Determination of free and total glycerol in biodiesel by spot analysis

The importance of alternative fuel production has increased in the lately years, and so has the need of methods of analysis to evaluate their quality. Biodiesel fuel is a valuable substitute to petrodiesel fuel. Among the side and byproducts that have to be determined as an indicator of biodiesel quality are free glycerol and mono-, di-, and tri-acylglycerol. A simple, economic, friendly, and green method to quantify free and total glycerol in biodiesel has been developed. Novelty of the approach is related to the new extraction method proposed and its combination with the further steps that allow to accomplish the determination fully in the field, with the results being read without any sophisticated equipment other than a smartphone. The method is based on two extractions, i.e., the conventional aqueous extraction and another one with detergent, both followed by four consecutive enzymatic reactions to produce a colored product, which can be quantified with a free downloadable smartphone application for digital image analysis. The methodology was assayed to determine free and total glycerol content in commercial biodiesel samples. The results obtained using the proposed method have been compared with those acquired by the standard, normalized gas chromatography-FID one, and turned out to be equivalent (P < 0.05). The proposed method was improved to minimize reagents consumption per test. A linear calibration curve was obtained between 8.34 10(-4) and 6.93 10(-3) % m/m (R-2 = 0.972), with 0.012 residual standard deviation. The limit of detection of the method was 8.34 10(-4) % m/m, whereas the limit of quantification was 2.41 10(-3) % m/m. The precision of the method was evaluated by the coefficient of variation, which ranged between 5 and 14%. Accuracy of the method was assessed by paired-sample t tests (P < 0.05), and the elliptic joint confidence region test. The potential interferences accompanying the analyte in biodiesel matrix or those introduced by the detergent used for extraction did not interfere with the enzymes or reagents used, the recovery having ranged between 90 and 96%. Our results support the suitability of the proposed method as an alternative tool for rapid and decentralized measurements of glycerol and total glycerol levels in biodiesel fuel with appropriate reliability and low cost.