Abstract
Immobilized enzymes were used as column reactors in a high-performace liquid chromatographic system for the specific detection of sucrose and glucose. Invertase (EC 3.2.1.26, Inv) and pyranose oxidase (EC 1.1.3.10, PyOD) were immobilized onto porous glass beads. The saccharides were separated on a TSKgel SCX column (30 cm × 7.8 mm i.d.) with a 0.01 M acetate buffer (pH 4.5) as a mobile phase. Inv was capable of hydrolyzing sucrose nearly quantitatively (98%) to fructose and glucose, which was oxidized to glucosone and hydrogen peroxide by PyOD. The produced hydrogen peroxide was monitored chemilurninometrically. The calibration graph was linear from 6.0 × 10-7 to 5.0 × 10-4 M sucrose or glucose. The detection limits for sucrose and glucose were 2.0× 10 -7 (3.4 ng) and 3.0× 10 -7 M (2.7 ng in a 50 μl injection), respectively.
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