When you calculate the %Inhibition (or either the %remnant) in DPPH method, is it enough use only the absorbances obtained (see formula 1 down)? or is better transform these absorbances to concentration units using of the respective calibration curve (DPPH absorbance vs. DPPH concentration), and then apply the formula 2? or is the same?.
% Inhibition = [(A0-A1)/A0] × 100 Formula 1
Where A0 is the absorbance of the control; A1 is the absorbance of test samples.
% Inhibition = [(C0-C1)/C0] × 100 Formula 2
Where C0 is the concentration (ppm, M, etc) of the control; C1 is the concentration (ppm, M, etc) of test samples.
I have made some calculates using absorbances and concentrations also, but I have obtained some differents values of percents. But I don´t know the correct form.
Hello Andrea. Here is my advice. What I normally do is use a number of concentrations of my sample material (eg green tea extract). So I'd make up concentrations containing 100%, 75%, 50% etc etc and test their DPPH activity. By using decreasing concentrations you can prevent a plateau because for all we know 100% sample may have same antioxidant activity as 25%, which allows you say for example 100% concentration scavenged 90% DPPH radicals, while 25% concentrate scavenged 30% DPPH radicals. Hope I didn't confuse you.
I think that I have not explained well, so I have edited the question. Anyway, I am really grateful with you for to take the time for answer me.
Dear Andrea
You take the absorbance and concentration, calculate the Ic 50 value using the formula.
To get standard curve you will need to measure absorbance of known concentrations of DPPH and the inhibitory agent. After measuring the absorbance of your sample, you will 'super-impose' on the standard curve to know the degree to which your sample can scavenge DPPH. Inhibition is usually measured in percentage..
Like you rightly stated in your question explanation, use the first formula i.e
% inhibition = (A0-A1)/A0 X 100
Where A0 is absorbance of reference/control and A1 is the absorbance of your sample.
I want to believe while carrying out your assay, the sample is concencentraion dependent (i.e varying volumes like 25ul, 50ul, 75ul, 100ul or 100ul, 200ul, 300ul, 400ul) depending on the protocol you used then now plot your graph (concentration against percentage inhibition) on microsoft excel and then use linear regression analysis to calculate the IC50 (i.e your sample concentration causing 50% antioxidant activity) . I hope you know how to calculate your concentration? If no, i will explain. Hope my input is helpful?
INHIBITION%in DPPH is:
%Inh=(Ao-A1)/Ao]x100,
Where: Ao is absorbance of standard and
A1 is isabsorbance of sample
I think this link is very useful,
http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/
Use the formula, %FRS=(Ao-A1)/Ao]x100,
Where: Ao is absorbance of negative control and
A1 is absorbance of the tested sample
FRS is free radical scavenging activity
Hope this can help you.
What is the dpph calculation formula for evaluating the antioxidants of fruit juices?
What is the difference between these two formulas?
DPPH activity, % = (1 - (Abs515nm Seaweed extract / Abs515nm Control)) × 100
or
(Ao-A1)/Ao]x100
Many thanks Victor Ndigwe Enujiugha for your information, that right the second formula much easy and more reliable
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