COLUMN LIQUID-CHROMATOGRAPHIC DETERMINATION OF SACCHARIDES WITH A SINGLE CALIBRATION GRAPH USING POSTCOLUMN ENZYME REACTORS AND COULOMETRIC DETECTION

Immobilized enzymes were used as column reactors in a column liquid chromatographic system for the specific detection of the saccharides stachyose, raffinose and sucrose. Invertase and fructose dehydrogenase (FDH) were immobilized onto poly(vinyl alcohol) beads and porous glass beads, respectively. The oligosaccharides were separated on a cation-exchange resin column with water as the mobile phase. Invertase was capable of quantitatively hydrolysing the oligosaccharides to fructose, which reacts with the hexacyanoferrate(III) ion in the presence of FDH. The hexacyanoferrate(II) ion produced was monitored coulometrically. A single calibration graph for fructose based on the peak area was used to determine each oligosaccharide. The limits of detection for stachyose, raffinose and sucrose were 27, 5 and 2 ng (in a 50-mu-l sample), respectively.