I’m performing a DPPH radical scavenging assay using Ascorbic acid as a standard antioxidant, but the inhibition results even for low concentration (25 ug/ml) around 90%, is this normal? however at higher concentrations ( 200 ug/ml) it is 92% . I’ve run the assay with the following Ascorbic acid concentrations (in u
g/mL): 12.5, 25, 50, 100, 250, and 400.
The DPPH solution (0.004 g in 100 mL methanol).
Blank : methanol.
control: 3 ml of the DPPH solution (0.004 g in 100 mL methanol).
control Abs at 517nm = 0.60
Ascorbic acid 200 ug/ml Abs at 517nm = 0.043
Ascorbic acid 25 ug/ml Abs at 517nm = 0.059
Is there a specific range of absorbance for ascorbic acid at 517 nm?
Because each reference has a different value for the same concentration of ascorbic acid, so I am confused. What the perfect value should be?
Many thanks
Although you think the ascorbic acid concentration you used (25 ug/ml) is low, it is quite high. In this case, the scavenging occurs in the dpph solution is a normal value. If you decide to work in unit of ug/ml, I recommend you start with values such as 0, 2, 4, 6, 8.
I present to you the method I used trolox (instead of ascorbic acid): DPPH stock solution is prepared with methanol at a concentration of 0.2 mmol/L. To create a trolox calibration curve, trolox (0, 10, 20, 30, 40 uM) is added to the DPPH stock solution at different concentrations, calculated from the ethanolic or methanolic trolox stock solution, incubated for 30 min at room temperature, centrifuged for 2 min (5000 rpm) and read at 515 nm on a spectrophotometer.
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