I am facing too many problems while isolating pure compounds. We don't have a facility for HPLC. How can I isolate pure compounds (Phenolics including flavonoids)?
Use XAD-4, 8, 16, HP-20 can use for prilimanry purification followed by C-18, sephadax LH-20 for pure phenolics.
Dissolve the extract in distilled water successively partition in pet-ether, chloroform, ethyl acetate and butanol. Ethyl acetate and butanol fractions contain the flavonoids. Subject both fractions to either tlc or column chromatography on silica gel as the adsorbent with chloroform/methanol solvent mixture.
Thank you very much for kind suggestion, @ Robin Joshi. Can you please tell me the proceedure how to separate with Sephadex LH-20, I prepared rich fraction with amberlite, so need to run with sephadex....pls send me any procedure and solvent system to be use...
I use ion-exchange (strong ion exchange) followed by LH-20, followed by C18.
Here's some examples I've run:
This one shows purification of flavanoids without HPLC:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
Other flavanoids with ion-exchange columns:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN87_RediSep%20Rf_SAX_Column_Applications.pdf
The type of solvent used for extraction is important for extraction of phenolic compounds. Various solvents generally used for extraction of different phenolic compounds include water, ethanol-water or acetone-water. For extraction of catechins, methanol-water or ethanol-water have been used and phenolic acids have been extracted with acetone-water, dimethylformamide-water. Methanol containing hydrochloric acid is used for extraction of condensed tannins. Since you have already prepared ethanol extract which may contain flavonoids either free or in glycoside form and you don't have HPLC facility, you can go for column chromatography followed by TLC examination of eluents and crystallization of TLC-homogenous eluents. Further, you may have to do repeat CC for the eluents with mixture of two or more compounds. Also, prior to CC you can opt for gross separation by liquid-liquid partition for which take/suspend the extract in distilled water and then successively partition in, chloroform, acetone, ethylacetate and butanol. Then CC of these fraction may result in good separation of pure individual compounds.
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