Ihave a paper with this protocol, you can try your work with this method:

DPPH Radical Scavenging Assay. Radical scavenging

activity of .... fruits extracts against 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical was determined spectrophotometrically. The method first introduced by Blois [11], developed by Brand-Williams et al. [12], and criticized by Molyneux [13] was employed. The principle of the assay is based on the color change of the DPPH solution from purple to yellow as the radical isquenchedby the antioxidant. Briefly, 100 𝜇L of methanol hawthorn fruits extract was added to 1.9mL of 410–3mM of DPPH in methanol up to completing 2mL. The free radical scavenging capacity using the free

DPPH radical was evaluated by measuring the decrease of absorbance at 517 nm every 2min until the reaction reached its state. Additional dilution was needed if the DPPH value measured was over the linear range of the standard curve.The

inhibition activity I(%) was calculated as follows:

𝐼 (%) = 100 ×(𝐴0 − 𝐴1)/𝐴0

where 𝐴0 is the absorbance of the control sample and 𝐴1 is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated from the graph.

In our lab for the DPPH assay we use the following protocol in a 96 well plate and it works quite well:

Stock solutions of DPPH were prepared by dissolving 24 mg of DPPH in 100 ml methanol. The solution was then shaken in a sonicator for 20 min. The working solution was then prepared by diluting 20 ml of stock solution with 80 ml methanol (5X dilution).

Trolox (1 mM) was used to make the standard curve, which was linear between 0 and 800 µM. To 15 µl of a sample, 285 µl of DPPH was added. The microplate was then left to stand for 15-30 min in the dark place.

The plate was then read at 517 nm and the data was expressed as % inhibition (%I), or µmol trolox equivalents per gram of sample (TE)/g.

Hope this helps.

Please find the attachment

Helo. How can  I calculate the Trolox equivalent per gram of sample if I had the % inhibition? If I didnt prepare my samples in series of concentration, can I still get the IC50 value?please help. Thank you.