Abstract

The aim of this study is to develop a method for determining full range of ethanol concentrations with high accuracy and low cost. Since applying other methods (e.g. non-enzymatic, optical...etc) suffer from difficulties, it was intended to develop method which overcomes them. A differential pH measurement device was used to achieve operation conditions of alcohol dehydrogenase reaction. Optimum operating conditions were temperature of 30°C, 10 µl of alcohol dehydrogenase enzyme volume (with a final activity of 563.75 units ml-1) per 50 µl of sample, NAD+ concentration of 0.05 mM and 20 mM glycine-pyrophosphate buffer solution of pH 9.1. In this method a range of ethanol concentrations from 0 - 99,985 %, which means 0.000001714 - 17.14 M, were used. The maximum obtained change in pH, delta pH, was (-33) mpH.

End point of equilibrium concentrations of reactants and products of ethanol oxidation reaction was measured within spectrophotometer. The results indicated 100 seconds to reach the end point for all ethanol standard samples. This required time was satisfied with results of measuring change in pH within differential pH analyzer system.

It seems that there is no method of determining a wide range of ethanol concentrations available yet. In contrary, this work was effective in quantifying full range of ethanol concentrations. Applying this method will reduce cost of assaying of samples as small amount of one enzyme, alcohol dehydrogenase, and low NAD⁺ concentration are used.

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