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.

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) overcomes the limitations other methods and delivers fast carbohydrate analysis. However, due to the high concentration of the biofuel samples, a several-fold sample dilution must be performed before analysis, introducing a source of error. In this study, 2 hardware modifications were applied to handle high-concentration samples: A reduced injection volume from 10 to 0.4 μL and an increase in spacer gasket thickness in the electrochemical flow cell to 62 mil to reduce detection sensitivity.

Wood, grass, and other lignocellulosic biomass are emerging as the preferred sources for biofuel and biochemical production. Compositional analysis of plant derived material is crucial for maximizing the yield of biofuel production. A single HPAE-PAD method, which uses the Dionex CarboPac PA20 column, can be used for the simultaneous determination of mono- and disaccharides and uronic acids.

Size-exclusion chromatography is used in carbohydrate research to determine molecular weight distributions of polysaccharides. Size-exclusion columns separate biological macromolecules by hydrodynamic size. Pulsed amperometric detection (PAD), provides sensitive and specific detection of carbohydrates. Here, fractionated pullulan standards were used to determine the separation range of linear carbohydrates.This application note shows how SEC can be coupled to PAD.