DPPH

DPPH, or 2,2-diphenyl-1-picrylhydrazyl, is a stable free radical widely used in laboratory assays to evaluate the antioxidant capacity of chemical compounds and complex mixtures. The molecule carries an unpaired electron on the hydrazyl nitrogen, which gives it a deep violet color in solution and a characteristic absorption maximum near 517 nm.

When an antioxidant donates a hydrogen atom or electron to DPPH, the radical is reduced to the non-radical DPPH-H, and the solution fades from violet to yellow. This color change forms the basis of the DPPH assay, a simple and widely employed method for assessing radical scavenging activity. In a typical protocol, a DPPH solution in methanol, ethanol, or acetone is mixed with the test sample and incubated for a fixed period. Absorbance is measured at 517 nm, and scavenging activity is calculated as a percentage: (A0 − A1)/A0 × 100, where A0 is the initial DPPH absorbance and A1 is the absorbance after reaction. Results may be expressed as IC50 values or as Trolox equivalents (TEAC).

Applications of the DPPH assay span food science, nutrition, and biochemistry, including screening plant extracts, fruits, vegetables, beverages, vitamins, and synthetic antioxidants. Limitations include the solvent dependence and lipophilicity of DPPH, potential interference from sample color, and the fact that in vitro radical scavenging does not always correlate with in vivo antioxidant activity. Proper controls and solvent choices are essential for reliable interpretation.