By means of serial dilution, I've prepared a range of aqueous ascorbic acid solutions (from 200 to 1 µg/ml). From each concentration I took 1 ml and mixed it with 1 ml of DPPH ethanolic (99.8%) solution (0.3 mm). I've also prepared a blank solution (1 ml of distilled water and 1 ml of ethanol) and control solution (1ml of DPPH solution + 1 ml of distilled water).
Using a spectrophotometer on a wavelength of 517 nm, control solution gave a value of 0.85 which is considered normal, but unfortunately all the other concentration of ascorbic acid is giving high values also close to 0.8 even with a concentration of 200µl/ml...
Does anyone know what can be the source of error?
Please note that I've already tried to change the ascorbic acid and used a higher quality.
Thank you
Angélique Nicolas Messi do you mean the calibration of the spectrophotometer ?
Ok cool !Elizabeth now know that the spectrophotometer comes with the ultra pure water that you have to calibrated before starting any experiment.
Angélique Nicolas Messi What is the calibration of water?
Elizabeth Aa Why have you ignored my question? I had a reason to ask it.
Please Prof. Yurii, It is not the calibration of water, but it is the zero of the spectrophotometer.
Dear Elizabeth; your question is not ignored and you have a Reason to ask it.
Angélique Nicolas Messi
Ma question a été ignorée, mais pas d’Elizabeth
Yurii V Geletii excuse me Mr I didnt want to ignore you question. I’ve used 1 ml of ascorbic acid in severa concentrations with 1 ml of dpph with 0,3 millimolar
You did not answer my simple question.
What is the molar ratio of ascorbic acid and DPPH after mixing?
In addition. the original DPPH protocol commonly uses Trolox as a standard solution in ethanol or methanol. You mix aqueous solution of ascorbic acid with DPPH in ethanol. You may respond in French if you have problem with English
Dear Elizabeth
If sample absorbance is giving high value than control then probably your incubation period might be the problem. The minimum incubation period for an antioxidant to sufficiently chelate DPPH free radicals under in vitro conditions is 30 minutes. You may increase your incubation time to 1 hr and check.
Properly mi the reaction mixture with the pipette and incubate in the dark
You also need to verify the concentration of DPPH solution, you can try 200uMolL-1
prepared in methanol ( 1.57mg in 20 ml of methanol)
Reference Thomas J Herald et al 2012
- Badges
- Science topic