I am working on crude methanolic extract of Amla which is further fractionated into chloroform, ethyl acetate , butanol fractions . I have got best respective activities in ethyl acetate extract . So, I want to purify a compund from the respective fraction . Please suggest some effective solvent systems for ethyl acetate fraction.
For TLC you can use Hexane : ethyl acetate, chloroform: Methanol, Water: methanol:Ethyl acetate(0.5:1.5:8), Ethyl acetate: methanol:acetic acid/formic acid in different ratios.
For purification on column you can start with non polar solvents like hexane/petroleum ether and increase the polarity with ethyl acetate.
Before all this, do you know which compound you are trying to isolate or finding the active molecule in the extract?
If you know the active molecule, then compare the TLC by spotting your EA extract and the compound. So, you will get some idea about its rf value. Based on that you can try the solvent system for isolation.
Yup, try Subheer's suggestions.
Also, your methanolic extract would contain diverse chemical compounds but in your ethylacetate fractions, phenolics, flavonoids, tannins, caffeates, benzoates, would be in abundance. Worth trying chloroform: methanol: formic acid (85:15:1) as a mobile phase for silica gel TLC F254 plates for their separations.
But, RP-HPLC of the ethylacetate fraction with commercially purchased standards would allow more identifications. MALDI-TOF-MS and LC-ESI-MS analysis would be helpful in identifying less abundant compounds and cases where the compounds are novel and no commercial standards available.
Thanks,
Biswa
Hi, there are too many different constituents you may have in this fraction! Will you try silica-gel, reversed phase (e.g. C18), or other stationary?
Also, you can develop your TLC with best mobile phase, but do you have any idea how you will observe your compounds (i.e. did you choose the staining method?).
I recommend for you a TLC Book with many applications in phytochemistry:
Thin Layer Chromatography in Phytochemistry
Monika Waksmundzka-Hajnos, Joseph Sherma, Teresa Kowalska
However, TLC is used as a preliminary analysis, the identification of your compounds should be performed by a more sophisticated technology as pointed by Misra, preferentially by LC-MS (MS2).
I hope I've helped you
Lauro
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