Hi,
Im doing the DPPH assay for my Tamarind Seed coat extract (color red as you increase concentration). First for my calibration curve, im using for each 1,6mL of extract at different concentrations (ascending) and 2,40 mL (DHHP solution - 2mg DHHP in 50mL of MetOH). Im measuring the control as methanol and the DHHP. Im getting ascending absorbance for ascending concentrations, and also my %inhibition DHHP (Abs control - Abs sample/Abs control)*100 is more the 100. Something is wrong here i think... please help.
Good day Jimena Arias
The issue lies with the colour of your sample. For instance where you obtained 0.271 abs for the control and 0.5844 for the sample, you will obtain a negative value ( - 53%). In my experience, this is due to the colour of the extract, as you mentioned the sample extract's red colour intensifies when you increase the concentration. The inherent sample pigments are interfering with the colour change from purple to yellow that takes place when the H is donated to DPPH, thus affecting how the light passes through during absorbance reading. This does not necessarily mean your sample does not possess DPPH radical scavenging activity.
To overcome this, you have to lower the concentration of the extract by further dilution. Depending on your sample, this will have to be a trial and error thing. In this instance you can adopt the EC50 (concentration required to obtain 50% DPPH radical scavenging activity) approach.
I hope this helps.
Hi Lusani, thank you for your experienced answer.
Perfect! I will try with lower concentrations but i dont have to much margin. To see what happens...
On the other hand, I was reading some papers, and in some cases they made a correction by measuring the Absorbance of a blank. They use the formula by ( (Abs of sample - Abs blank)*100/ Abs control ), but im not sure if theses if physically correct...
Hi Jimena
I am glad I could be of help to you.
I am not familiar with the formula you mentioned above; try it, and see if it will work for your results. Another formula which is similar to the one you proposed/applied initially is:
[1 - (abssample / abscontrol )] x 100
All the best
Dear Jimena Arias
If the sample to be measured on a start basis has already its color, you have to prepare each sample concentration has each control;
sample mixture
- Control x: sample concentration x ug/mL with vol a (mL) + solven sample b (mL) --> total volume for example 2 mL ---> Absorbance A
- Sample x: sample concentration x ug/mL with vol a (mL) + solven b - c (mL) + DPPH volume c mL. ---> total volume 2 mL ---> Absorbance B
also prepare: control mixture
- Control 0: sample 0 ug/mL (solven only) = total volume 2 mL (Absorbance C)
- Sample 0: DPPH volume c (mL) + solven 2 - c = total volume 2 mL (Absorance D)
Note: Final volume of control and sample are the same, the concentration of DPPH in sample x and sample 0 is the same
%Inhibition = [(D-C)-(B-A))/(D-C)]x100% (% inhibition sample with x concentration)
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