Hello everyone,
I was conducting the total phenolic content assay by using Folin-Ciocalteu's method. During mixing my microalgae extract samples (ethyl acetate, hexane, and ethanol extracts) with Folin-Ciocalteu's reagent, my sample mixtures did not change their color from yellow to blue as expected in many journals. Instead, my samples turned into two-layer mixtures which the top layer was my extract. My procedures were starting from;
1) add 0.5 ml of extract (1mg/1ml) into 2.5 ml of 10-fold diluted F-C reagent (diluted with distilled water)
2) incubate for 5 minutes at room temperature
3) add 2 ml of 7.5% Na2CO3
4) incubate for 1 hour and 30 minutes at room temperature
5) measure absorbance reading at 765 nm
All procedures were conducted in dark condition.
Thank you very much and have a nice day!
You can try the following points:
1) use 20% Na2CO3 instead of 7.5%. The pH value of sample with sodium carbonate must be around 10.
2) use undiluted FC reagent (0.5 mL) instead of diluted one.
3) use more concentrated sample.
Dear Nor Shafiqah Binti Nor Shahril,
You will find in attached the protocol which I generally use to perform this technique, I hope it will help you in your research.
If you need any help, don't hesitate to contact me.
Best wishes,
Sabri
Dear Nor Shafiqah Binti Nor Shahril thank you for sharing this very interesting technical question with the RG community. I think that the total phenolic content varies with the solvents which you used for extraction. Chances are that especially the hexane extract (and perhaps the ethyl acetate extract as well) did not contain measurable amounts of phenolic components. In addition, you should make sure that your extracts are miscible with the Folin-Ciocalteu reagent and do not form two layers. Thus I would suggest to use only the ethanolic extract and add some water for better results.
Good luck and best wishes!
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