α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method offers the first approach for evaluating the antioxidant potential of a compound, an extract or other biological sources. This is the simplest method, wherein the prospective compound or extract is mixed with DPPH solution and absorbance is recorded after a defined period. However, with the advancement and sophistication in instrumental techniques, the method has undergone various modifications to suit the requirements, even though the basic approach remains same in all of them. This article presents a critical review on various developments to the DPPH method
This method was developed by Blois (1958) with the viewpoint to determine the antioxidant activity in a like manner by using a stable free radical α, α-diphenyl-β-picrylhydrazyl (DPPH; C18H12N5O6, M = 394.33). The assay is based on the measurement of the scavenging capacity of antioxidants towards it. The odd electron of nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants to the corresponding hydrazine (Contreras-Guzman and Srong 1982).
DPPH is characterized as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole (Fig. 1), so that the molecules do not dimerise, like most other free radicals. The delocalisation also gives rise to the deep violet colour, with an absorption in ethanol solution at around 520 nm. On mixing DPPH solution with a substance that can donate a hydrogen atom, it gives rise to the reduced form with the loss of violet colour. Representing the DPPH radical by Z• and the donor molecule by AH, the primary reaction is