Solution turning cloudy during DPPH antioxidant assay?

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A. M. Naim O'Sullivan

It can be caused by numerous factors. Some samples have undissolved solids that float making it look cloudy etc. You can centrifuge your samples and hopefully the solids sediment at the bottom and decant the desired solvent. If it's oil you have an issue as DPPH is for polar compounds mainly.

Prakhar Chatur

Hi Naim, Thanks for this information. I did try centrifuging my samples but it is still giving a cloudy mix. I have extracted polyphenols from legumes which have about 6-8% fat content. During the extraction process, I could see that there was fat like substance present in the solution, so I segregated my extract from it. But when I mix DPPH in the extract, it turns cloudy and I cant figure out why is this happening.

Ayorinde Victor Ogundele

Hi, Prakhar Chatur , what you are experiencing might be due to high concentration of the extract and difference in polarities in solvent . which solvent did you use for the assay? what is the concentration of your extract?

Hi Ayorinde, I use 10mL methanol to dissolve bound polyphenols. My free polyphenol extract doesn't turn cloudy and it is in 70% methanol.

I use 10mL methanol to dissolve bound polyphenols !! 10 mL to dissolve what mass of extract? I assume this 10mL of the methanol extract was your stock solution, so what range of concentrations did you use for the actual experiment, i mean something like 20 ug/mL, 40 ug/mL, 60 ug/mL, 80 ug/mL etc. Please, clarify

I used the exact procedure from - "Phenolic profile and content of sorghum grains under different irrigation managements". I extracted free polyphenols from 2 g sample. After this, the residue was used to extract bound polyphenols and the extracts were used as i is.

Davide De Angelis

Hi, i am working on legume antioxidant activity, I think that you should centrifuge the sample after the reaction with DPPH and then read to spectrophotometer. Wich method are you currently using?