Enzyme-based biosensor as a selective detection unit in column liquid chromatography.

A reagentless enzyme electrode based on co-immobilized alcohol oxidase and horseradish peroxidase was used as the working electrode in an amperometric flow-through cell connected to a column liquid chromatographic (CLC) system for the selective detection of methanol and ethanol. The enzymes were covalently immobilized in carbon paste (graphite-phenylmethylsilicone oil) in the presence of polyethylenimine. Electrodes prepared from the enzyme-modified carbon paste were optimized with respect to their sensitivity and selectivity. Different membranes were cast or electropolymerized directly on the surface of the electrode to increase the long-term stability of the biosensor. The compatibility with the reversed-phase chromatographic system was established. A PLRP-S polymer-based separation column was used with phosphate buffer as the mobile phase. The selectivity of the enzyme electrode was also determined by injecting some easily oxidizable and possibly interfering species normally present in biological samples. The enzyme electrode was also used in an on-line system, consisting of a microdialysis probe as the sampling unit, the CLC system and the biosensor detection device, for the selective following of the ethanol produced when a paper pulp industrial waste water was fermented with Saccharomyces cerevisiae.