Hello Sir,

you can use wavelength of 500-520nm for DPPH radical where you can get strong absortion band. but it is not possible at 490nm.

This the qualitative assay for antioxidant if you cant measure wavelength.For qualitative assay crude extract (10 mg/ml) was dissolve in methanol. Required size of the TLC plate was excised, on this 1 µL of extract was loaded with the aid of capillary, quercitin (0.5mg/ml) was loaded as positive control and methanol as negative control. The sheet was marked lightly with pencil making sure the sample was loaded at the correct position. The plate was then kept in oven at 500C for 20 minutes for drying. DPPH solution (concentration 80 µg/ml) was prepared and then sprayed onto the TLC plate with the aid of atomizer. The system was left for drying for 30 mins. Antioxidant activity can be observed as white spots against the purple background. Color intensity determines the quality of antioxidant present.

Hi, you just need to record a spectrum (e.g. 250 to 800 nm) with your assay mixture, including the desired DPPH concentration (e.g. 150 µM) in the cuvette, to find the peak. Usually it is at 520 nm. So, then find out if the nearest wave length you can measure with your photometer is still on the peak or close to it. That's it. Cheers.

Hi All,  Please find the attached articles    (DPPH  OD at 490nm)

Hi Ahmed,

I have the same problem!!

Have you tried at the wavelength 490nm

is it working?

Dear all,

Best is to read your assay mixture between 400 and 600 nm in a spectrophotometer to determine whether or not a wavelength of 490 nm is working.  This is the only way to find it out.

According to Wikipedia (https://en.wikipedia.org/wiki/DPPH) it seems to be possible to read your samples at 490 nm (instead of 520 nm) but the difference in absorption between the DPPH radical (deeply violet) and the its neutralised form (yellow) won't as high as at 520 nm.  This might cause some trouble with your results.

Cheers,

Ralf

Hi all,

I did it and it seems working

I Tried with blank and BHT(at different concentrations) and the DPPH radical and the neutralized form absorbs at 490nm

Hi Safa,

That sounds great.  As long as you can clearly distinguish between both DPPH forms by sufficiently different absorbances, I can't see at the moment why you shouldn't measure at 490 nm.  A clear communication in your manuscript can help both the reviewers and the readers to understand your decision to measure at 490 nm instead of 520 nm.

Cheers,

Ralf