I am working with plant extracts. I need to purify by solvent fractionation and perform antioxidant assays like DPPH ABTS. I am not able to make a clear solution of those non polar fractions. Whenever I try performing the Assays, the solution containing the reagents and my sample turns milky. The sample precipitates out. Please suggest any solution. Thanks in advance.
Dear Ekta,
You didn't mention what solvent you used. There are many non polar solvent. All of them have different polarity. Therefore, based on your solvent used, answer might be appropriate.
For non polar, DMSO might also work but in some extract, it forms ppt. Olive oil might also be good alternative.
Good luck
First, after solvent partitioning, concentrate the fraction to dryness using rotary evaporator. Then prepare a stock solution of 1mg/ml using methanol. If you have problem with dissolution, first use two drops of the partition solvent to dissolve it before adding methanol. From the stock solution measure 0.2, 0.4, 0.6, 0.8, and 1.0ml into a 10ml measuring cylinder and make up the volume of each to 10ml. This will result in 20, 40, 60, 80 and 100ug/ml concentrations. Now measure let say 3ml of each into another test tubes and add 2ml of DPPH reagent and incubate in a dark chamber for 30min before reading your absorbance at 540nm. The same procedure can be use to prepare the samples for other assay.
If you have non polar fractions, use non-polar or less polar solvents to dissolve it (DMSO, methanol, concetrated ethanol etc.). It is also important that your DPPH radical be dissolved in less polar solvent (for example methanol or ethanol are good as solvents for DPPH and ABTS radicals). In this case there should be no blurring or occurrence of precipitation.
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