Hello, for a part of my thesis work I´m doing a DPPH assay on a food product extract, and I´m running into a situation where the absorbance rate of DPPH+sample during the first measurements ends up higher than the absorbance rate of the DPPH reference. Does anyone know why this phenomenon occurs? The extract is colorless so it sohuld initially dilute de dpph when introduced, thus giving less absorbance from the start.
The reaction product might have higher absorbance than DPPH. Try to follow the formation of reduced DPPH (yellow colored)
What are the absorbance values of DPPH and DPPH+Sample? If they are above 2, that could be the cause as absorbance is not linear with amount of test substance (Beer-Lambert Law) for abs above 2 for most spectrophotometers. If this is the cause, try diluting the DPPH. Also test whether Sample has abs as well.
mostly you have turbidity issues, try centrifuge your samples and use only clear solution for spectrophotometer measurements.
Ken Ng We always work with absorbance values below 1 to maintain linearity. The protocols we use detail to dilute the DPPH until we reach an absorbance between 0.800 and 0.900. The problem we are having is that when sample is additioned (50 microliter sample in 1,95 ml DPPH), absorbance rates shoots up to above 1.
Omar Alajil we centrifuge the samples for abour 20 minutes and then collect the clear solution from the top of the tube.
Omar, if the solution is not clear or turbid diluting will reduce the absorbance not increases.
Matias, I presume you have accounted for sample absorbance itself. If so, the other effect I can think of is the chemistry. DPPH can generated long life intermediates that is more absorbing (check the literature on this issue with DDPH). That is why I prefer using APTS for this and many other reasons.
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