Analysis of Free and Total Glycerol in Biodiesel Using an Electrochemical Assay Based on a Two-Enzyme Oxygen-Electrode System

In this work, an electroenzymatic methodology based on two coupled enzymatic activities (glycerokinase and glycerol-3-phosphate oxidase) was developed using an oxygen Clark-type electrode for the determination of free and total glycerol in biodiesel samples. The enzymatic conversion of glycerol consumes oxygen, which is measured amperometrically in a Clark-type electrode and correlated with the concentration of glycerol in the sample. The electroenzymatic method proposed showed a good linear correlation coefficient (R = 0.9990) with a linear response in the concentration range of 6.25 x 10(-5) to 6.25 x 10(-4)% (w/v) and limits of detection and quantification at 1.0 x 10(-5)% and 3.0 x 10(-5)% (w/v), respectively. Good correlations were found between the results obtained in this work and those by the gas chromatography technique (R = 0.9994). The proposed method was shown to be promising for the analysis of glycerol in biodiesel samples, with a simple and inexpensive methodology compared with the gas chromatography technique.