Hi, I am checking the antioxidant activity of polyphenols using the DPPH reagent. I'm taking a 0.1mM reagent. I tried two 3 things. I'm using ethanol as a solvent for my extract.
1. I made DPPH SOLUTION USING ethanol as my extracting solvent was ethanol and checked the absorbance but my standard values are coming negative while the test sample is giving +ve.
2. I also tried methanol. Made DPPH solution using methanol. here for control, I took DPPH standard solution + ethanol (s my extracting solvent was ethanol ) and test samples as meth DPPH soln with the extract. still, the absorbance values are either less or -ve.
I have tried both methods on three different instruments. Please suggest something to do?
thanks in advance!
Hello Mandeep Gulati
thanks for you question,
it would be interesting to know how long did you incubate the samples after you have reacted with the DPPH. Did you observe any colour change on the samples vs the control?
Normally, the DPPH solution gives a purple control and the presence of an antioxidant in a sample results in a colour change to Yellow.
Secondly you can also check the wavelength of the spectrophotometer - we used 517 nm. Please see our paper here wheere we have used the assay hope it helps
https://www.hindawi.com/journals/ijfs/2021/5572554/
Dear Mandeep
First of all I will suggest you to calibrate your UV Spectrophotometer before experiment. Negative value of absorbance might be due to some errors in measurement process. Negative value means according to the formula that blank had higher absorbance and its its not showing the properties of blank. You can go with the link for calibration.
https://www.pharmaguideline.com/2010/05/calibration-of-uv-visible.html
And here I am sharing a protocol for DPPH. You can modify the given procedure as per your solvent, standard and experiment.
DPPH solution (100 µM) was added to 1 mL of extracts with 1 mL of methanol. The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 10 min. The resulting solution was monitored at 517 nm after 10 min. XY can be used as standard control. The percent of DPPH discoloration of the sample was calculated according to the equation:
% Scavenging [DPPH] = [(A0-A1) / A0] X100
where, A0 is the absorbance of the control and A1 is the absorbance in the presence of the samples or standard.
I hope it will help you.
Regards
1. What is the incubation time?
2. Calculate the IC50 value
3. Try using methanol once (i used methanol for extraction).
Hello,
As I performed DPPH in my laboratory, 0.1M DPPH (powder) dissolved in 100% methanol (we prefer methanol over ethanol). Incubate it in dark for minimum 30mints. If you are using standard DPPH then see the protocol and make the doses according to the standard one. While applying with the Extracts, take 200 µl sample+ 1.5ml DPPH. again incubate for 30mints in dark. It's not mandatory to use the methanolic extract as the DPPH is dissolved in methanol, you can use any.
Calibrate the Spectrophotometer properly and measure the OD at 517nm.
While taking the reading try to keep the room dark as it affects the result further.
Use the prepared DPPH as blank (purple), whose OD will be higher, and the samples containing antioxidants will fade out the purple color up to yellow. if the saturation of DPPH comes (OD will be similar in higher and lower doses), you have to increase the molarity of DPPH.
I hope it will help you.
Thank you,
Thank you, everyone! I got the desired results. Naveen Dhingra Esha Sarkar thanks!
1. Calibrate the UV-vis
2. Change the cuvette, better to use fresh quartz cuvette
3. Go through any of the above-mentioned protocols.
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