Simultaneous determination of glucose and sucrose using a flow system with two enzyme reactors and an octadecylsilica column in one line

ABSTRACT Simultaneous determination of glucose and sucrose was achieved with an apparatus containing two enzyme reactors and an octadecylsilica column positioned between the two reactors in tandem in a single flow line. The enzymes used were mutarotase and glucose dehydrogenase for glucose analysis, and sucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate dehydrogenase for sucrose analysis. The pH and the concentration of the phosphate buffer used as the medium for immobilization of mutarotase and glucose dehydrogenase to activated aminopropyl glass were studied. Several aqueous buffers and their pH values were also examined for use as the carrier medium, and HEPES buffer (pH 7.5) was the most favorable. In this apparatus under these conditions, NADH due to glucose and that due to sucrose were observed separately. The calibration curve for glucose was linear in the range of 1 - 500 μM, and that for sucrose, 5 - 500 μM. This method was applied to the analyses of glucose and sucrose in several ...