This work describes an HPAE-PAD method for monosaccharide composition analysis using manually prepared eluent. Monosaccharide analysis using electrolytically generated eluent has been described before. Here, three commercially available proteins, IgG, fetuin, and alpha-1-acid glycoprotein, were individually subjected to two hydrolysis conditions using 1)HCl, for the amino sugars galactosamine and glucosamine, and 2)TFA, for the neutral sugars mannose, glucose, and galactose. Results for method linearity, robustness, and accuracy are presented here. An ICS-6000 can be used for this application.

This is the first document showing the use of eluent generation for HPAE-PAD monosaccharide analysis of glycoproteins. Those interested in an updated version should see TN40. This record is provided for those with an EG40 and a Dionex CarboPac PA10 column that would like to do this application. This document also has useful sample preparation information for HPAE-PAD glycorprotein monosaccharide analysis.

Characterization of glycoproteins routinely involves carbohydrate analysis. Studies show that HPAE-PAD is a simple and effective way to determine glycoprotein monosaccharide composition without derivatization. In a typical method, monosaccharides are separated on a Thermo Scientific Dionex CarboPac PA10 or PA20 anion-exchange column, preceded by a Thermo Scientific Dionex AminoTrap guard column, using a hydroxide eluent prepared manually or generated by an eluent generator. This TN reports such a method.

Free amino acids and small peptides can interfere with HPAE-PAD monosaccharide analysis. An AminoTrap column in front of a CarboPac column can remove the aforementioned interference. This TN describes how to use the AminoTrap and how to test if it functioning correctly. The record reports an AminoTrap test method.

In this application note, the organic impurities in heparin are determined by the HPAE-PAD method using the CarboPac PA20 column following the USP monograph method. This method was repeated using manually prepared eluents and an electrolytically generated eluent, with both eluent preparation options providing data that exceeds the system suitability requirements in the monograph.