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.
Highlights
Ethanol is one of the most important substances with different application
A calibration curve of logarithmic values of ethanol concentrations against change in pH for standard ethanol samples was done. Since this calibration curve is a linear with a correlation coefficient (R) of 0.998, this calibration curve can be used in quantification of ethanol concentration
50 μl of sample were well mixed with 10 μl of alcohol dehydrogenase enzyme to get 60 μl of enzyme-ethanol complex in glass tube. 250 μl of Magic N50 solution was injected into the mixing chamber which contained 1090 μl of buffer
Summary
Ethanol is one of the most important substances with different application. For any application of ethanol it is necessary to detect and quantify ethanol with high accuracy. The available techniques of ethanol detection include gas chromatography, electrochemical and enzymatic assay. The most important methods can be found in previous literatures (Goodman and Jacksonville, 1975; Bauer and Magers, 1985; Watanabe et al 1985; Laccheri, 1987; Moldowan, 1987; Stefan and Luc, 1997; Kempa, 2004; Hernandez, 2005; Olt, 2007; Beutler, 1984; Bernt and Gutman, 1974; Majki and Berkec, 1980)
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