I'm trying to do a DPPH antioxidant test but when I mixed all the reagents with my polyphenol extract, some of the samples turned cloudy. Why does it happen and how can I prevent it? I am working on Dong Quai ginseng with different drying conditions.
DPPH is a stable free radical with a deep violet color in solution. When it encounters an antioxidant, it reacts by donating an electron to the antioxidant molecule, reducing DPPH to a colorless compound. Depending on the concentration of antioxidants present in the solution, this reaction can lead to a decrease in the intensity of the violet color and may appear cloudy.
Control sample concentration: Dilute samples if necessary to prevent excessive interactions with DPPH or other components in the solution. This can help minimize precipitation or aggregation that leads to cloudiness.
If you have a mixture of solvents (extraction solvent and DPPH solvent) or your extract is more concentrated, it's best to use dilution.
DPPH solution must have an absorbance in the range 0.1-1.5. The concentration of antioxidant must be about the concentration of DPPH. Therefore, the dilution is not the solution of the problem. The DPPH assay is perfomed in EtOH. If your antioxidant is badly soluble in EtOH, you can't use DPPH assay. Alternatively, the reason of claudness might the bad solubility of the reaction products. In this case you may filter off the precipitate.
Ethanol is used to precipitate proteins during various processes, including purification and crystallization. This may be a cause.
I tried to solve this problem and when I used a solvent for the sample and DPPH, the cloudiness disappeared. So, I think because of some compounds in my extrect is not disolve in Ethanol 99,5% (this is the first solvent for DPPH) and lead to the cloudy
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