On practical problems in estimation of antioxidant activity of compounds by DPPH method (Problems in estimation of antioxidant activity)

2,2′-Diphenyl-1-picrylhydrazyl (DPPH) method is commonly applied for the estimation of antioxidant activity of single compounds and their mixtures. The method is based on the spectrophotometric measurement of DPPH concentration changes resulting from the DPPH reaction with an antioxidant. The amount of remaining DPPH in the examined system is a measure of the antioxidative activity of compounds. The study shows that the type and amount of solvent used for the antioxidant dissolution, water content, and hydrogen or metal ion concentration in the measuring system significantly influence the differences in the amount of unreacted DPPH. The presence of ethyl acetate or dioxane or metal ions in the system decreases the kinetics of the reaction between DPPH and butylohydroxytoluene in relation to its kinetics in the system containing only methanolic solutions. The opposite effect is observed for water, low concentration of chloroform or hydrogen ions. The presented relationships confirm the necessity of standardising the DPPH method and show the complexity of the problem, even in very simple DPPH/antioxidant systems.