Need help with the DPPH antioxidant activity experiment
In the first attempt, the blank was ethanol only. With an increase in the concentration of the sample, the absorption value was higher than the control value (DPPH + Ethanol)! Although very visible to the eye, the violet color lacked intensity
I made another attempt keeping in mind that the blank was the same concentrations of the samples with ethanol without DPPH
Plz find he attached photo
Thanks for the help
We have to make sure that the sample doesn’t have an absorbance of 515 – 517 nm. It would be better if we use the same solvent for DPPH and the sample; moreover, every sample has its own control, including the DPPH solution. So, we have to measure 4 absorbances for each sample test.
The absorbance DPPH + solvent = D1
The absorbance of solvent only = D0
Absorbance of DPPH and sample in a solvent = S1
The absorbance of the sample in a solvent = S0
% Inhibition DPPH of the sample = [(S1 – S0)/(D1-D0)] * 100%
@Dear Amany Hamad,
I think the suggestions by Harto Widodo should sort out the problem and would increase the accuracy of this assay.
By the way what was the lambda max chosen for recording the absorbance?
Regards,
Assay,
1.0 ml DPPH (3 mM) in ethanol
1.0 ml Extract
1.0 ml Ethanol
total 3.0 ml Assay
10 min Incubation
O.D. at 517 nm
Calculation,
% Inhibition = Control abs - Test abs / Control abs X 100
Where,
Control abs = absorbance of DPPH + Solvent (Ethanol) at 517 nm
Test = Sample + Alcohol + DPPH
Key points,
1) Try to set DPPH concentration (control) at 1.0 O.D. (Absorbance)
2) Set Solvent (Ethanol) as Blank
3) take O.D. of sample blank (Sample color without DPPH)
Harto Widodo
Many thanks for the help
For clarification: the solvent used was ethanol, and the ethanol was considered as a blank, and the zero button was pressed on the device
So do I neglect to compensate with the value of D0 (the absorption of the solvent only which is ethanol)
Arun Padhye
Many thanks for the help
Measurements were done at 517 nm
For clarification: the solvent used was ethanol, and the ethanol was considered as a blank, and the zero button was pressed on the device
So do I neglect to compensate with the value of D0 (the absorption of the solvent only which is ethanol)
@Dear Amany Hamad,
I think my answer is yes.
So you may neglect to compensate with the value of D0 (the absorption of the solvent only which is ethanol).
Regards,
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction