Hi everybody,
I found in many articles for the antioxidant estimation using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method a mistake that I think everyone has to take care about. all the papers that I read considered the prepared stock solution of DPPH as a blank to subtract the results after a period of incubation (after adding the antioxidant agent). I think that the reading of blank (by spectrophotometer) should be the DPPH+ the antioxidant agent before incubation and the reading to be subtracted from this blank should be after the incubation.
You are welcome to discuss.
Hope you all the best.
Do you mean that the antioxidant molecule itself might absorb light at the same wavelength of DPPH?
The problem with the approach you described (i.e., measuring "time zero") is that many antioxidants act readily just after mixing with DPPH. Although it usually takes 30 min to stabilize the absorbance values, one can readily see the color shift right after mixing DPPH and the antioxidant.
An alternative approach, especially for those samples that can absorb light at ~515 nm (e.g., vegetal extracts), is to simply read the sample at the final concentrations without DPPH and account for this interference.
What do you think?
Yes, I found that the antioxidant molecules that I used absorbed light at the same wavelength of the DPPH, these molecules are derivatives of proteins.
I understand that the reaction starts immediately after mixing the antioxidants with DPPH, so I suggest that the reading of blank should be taken immediately after mixing.
on the other side, considering the stock solution as a blank gives wrong calculation, because the resulting value will be higher than the actual.
Hi,
Proteins absorb the light wavelength far below 515 nm.
Adding proteins to the DHHP reagent and taking a reading immediately doesn't have a significant effect on absorbance value.
I guess
Sorry, if I'm wrong.
Hi Rohit,
derivatives of proteins can absorb light at the same wavelength of the DPPH. so if you added these derivatives to the DPPH and measured the absorbance you will get a reading, after incubation you will get a lower reading. I think that this makes sense. However, considering the stock solution is a blank is not right, because the difference between the two readings will be higher than the actual.
Ok.
When u added protein derivatives to the DPPH reagent and immediately take the absorbance the reading will be similar to the Blank i.e. only the absorbance of the DPPH reagent because the chemical reaction did not start.
That is not right, after adding the antioxidant agent to the DPPH the reaction starts immediately. Also, your opinion is proofing my hypothesis.
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