My DPPH assay using plant extracts even ascorbic acid constantly reduces absorbance on keeping inside instrument, get stable value in 10-15 minutes?.
If your solution is not stirred, even the low intensity light from the instrument may result in photoinduced chemical reactions. As a minimum you should put a cut-off filter in front of your cell to protect your sample from UV-light. A common borosilicate glass is not transparent for UV. Some cheap glass (not quartz) optical cell could be good cut-off filters.
Thank you Dr. Geletii. Please elaborate a bit more on this. I am confused as in if I put a filter before my sample, it will impact the analysis?
It is necessary to notice that to measure DPPH absorbance must be directly after incubation time reach. If we measure DPPH absorbance in different time, the absorbance will be different
Thanks Andi Suhendi.. but my concern is that. the absorbance does not even stay stable at all. So which value to take as reading?
It depends on the UV-vis instrument. If you record several spectra, you do not need to worry about a filter. DPPH assay is based on measurements of the reaction rate between DPPH radical with an antioxidant. Compare the rates dA/dt, measured as the slope of A plot versus time.
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