Does one use only one concentration of ascorbic acid and measure over time to get the linear equation? Or use several concentrations? Is it advisable to use dilutions of blank DPPH solutions as a standard curve and ascorbic acid as a positive control?
Seems like everyone does this assay differently. I guess I will just be working from the papers:
EVALUATION OF THE METHODS FOR DETERMINATION OF THE FREE RADICAL SCAVENGING ACTIVITY BY DPPH by G. MARINOVA and V. BATCHVAROV 2011
DPPH antioxidant assay revisited by Sharma and Bhat 2009
Hi Arwen M Milroy
it is always better to use several concentrations of a standard. In this way you can calculate the precision of your assay . By calculating the coefficient of the determination you can check the robustness and degree relationship through regression analysis. And once R square value is more the 95% your assay, handling and concentration vs absorbance relation graph is highly accurate. Hope this will help you all the best
Perform Assay by MCCune and Johns method In which take different concentration (1 to 15 microgram per ml) of ascorbic acid then plot graph and u can get IC50 value
Hi Arwen M Milroy
It seems to me that you are confusing the principle of scavenging activity. It is always best to optimize the protocol and test for sample of any. To get standardized protocol, different concentrations is advisable for known compound like ascorbic acid.
It is not Ascorbic vs DPPH but by doing ascorbic acid scavenging activity on DPPH by different concs. You can generate the equation. but if want to use AA as positive control, you may need use other known compound for standard instead.
What I did before was preparing the gallic acid (function same as ascorbic acid) in different concentration, then add in a same concentration and volume of DPPH solution into those prepared gallic acid to test the scavenging activity of gallic acid. Then I can plot a standard curve using the scavenging activity of gallic acid vs concentration. The R2 of this standard curve is usually be 0.99.
According to DPPH antioxidant assay revisited (by Om P. Sharma and Tej K. Bhat, Food Chemistry 113 (2009) 1202–1205), the range of accuracy for spectrophotometric measurements falls within an absorbance of 0.221–0.698 which equals a transmittance of 20–60% (Ayres, 1949). This corresponds to the DPPH concentration of nearly 25–70 microM. Just sharing.
Pardon my english... This article uses a calibration curve for DPPH... I was looking information about it because I have to use the method to analyze some samples... The article is Vitamin C Equivalent Antioxidant Capacity (VCEAC) of Phenolic Phytochemicals 10.1021/jf020071c DOI...
I did last week a calibration curve for DPPH with Gallic Acid and obtained a correlation coefficient of 0,997 between the concentrations from 2,5 to 100 mg/L...
https://www.banglajol.info/index.php/JBAS/article/view/48561
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