Hi there, in order for you to have a reliable reference for the absorbance of DPPH at 60uM concentration, why not scan a 0.1M DPPH solution first from 200 nm to 800 nm, through scanning you can detect at what wavelength will be the maximum wavelength of absorption for DPPH, after establishing the maximum wavelength of absorption or the Lambda Max, test the lambda max if it really jives to BEERS LAW by preparing an increasing solution of DPPH from 10uM to 100uM DPPH Solution with 10uM increments. then use the previously obtained Lambda max to determine the absorbance for each concentration prepared.

using the absorbance data gathered, plot the absorbance vs. its concentration using excel, then obtain the equation of the line together with the regression or the R2. if you will obtain an R2 of about 0.99999 ( seen in the graph is a straight line), then this will prove that the wavelength you've obtained as its maximum wavelength of absorption follows BEERS LAW, by which the concentration is directly proportional to the absorbance of the sample

through this you can have a reliable result on antioxidant activity assay using DPPH as a radical scavenging agent by establishing a good lambda max for DPPH.

note: it is important for you to obtain the maximum wavelength of absorption (Lambda Max) because at at this wavelength, the probability of the molecules that absorbs the incident radiation and transmits the radiation is very close to 1 which means that all the molecules in the solution absorbs the incident radiation and transmits radiation which is then transformed into an absorbance reading, as a result the abosrbance obtained will be most likely equal to the total concentration of the sample given by the equation below.

A= Ebc.

A= absorbance

E= absorptivity constant

b= path length of the cuvette

c= concentrataion of the solution.

Dear Farid Abrigach, 

You can try with 4mg of DPPH dissolved in 100 ml of pure Methanol. this will give absorbance above 0.6 at 517nm

Hope this will help you

DPPH has a absorption band centered at about 520 nm, is deep violet in color and looses color when netralised, it is also very sensetive, are you using the same blank as and reaction time as the refence papers - Abs values  do seem do deviate a little between experiments and on using different spectrophotometers -  also check whether your Abs values for the standard remains constant with time or changes.

DPPH scavenging activity was carried out using the method of Braca et al. [12]. Different concentrations (400, 200, 100, 50, 25 and 12.5 μg/mL) of Steudnera colocasiifolia extract were dissolved in methanol and placed in different test tubes, and 3 mL of a 0.004% w/v methanol solution of DPPH was added to each test tube. Absorbance at 517 nm was determined after 30 min against a blank, and the percent inhibition activity was calculated from [(A0 – A1) / A0] × 100, where A0 is the absorbance of the control and A1 is the absorbance of the sample. Ascorbic acid was used as a reference standard and dissolved in methanol to make the stock solution with the same concentration. The control sample was prepared containing the same volume without any extract or reference drug. Methanol served as a blank. The inhibition curves were prepared and the half maximal inhibitory concentration (IC50) values were calculated using linear regression analysis.I hope you can done experiment in this way you will get desired result

prepare 0.002 % (w/v) of DPPH solution by dissolving 2mg in 100 ml of pure Methanol. this will give absorbance above 0.235 at 517nm.

Hi !

I used DPPH in my thesis ! the attached file helps you figure out more detailed information about DPPH assay and compare its results in different conditions.

Also you should note that DPPH is a free radical and very sensitive... so you should only focus on the inhibition percentage.

You are not supposed to report absorbance of dpph but the inhibition percent and Ic50 matter.

Best regards

There are issues working with DPPH. First of all make sure that DPPH is fully soluble in your solvent. There is always some deviation in the absorbance of the your DPPH solution. The blank will be of ethanol. You can work out with 0.3mM solution as well. working at 517nm is quite fine. Bst of luk

Mostly I worked with a concentration of 0.06 mM DPPH in pure methanol. It gives me an absorbance about 0.5-0.6 at 517 nm. I performed this method according to

Brand-Williams W., Cuvelier E., Berset C.M., Use of free radical method to evaluate antioxidant activity, Lebensm.-Wiss. U.-Technol., 1995, 28, 25-30.

Good luck

Thank you for everyone, but I also want  to know at what range  I must work 600-400nm or 800-400nm because the absorbance values are different for the same DPPH solution

Keep 517 nm wavelength value as this is the highest absorbance value for methanolic solution of DPPH

517 is the max absorbance value in the absorption spectum

I tried 570, 523 and 517nm for 0.2mM DPPH in methanol by different instruments based on literature available. Among these, 523nm give max and stable absorbance in different instrument used.

In my opinion 517 nm.

If you are using a spectrophotometer, you should select 517 nm, and you should get an absorbace ~0.6, for a 60 uM solution (2.4 mg/100 mL). DPPH solubilizes better in MeOH than in EtOH. However, in both cases, you should use ultrasound to achieve a complete solubilization. Otherwise, the absorbance will be lower.  

We measure the absorbance at 515 nm (Brand-Williams W. et al. Food Science and Technology 1995;28:25-30 - Bondet V. et al. Food Science and Technology 1997;30:609-615 - Sanchez-Moreno C. et. al. J Sci Food Agric 1998;76:270-276. - Villaño D. et. al. Talanta 2007;71:230-235.) , as Joao Barreira say, you get an absorbance of 0.693 (molar extinction coefficient 11563 M-1 cm-1). Probably the DPPH is decomposed.

DPPH absorbance valueis 515 nm. best way One can preare dilute solutions of DPPH in ab. alcohol &  measure absorbance at various wavwlength . Plot a graph of O.D. Vs wavelength. fndout the wavelenth at which there is maximum absorbance.

I think this link is very useful, 

http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/

I have the same problem

Hello dear colleague,

Check your balance, check your DPPH, check the dimensions of your tank (it must be 1 cm).

Good luck.

Article Chemical Composition and Antioxidant Capacity of Eggplant Pa...

dear

this article us good for your question

best regards

I used 515 and 517 they all work perfectly even to the standard

I used 517 nm. you should check unpaired electron in DPPH.