The flavonoid of interest is polar in nature. The flavonoids were adsorbed onto acid pre-treated activated charcoal (packed in column) from ethyl acetate solution. I tried recovering with methanol, water, 7% aqueous phenol solution, 0.1N HCl and also 0.1N NaOH but couldn't recover it. Could someone suggest me solution?
Adsorption of polar flavonoids (e.g., glycosides) on charcoal maybe of some difficulties, as those interactions can be very rigid, as you observed...it would be better to use polyamid to enrich the flavonoids....then you can easily recover them....
I agree with Dr Rohn. Flavonoids, as well as phenolic compounds, are strongly retained by activated charcoal. I would suggest to use hot ethanol to try to desorbe the flavonoids but I do not assure that will work. Acidic solutions should be avoided since may hydrolize glycoside derivatives.
There is one problem. Actually, I had isolated this flavonoid from column chromatography with silica gel as packing material. There are 2 other compounds with almost similar retention factors to that of the flavonoid due to which they always get eluted together. Since I have extracted them from aqueous extract, those other compounds are also polar. I have to separate the flavonoids from those componds. But if I use Polyamide, there is chance that those impurities will also bind to the polyamide along with the flavonoid. what to do in such case?
Dear Sanjana, changing the stationary phase also the retention indices may change.
If you will separate the mixture using column chromatography you may first check on TLC with different eluting systems what are the best conditions to resolve the mixture.
Otherwise, if you planned to use adsorption/desorption technique you may use desorbing solutions at different polarity, starting from the more polar e.g. ethanol 30% and then use less polar solutions (60 % and 95%). May be that the contaminant will be desorbed by a different mixture of solvents in respect to the flavonoid of interest.
For the polyamide column and flavanoids, I like a gradient starting with water to 100% methanol, followed by another gradient from methanol to 100% acetone, then acetone to 100% water containing 5% ammonium hydroxide (3 gradients total). When I know where my desired compound(s) elute, I can just run the gradient where the compounds elute rather than all 3 gradients. This seems to elute most compounds, but tannins may remain bound.
I agree with you sir. I have tried out various elution systems and optimised that with TLC. Also, I have tried with two stationary phases - Alumina gel and silica gel. But the flavonoids and the contaminants Rf values are so close that it is being difficult to resolve them with column chromatography even with different elution systems. Since polyamide is also a polar stationary phase like alumina and silica, I suspect I would be facing the same problem. I will try it out anyways to see if there is better resolution.
To overcome this problem, I had chosen Activated charcoal as an alternative for purification. In this case, it is observed that only the flavonoid is binding to it and the contaminants are eluting out. Due to this, if I have a desired solvent system which can recover the flavonoids, I can obtain it with high purity. I will try it out with ethanol at different polarity.
If there is any other adsorption/desorption systems which have particular affinity to flavonoids/phenols, I will be very happy to try that as well.
Remember that polyamide has the amide linkage, depending on the type of contaminant, the selectivity of polyamide may be different from that of silica or alumina. What solvents have you tried with silica and alumina? Any modifiers?
Another possibility may be amine, which can be run like silica or alumina, and one can test it with TLC plates before buying the media.
Sephadex LH-20 may also clean up your flavanoid too. Also depending on the nature of the impurity, ion exchange may work too; both techniques were used in this poster: http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
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