Total glycerol analysis in biodiesel samples using solid phase extraction coupled with enzymatic-spectrophotometric determination

In this work, a method was developed for the quantification of total glycerol in biodiesel using solid phase extraction (SPE) coupled with enzymatic-spectrophotometric determination. An aminopropylsilane stationary phase was used for the separation of the main contaminants of biodiesel (monoacylglycerol (MAG), diacylglycerol (DAG), triacylglycerol (TAG) and free glycerol (FG)) from its main components, fatty acid methyl esters (FAMEs). A biodiesel sample from a high-conversion transesterification reaction was used, and the separation of contaminants was monitored by non-aqueous, reversed-phase high-performance liquid chromatography (HPLC-NARP). After SPE, the samples were analyzed by spectrophotometry using a commercial enzyme kit widely used to measure total triglyceride levels in blood. The method showed good analytical performance for linearity (R2 = 0.9919), limit of detection (9.81 × 10−4%, w/w), limit of quantification (3.24 × 10−3%, w/w), repeatability (RSD% ranged from 1.22% to 4.80%), intermediate precision and recovery (96.8% to 100%). The proposed method efficiently determined the total glycerol in biodiesel, and the paired t-test demonstrated strong correlation with the reference method (GC).