Immobilization of glucosidase onto silica-based, amino functionalized beads for enzymatic hydrolysis of urinary phenol prior to liquid chromatographic analysis

Abstract

An amino-functionalized silica commercial liquid chromatography packing material was used instead of controlled-pore glass as the support to react with glutaraldehyde and then to directly immobilize the β-glucosidase. After enzymatic hydrolysis, the urinary phenol was separated by reversed-phase liquid chromatography and monitored by an electrochemical detector at 0.85 V (vs. Ag AgCl ). Factors that affect the immobilization and hydrolysis were investigated. The proposed method provides a simpler procedure with a less expensive material for glucosidase immobilization, and the electrochemical detection avoids the interference from other urinary species and hence simplifies the chromatogram obtained. The detection limit was 0.2 ng with a 20 μl injection. The relative standard deviations for 1.2 ng and 2.0 ng levels of phenols were 1.16% and 3.38%, respectively.