Analytical Aspects of Immobilized Enzyme Columns

The fundamental principles of operation of an Immobilized enzyme flow analyzer have been studied. In particular, models have been developed to explain the dependence of peak height, area, and half-width on sample concentration (0.1 to 2000 mM), sample volume (40-2500 μ), system flow rate (0.3-4.5 mL/min) and column size (0.4-0.9 mL). The system employed in this study Is an Improved high speed thermochemical analyzer based on the use of the enzyme urease which is Immobilized on porous glass by covalent attachment. A sample throughput of over 60 per hour can be achieved In small (0.5 mL) columns packed with approximately 1000 units of enzyme when operated at a flow rate of 2 mL/mln. In general, we find that the dependence of the peak height, area, and width upon sample volume, flow rate, and column configuration are analogous to the behavior of a concentration sensitive chromatographic detector.