic amino groups of the AAH column enhanced the resolution of sialylated complex glycans that could not be separated on the amide HILIC column. In the reversed-phase separation mode, the retention of glycan derivatives was mainly dependent on the hydrophobicity of the labeling groups. In contrast to the conventional packed ODS column, the monolithic capillary ODS column showed good resolution for neutral glycans.