Development of Flow Injection Analysis System for Amperometric Determination of Cholesterol Using Immobilized Enzyme Columns

Min-Chol Shin and Hak-Sung Kim

A flow injection analysis(FIA) system was developed for the determination of cholesterol using immobilized cholesterol oxidase and cholesterl ester hydrolase. The enzymes were immobilized on controlled pore glass(CPG) by the glutaraldehyde method. The glass columns packed with immobilized enzymes were found to contain 3-5 I. U. for each enzyme. A hydrogen peroxide sensitive electrode was constructed and applied to the FIA system. The operational conditions for FIA response were investigated and optimized with variation of sampling volume, flow rate and composition of carrier solution. The FIA response were linear upto 60 and 400mg/mL for free cholesterol and cholesterol ester, respectively. All samples were analyzed with a good precision(<2.5% CV) and accuracy. 23 samples were measured succesively within about an hour. Intermittent assays if more than 500 times caused 50% decrease in response sensitivity.