I want to perform Antioxidant assay for Iron oxide NPs synthesized through green synthesis method from leaves extract and also wants to check the antioxidant capability the plant extract which is used the synthesis of these NPs. I have also used Ascorbic acid as standard antioxidant.
Thanks!
The control value in an antioxidant assay is the absorbance of a solution that does not contain any antioxidants. There are a few reasons why your control value might be lower than your sample values.
One possibility is that the methanol in your control solution is not pure.
Another possibility is that the DPPH in your control solution is not stable. DPPH can decompose over time, resulting in a lower absorbance.
The volume of the micro plate wells might be lower than you think. This can happen if the wells are not filled to the same levels or if the solvent evaporates during the assay. A lower volume of solvent will result in a lower absorbance.
The methanol in your control solution might evaporate during the assay. This can happen if the assay is performed in a warm environment or if the microplates are not sealed properly. Evaporated methanol will result in a lower absorbance.
Here are some additional tips for troubleshooting antioxidant assays:
- Make sure that all of your reagents are fresh and of high quality.
- Use a clean microplates for each measurement.
- Avoid exposure to light, as this can degrade DPPH.
- Seal the microplates tightly with plastic wrap or aluminum foil to prevent evaporation
- Add a blank under each sample. This will help to remove the contribution of the color of your sample to the absorbance measurement. The blank should be made up of the your sample, but without any of the antioxidants.
It's difficult to answer without seen the spectra.
Nimashi Herath "Impure methanol can contain free radicals." I can't find words to comment that.
The DPPH assay is "unusual" because the antioxidant sample causes a decrease not an increase in absorbance. OK. Let me speak about normal antioxidant assays where antioxidant increases color. In that case "your problem" would be restated.. [Why i get control value(2ml methanol+1ml **** reagent 0.1mM SOL) higher then our sample ie, plant extract and Nanoparticles) in antioxidant assay?]. In this case the control would be exhibiting a reaction (apparent antioxidant content) which should not be. A faulty control is usually quite difficult to produce - provided THE CONTROL is identical in every way as the test sample. The only difference should be that the test sample is replaced with the same volume of solvent (which you seem to do). You also seem to be using 3.1 ml total volume (cuvette or test tube) not a microplate. I agree with Dr Nimashi Herath that DPPH degradation (apparent antioxidant results) occurring in the control perhaps due to sunlight exposure. Another point is that: impure methanol contains trace metal ions which are redox active. However, provided that the control is made-up exactly like the general assay using all the same reagents, these errors should cancel out. I agree with Dr Yurii V Geletii that this is truly perplexing. My short-hand advice is, to prepare a control experiment alongside of each and every assay: same solvents, same reagents, same cuvettes, same instrument (please allow to warm up and check the zeroing with water frequently). Check the control gives the same absorbance readings (vs. air or water blank) at the start and END of your analysis. Good luck.
@Yurii V Geletii, Sir I will share the complete experiment data i.e., the absorbance of the test sample (plant extract + NPs), and the control i.e., DPPH, if you could check and to find the answer to solve the issue. thanks!
@Nimashi Herath thanks madam, as I used test tubes and cuvettes in this experiment not micro plate, yes I will double check the purity of methanol next time my experiment.
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