Please give me some pointers and things to consider in phenolics separation using column chromatography. What are your experiences in doing so? What specific solvents did you use to elute your phenolics? Isocratic or gradient elution? All info regarding my question is welcome.
What type of compound are you looking into. If your compounds are light sensitive they might decompose in the column.
Phenolics cover a wide range- from non-polar tocopherols through anthocyanins and flavanoids. I use ion echange for the initial purification from natural sources.
For more informaiton, see:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Facile_Enrichment_of_Tocopherols.pdf
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN87_RediSep%20Rf_SAX_Column_Applications.pdf
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN27_Rapid_Purification_of_Tocopherols.pdf
I am more into characterization of phenolics, then later on I am planning to isolate and identify other unknown phenolics in my sample, if there are any. I am sure that most of the compounds I am looking for are not light sensitive. Thanks for the idea by the way.
Thanks Sir Jack. As of now I am looking into a wide variety of phenolics in my sample and your contributions will greatly benefit me.
Check out the grape and wine literature and you'll find plenty of ideas. Even multi-step approaches to fractionate different classes to do further work with.
I normally apply the aqueous/methanolic extract over a polyamide column using (water / methanol) mixtures in decreasing polarities (start with water to elute free sugars then 10 % methanol then 20 % Methanol.... till you reach 100 % methanol to elute aglycones ).... you can apply the fractions on a sephadex LH20 subcolumn (50% Methanol or Butanol water saturated) or use a preparative paper chromatography according to the composition of your fraction... always trace your fractions and subfractions using 2DPC or CoPC ... use spray reagents (ferric chloride for phenolics in general & other more specific reagents for tracing flavonoids & tannins or any other compounds u wish
Note: if you have sulphated phenolics you can't apply your extract on a polyamide column because they are adsorbed on the stationary phase and you can'y elute them... better start with a Sephadex LH20 column and re-purify using Sephadex LH20 subcolumn with a different mobile phase or prep. paper chromatography
You can put your column in a dark room and check the bands under long UV light , it is always detectable when using Sephadex LH20 columns ... and of less use in case of Polyamide columns... best wishes
Thanks Reham and David! I will consider your inputs. Regards!
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