I'm doing my experiment of total antioxidant on plant rhizome.
3g rhizome were ground in 10mL extraction buffer (2mM NaF dissolved in 800mL and 200mL dH2O)
DPPH as stock solution by dissolving 24mg DPPH in 100mL methanol. Diluted stock solution (1:4v/v) was prepared as daily solution
50microlit of supernatant was mixed with 950microlit daily solution. The OD was read at 515nm
And the OD I must have in the range of 0.6-0.7, if i get 0.7 the dilution must be done
I didn't understand why we cannot just accept the OD value we get even not in the range. is there specific range for total antioxidant of any sample?
I think there is no specific range of OD value for total antioxidant in any samples, it is calculated with reference to OD value of DPPH solution.
Two reasons which I could think to the best of my understanding.
1. Limitation of spectrophotometeric reliability beyond OD values >1.5 absorbance units (0.6 < Ideal range < 1.5 for accurate colorimetric estimation). OD > 1.5 presents certain degree of uncertainty
2. DPPH protocol - higher [DPPH] may require longer incubation, as antioxidants in test extract need adequate reaction time to scavenge DPPH free radicals.
It is always wise to quantify antioxidant capacity in equivalent units of standard antioxidant compounds per weight basis or EC50, instead of just %inhibition.
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