Liquid chromatographic determination of trace amounts of saccharides typically needs derivatization to improve the detectability due to their low UV-absorptivities. Pre-column derivatization of saccharides, followed by reversed-phase chromatography is a sensitive methodlp4. However, quantitation is one of the problems associated with the methods.
Post-column derivatization has been employed for the quantitative analysis of saccharides and a number of different fluorescent reagents have been examined for reducing saccharides'-". However, very few fluorescent reagents are known for nonreducing saccharides' l,12.
Galactose oxidase (D-galactose:oxygen oxidoreductase, E.C. 1.1.3.9) (Gal-OD) catalyzes the oxidation of galactose by molecular oxygen. Oligosaccharides containing galactose such as raffinose and stachyose are oxidized much more rapidly than galactose itself. Gal-OD has been used as a reagent for the determination of galac-tose13-16. It has also been immobilized on glass beads and used as a reactor for the determination of galactose in flow injection analysis"-i9.
This paper describes the use of immobilized enzyme as a column reactor in a liquid chromatographic system for the specific detection of stachyose, raffinose, melibiose and galactose. Gal-OD and peroxidase (doner:hydrogen peroxide oxidoreductase, E.C. 1.11.1.7) (POD) were co-immobilized onto hydrophilic vinyl polymer beads. The saccharides were separated on a cation-exchange resin column with water as the mobile phase. In the column reactor, the saccharides were converted into hydrogen peroxide by Gal-OD, which reacts with p-hydroxyphenyl acetate in the presence of POD, and the fluorophore produced was detected. The fluorophore (6,6'dihydroxy-3,3'-biphenyldiacetate) is formed in the following reaction sequence: D-Galactose + O2 ?F?$ D-galatohexodialdose + H202 H202 + p-hydroxyphenylacetate '2 Hz0 + fluorophore This method is sensitive and highly specific for the detection of stachyose, raffinose, melibiose and galactose.