HPLC ANALYSIS OF CARBOHYDRATES ON POLYSPHER(R)CH 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 p ulsed 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-ace tylgalactosamine (GalNAc). As carbohydrates dissolved and eluted with pure water are present in the neutral form they are not detectable wit h PAD in contrast to carbohydrate anions formed at high pH values. The refore an additional NaOH post column reagent has been continuously pu mped through a mixing chamber into the mobile phase to form carbohydra te anions resulting in improved detection limits. Monosaccharides as w ell as glycoprotein carbohydrates could be detected in the mu g/ml-ran ge. This method has been applied successfully to the analysis of sugar s in fruit juice. With only 2 mu l of juice per 50 ml water, the deter mination of the main constituents, sucrose, glucose and fructose, was possible in a few minutes without sample preparation.