Total compositional analysis by high-performance liquid chromatography or gas-liquid chromatography.

Once the presence of carbohydrate in a glycoprotein has been confirmed, the next step is to determine the precise molar ratio of its monosaccharide constituents. The analysis involves two major phases. The release of the individual monosaccharides is achieved by methanolysis (described here), total acid hydrolysis, or enzymatic release of sialic acids. The resulting mixtures of monosaccharides are then analyzed by methods described in this unit: fractionation, characterization, and quantitation by high-performance liquid chromatography using anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and other HPLC systems or by gas-liquid chromatography with flame ionization detection. The identity of the individual monosaccharides is determined by comparison with known standards processed and analyzed in the same way.