HPLC analysis of carbohydrates on POLYSPHERCH(R)OH columns using pulsed amperometric detection (PAD) with sodium hydroxide as post column detection reagent.

Carbohydrates have been separated on POLYSPHER(R)CH OH columns using pulsed amperometric detection (PAD) and UV detection (lambda =196 nm) in series and pure water as mobile phase. Nearly baseline separations have been obtained for the glycoprotein carbohydrates of sialic acid ( N-acetylneuraminic acid, NANA), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). As carbohydrates dissolved and eluted with pure water are present in the neutral form they are not detectable with PAD in contrast to carbohydrate anions formed at high pH values. Therefore an additional NaOH post column reagent has been continuously pumped through a mixing chamber into the mobile phase to form carbohydrate anions resulting in improved detection limits. Monosaccharides as well as glycoprotein carbohydrates could be detected in the microg/ml-range. This method has been applied successfully to the analysis of sugars in fruit juice. With only 2 microl of juice per 50 ml water, the determination of the main constituents, sucrose, glucose and fructose, was possible in a few minutes without sample preparation.