*3.1.2 Free and total glycerin*

Free or total glycerin in biodiesel can be determined by spectrophotometry, since the reactions shown in **Figure 8** result in a colored compound, as initially described by Greenhill [37]. Several publications have used similar methods, taking advantage of kits used to analyze triglycerides in blood [36, 38–40]. In the presence of adenosine-5'-triphosphate (ATP) and the enzyme glycerol kinase (GK), glycerol produces diphosphate adenosine-5'diphosphate (ADP) and glycerol-3-phosphate (G-3-P; reaction 1), which, in the presence of glycerol phosphate oxidase (GPO) and oxygen, produces dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H2O2; reaction 2). Then, in the presence of a peroxidase, an oxygen acceptor (OA), and 4-aminoantipyrine (4-AAP), water and a colored compound (CC) are formed (reaction 3):
