I am performing DPPH assay test of citrate and ammonium based compounds. While performing DPPH assay test color of solution changes from violet to brown after incubation. Peak at 417 nm disappears and shifted 425 nm while increasing overall intensity at 417 nm. now colour change is happening BUT why intensity is increasing after adding my samples to DPPH solution. Any particular reason?
P.S using absolute EtOH as solvent. and incubation time is 60 min.
Decrease the concentration of your sample to the extent that after the incubation period color intensity of DPPH (violet) gets reduced i.e. test tube containing sample conc + DPPH should have fade color than the color of reagent blank. Then increase your conc sequentially until color of the mixture solution is completely gone.
You can decrease the conc of your sample to a great extent and then make the ratio of sample : reagent to 3:1(if problem persists with current ratio & decreasing conc) with the incubation period of 30 min.
@ Proshanta Majumder
Thanks for the answer. However my problem is that sample+DPPH is changing colour to brown instead of yellow. Also, i am using 0.1mM of DPPH which i think very low.
The change of color is probably not related to the reduction of DPPH, but to reactions of your sample. Do a simple control experiment, incubate your sample without DPPH and see the color changes.
dear yurii, i did control experiment but there is no effect. control solution remains colorless.
Then, the color changes are related to products of oxidation of your sample. You need to perform a full analysis of spectra after the reaction with DPPH, not just at several wavelengths.
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