I am trying to introduce the Folin-Ciocalteu method in the extraction of phenolic contents with NADES. The problem is that when I trying to measure TPC, Folin precipitates, specifically using solvents that contain Choline Chloride, generating a yellow precipitate when the Folin reagent is added (without adding Sodium Carbonate). When I add the Sodium Carbonate, the precipitate is blue. Can this be fixed? Do I have to find another method? Has someone else precipitated the sample by adding Folin-Ciocalteu?
Hi,
It seems to be the precipitate you get include phosphomolybdate which gives the yellow color to Folin-Ciocalteou’s (FC) reagent. In this case, there are some possibilities. Phosphomolybdate has an affinity to compose coordination complex with ammonium salts. Ionic liquids prepared as DES/NADES with choline chloride as proton acceptor can serve as a quaternary ammonium salt functionality to observe bulky ammonium phosphomolybdate precipitate which is typically insoluble in DI water. This is a possibility since the DES/NADES is formed by quaternary amine salts in this query. You are altering the molecular interactions (primarily H bonding) between the DES components by forming a type of octahedral or tetrahedral coordination complex. You can also break DES structures by diluting them using water or organic solvents.
As another assumption, disrupting the molecular web of DES may cause the loss of solubility for phenolics thus hydrophobic ones can precipitate if they are abundant in your matrix. Folin C reagent conjugated phenolics may form a precipitate and your total phenolic assay may be occurring at the solid phase. Adding Na₂CO₃ and increasing the pH will catalyze the reduction of bound metals thereby color changes could be seen at the precipitate. It would be clarifying to know which constituents and ratio you have in DES formulation, did you directly mix the sample with FC reagent, or did you apply any dilution prior to assay and which matrices are you working on. Another co-eluent from your matrix or DES subunits may also induce this reaction.
By implementing some modification to the technique, accurate results may be acquired. I would try to add organic (EtOH/MeOH) or water dilution steps to experiment to improve the solubility and reducing the amount of choline. Optimize your sample prep for TPC as follow and give a try; 0.1 mL of extract, 7.9 mL DI water, and 0.5 mL of Folin-Ciocalteu reagent. Always use Folin R. considering its expiry date. Confirmatory results could be obtained by performing alternative approaches such as radical introducing (e.g. DPPH, ABTS) or non-redox-based assays such as chromatographic elution and UV monitoring.
Good luck,
Emir
Hello, Nicolás.
I've also been working with DES comprising choline chloride and the same happens.
I think Ismail gave a nice answer to the issue and as I tried, dilution mitigates the effect. If you say it's specifically for those with choline chloride, maybe you tried other compositions?
I also used a mixture where choline chloride is present but no precipitate was formed as it was previously diluted.
I would advise for you to measure the pH of the mixtures and before Folin assay, if possible, add a dilution step. Of course it will depend on how concentrated is your extract and if you'll be able to acquire enough absorbance.
Sometimes even by dilution is not possible to avoid precipitate so complementary assays would be advised.
Hope it helps.
Regards
Hugo
Maria Carla Marcotullio.... Hi, I faced the same problem. But my problem was solved when I added NaOH after the addition of sodium carbonate. According to your sample, you can standardize the amount of NaOH that will help to rectify the problem of precipitation. I am recommending this research paper which was helpful to me. I hope it will help you. Article Recovery of Bioactive Extracts from Olive Leaves Using Conve...
Regards
Taru Negi
Taru Negi Which is NaOH concentration? The paper doesn't mention it. What do you mean by "you can standardize the amount of NaOH that will help to rectify the problem of precipitation"?
I really appreciate any help you can provide...
Carla
Maria Carla Marcotullio I have used 4 % NaOH. After adding sodium carbonate, I added NaOH in different concentration like 100 microlitre, 200 microlitre and so on (done hit and trial). Then, I have vortexed the sample and kept it for 1 minute in waterbath. And then checked the precipitation. For my samples, 1500 microlitre NaOH worked (no precipitation found). I hope it will be helpful for you.
Regards
Taru Negi
Hello!
We had the same problem. When we used choline chloride-based NADES Folin-Ciocalteu always precipitated.
Our solution was to put sample tubes with all the solvents (Sample + Sodium Carbonate + Folin) in a rotator shaker in ambar tubes for 1h.
After that time, centrifuged and measured the absorbance as always.
Hope you find it useful!
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