High pH Anion Exchange Chromatography (HPAEC)

Purpose:
Quantitative determination of the monosaccharide composition of biomedical samples and samples originating from animal cells. Specifically, HPAEC test includes the following sugars:

Fucose (Fuc)
N-acetyl-galactosamine (GalNAc)
N-acetyl-glucosamine (GlcNAc)
Galactose (Gal)
Glucose (Glc)
Mannose (Man)

HPAEC is the method of choice for quantitative determination of sialic acids.  Furthermore, HPAEC is used for quantification of oligosaccharides, and collection of oligosaccharide fractions intended for structural profiling.

Methods Used:

For neutral and amino sugar analysis, the sample is hydrolyzed with trifluoroacetic acid, whereas for sialic acid analysis, the sample is hydrolyzed with acetic.  Hydolysates then are dried, resuspended in water, sonicated, and analyzed by HPAEC-PAD.

Amount of sample required for analysis:
For glycoproteins, a minimum of 100 to 200 micrograms is required but preferably more to obtain a conclusive and meaningful data.

General criteria for sample preparation:
Preferably, a purified sample is desired for quick turn-around analysis.  However, the laboratory has some procedures to handle less pure samples such as those containing free sugars or detergents before analysis.