Hello
I wish you good health
I have a question:
In different articles, different volumetric ratios have been used for DPPH testing. For example, one paper used 0.5 cc of sample and 2.5 cc of DPPH solution, and another article used 1 cc of sample (ascorbic acid) and 1 cc of DPPH solution.
What is the reason for these different ratios and how do I know which method is correct?
Thanks for your response.
Sahar Kelvani
Dear Researcher,
Thank you for the interesting question.
I must admit in the beginning that I am not absolutely sure about this. But, I guess that different researches are having different research methodologies and different research questions. That is why, different volume ratios have been used in different articles.
I am eagerly waiting for the replies to be written in this thread by the eminent scientists and researchers from around the world.
Best regards,
Anamitra.
It does not matter the volume ratio, you have to worry about final concentrations after mixing.
Dear Dr Sahar, please kindly follow the attached article, hope it can be useful for you!
" according to reaction time (slow, fast kinetics), the linearity range of DPPH radical depending on the detection conditions as well as the kind of the investigated antioxidants (reference chemicals and the ground elder prepared from fresh and dry plants"
The percentage of DPPH scavenging effect was calculated by following equation. DPPH scavenging effect (%)/% Inhibition=A0-A1/A0 × 100 Where A0=The absorbance of control. A1=The absorbance of sample. As DPPH was soluble in methanol, it was taken up as organic phase.
Dear Sahar Kelvani many thanks for posting this important technical question on RG. This is certainly of broad interest as many RG members also use the DPPH (= 2,2-diphenyl-1-picrylhydrazyl radical) assay. There are many variations in the experimental procedure possible. For a very useful literature reference in which the different protocols are listed and compared, please have a look at the following article:
EVALUATION OF THE METHODS FOR DETERMINATION OF THE FREE RADICAL SCAVENGING ACTIVITY BY DPPH
https://www.agrojournal.org/17/01-02-11.pdf
This paper is freely available as public full text on the internet (please see attached pdf file).
Good luck with your research work and best wishes, Frank Edelmann
Dear Sahar,
it's a technical issue. To get IC50 you need to get absorbance ratio from 0-100, you would increase the volume of extract to increase the ration toward 100 and vise versa.
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