Observations on the estimation of scavenging activity of phenolic compounds using rapid 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH•) tests

Rapid 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH•) tests are often applied to classify the scavenging activity of phenolic compounds (AH). Published analytical protocols differ in more than one experimental condition, and results for the relative order or magnitude of activity are often contradictory. In this work, parameters such as duration of test, [AH]/[DPPH•] molar ratio, and solvent effects were examined and discussed. The test duration and the value of the [AH]/[DPPH•] ratio did not influence the order of activity among tested antioxidants. Ethanol, commonly used as solvent in such tests, was compared with acetonitrile and tert‐butyl alcohol. Solvent properties such as the ability to form hydrogen bonds with the AH seem to influence the level of the relative activity (%RSA). Higher %RSA values were observed in ethanol. The activity of the most polar compounds was affected the most, and in some cases (caffeic, dihydrocaffeic, and rosmarinic acids) the order of activity was changed owing to different kinetics. Standardization of the analytical protocol should include a 20‐min reaction period and a molar ratio that permits attainment of a 60–80% RSA value for the most potent antioxidant. Solvent choice is critical for classifying activity. Safe classification can be based only on results from kinetic studies.