A New Amperometric Carbon Paste Enzyme Electrode for Ethanol Determination

Abstract

In this study, a new amperometric carbon paste enzyme electrode for determination of ethanol was developed. The carbon paste was prepared by mixing alcohol dehydrogenase, its coenzyme nicotinamide adenine dinucleotide (oxidized form, NAD⁺), poly(vinylferrocene) (PVF) that was used as a mediator, graphite powder and paraffin oil, then the paste was placed into cavity of a glass electrode body. Determination of ethanol was performed by oxidation of nicotinamide adenine dinucleotide (reduced form, NADH) generated enzymatically at +0.7 V. The effects of enzyme, coenzyme and PVF amounts; pH; buffer concentration and temperature were investigated. The linear working range of the enzyme electrode was 4.0×10⁻⁴–4.5×10⁻³ M, determination limit was 3.9×10⁻⁴ M and response time was 50 s. The optimum pH, buffer concentration, temperature, and amounts of enzyme, NAD⁺ and PVF for enzyme electrode were found to be 8.5, 0.10 M, 37°C, 2.0, 6.0, and 12.0 mg, respectively. The storage stability of enzyme electrode at +4°C was 7 days. Enzyme electrode was used for determination of ethanol in two different wine samples and results were in good agreement with those obtained by gas chromatography.

Keywords:

• Alcohol dehydrogenase
• amperometry
• enzyme electrode
• carbon paste
• ethanol
• poly(vinylferrocene)

Acknowledgments

We gratefully acknowledge the financial support of T.R. Prime Ministry State Planning Organization (Project No: 98‐K‐120830), Ankara University, Biotechnology Institute (Project No: 155) and a scholarship by Scientific and Technical Research Council of Turkey for P. E. ERDEN.