DPPH is a stable radical and its reactions usually take a longer time when compared with the much more reactive in situ generated ABTS radical (its reactions are usually instantaneous). The rate of the reaction with DPPH is also dependent on the pH of the medium and the solvent itself, and it takes sometimes several hours (or longer) to reach steady-state. That means that you could get negative results, such as yours, if the antioxidants present in the sample are not as "strong" enough as the positive controls (ascorbic acid etc.), that you have to include in your experiments as pointed by Salam, should be.

hi Olivia .... I think the use of positive control in both experiment will be helpful ... to compare your results with

DPPH is a stable radical and its reactions usually take a longer time when compared with the much more reactive in situ generated ABTS radical (its reactions are usually instantaneous). The rate of the reaction with DPPH is also dependent on the pH of the medium and the solvent itself, and it takes sometimes several hours (or longer) to reach steady-state. That means that you could get negative results, such as yours, if the antioxidants present in the sample are not as "strong" enough as the positive controls (ascorbic acid etc.), that you have to include in your experiments as pointed by Salam, should be.

Hi, thanks for the answers:

-Is there a specific pH range of the medium that is er more suited for DPPH testing?

-I increased the concentration of the sample as in trial 2 (0-5mg/ml) compare to trial 1 (0-3mg/ml), but my absorbance value is around 1.0+ (which I heard from my friends that is my concentration is too high?)

-Several hours to reach steady state? Hmm, Maybe I should try again with 1-3 hours? Or any advice of a range of time I should try?

-Positive control. I think I will try with Trolox, maybe with concentration 0-3mg/ml?

Yes Olivia, the pH you dissolve your samples in should be around 7. Depending on the solubility of your sample, you can use Phosphate buffer containing 1% Triton X(pH 7.0). I hope that helps

Some compounds in the extract can absorb in the same range of DPPH, and the solution must be clear without suspended particle. On the other hand, concentration of the extract (used) is too high, you must try dilutions.

Isnt higher concentration a higher chance for reaction to happen?

Actually the higher the concentration of your samples, the less likely you are to get accurate results if your samples are able to scavenge the DPPH radical because the high concentration of samples scavenges the radicals almost immediately when you had it to DPPH even without incubating. So it is better to always start from a lower concentration and work upwards if necessary. However, you have stated that their is no change in the purple colour even after incubation, so I don't think it is the high concentration of your samples that is the issue.

So by right higher conc should be able to scavenge DPPH even before incubating. In my case, without any reaction, I should try lower concentration.

So far, I have tried

Trial 1: Sample: 2ml extract+ 2ml DPPH (0.135mM), 30 mins in dark, Abs at 517nm (methanol blank)

Trial 2: Sample: 1ml extract+ 2ml DPPH (0.059mg/ml), 30 mins in dark, Abs at 517nm (methanol blank)

Any other procedures that anyone can recommend. Like DPPH concentration?volume of extract to volume of DPPH?Incubation period etc

Jayanta Kumar Patra recommended concentration of sample in terms of its final concentration instead. Hmm

Positive control. I was thinking trolox. For standard curve, I should try to aim to get the absorbance of the trolox to fall within my sample absorbance range? For example, lets say if my sample conc (1-5mg/ml) and abs is arnd 0.15-0.35. My trolox abs should fall within that range as well?

Try this: Antioxidant properties of Australian canola meal protein hydrolysates.

I used 100uM of DPPH in methanol. I see, the concentration of your DPPH is quite high. Goodluck

Some DPPH protocals could take up to 24 h for the reaction depending on the purpose. Olivia..Please try to increase the reaction time and or increase the concentration of sample.

water content in the mixture, surfactants, and too acidic or too alkaline pH of the solution..they affect to the reaction rate,

How did you extract the sample? What was your solvent in the sample ?

you can Try this...... Sample: 1ml extract+ 4ml DPPH (42ug/ml DPPH ), 30 mins in dark, Abs at 517nm (methanol blank) and for positive control (gallic acid,ascorbic acid,BHA,Trolox) can be used with very low concentrations (ranging from 0.0024 mg/ml to .156 mg/ml) or lower to avoid complete scavenging activity and to obtain better standard curve in the presence of variation of abs according to concentrations

I think you should to turn your attention to the equation you will obtain from the positive control standard curve more than the abs values ,which you will depend on for your calculations ...

Very good answer Salam! Thank you

Dear Olivia Ho,

I think your antioxidant structure doesn't have marginal active sites and surrounding neighbors of these active sites may have negative electrostatic potential.