Using DPPH assay, most of my sample at higher concentration at 10 mg/ml has negative percentage or above 100 percentage and what should i do about it?
This unexpected outcome can occur due to several reasons like some compounds exhibiting pro-oxidant properties leading to an increase in the absorbance of the DPPH solution. High concentration can also interfere with assay components or chemical interactions with the DPPH radical giving you negative inhibitions.
So I suggest that you carefully optimize the concentration range of the test sample to ensure that it falls within the linear range of the assay. Also testing different solvent concentrations may also help identify the optimal conditions for accurate assessment of antioxidant activity. There after use complementary assays or methods to validate the antioxidant properties or results of the test sample.
Maybe something in your sample interferes with the measurement and absorbs at a wavelength typical of the DPPH radical, hence the results. Then you need to change the method to another one, e.g. ABTS, in which the measurement of the radical is carried out at a high wavelength (less exposed to interference) or use chromatographic measurement (test the depletion of the radical after separating it from the components of the sample that make measurement difficult). You can read more in my publication Is it possible to use the DPPH and ABTS methods for reliable estimation of antioxidant power of colored compounds?" Chemical Papers, 72 (2018) 393-400.
Regards,
Małgorzata
above 100% and negative results may occur due to errors in the procedure for DPPH blanks measurement and % inhibition calculation
Every time we make a DPPH solution we have to measure the absorbance of the blank. where the blank can only used to calculate the sample that was diluted with those DPPH solution.
If the sample diluted by another DPPH solution which we made afterwards, but using the first blank's absorbance for the calculation, there will definitely be an error in calculating the inhibition percentage (it could be more than 100% or negative)
The other reason could be your sample solution. When you have cloudy solution, you have to filter it with millipore, before reacted with DPPH solution
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