I have prepared methanol extract from dried leaves and bark, then dried them at room temperature. During drying I observed formation of very thin film over the surface of solvent in case of leaves extract. After complete drying I got semisolid sticky material plus sticky shiny thin rubber film-like mass in methanol extract of leaves.
As I wanted to focus on isolation I have planned to fractionate total methanol extract using different solvent. As we know fats, waxes, oil etc come out with petroleum ether, I have first partitioned the collected methanol extract with petroleum ether.
Now, my query is: which other fractions should I prepare from the same methanol extract in order to isolate compound?
I would like to mention that the rubber film-like mass we have in methanol extract of leaves is not affected by petroleum ether partitioning. So what should be done with this kind of mass present in methanol extract?
Partition your methanolic extract with hexane, chloroform, ethyl acetate and methanol. Your sticky mass may be polysaccride types of compounds.
Do you know what sort of compounds exhibit activiety in your assay? If acidic or basic, you may be able to partition them. For example, for basic compounds, I partition between organic solvent and 0.1N HCL. I then make the aqueous layer basic, and partition again. The alkaloids are free bases in the organic layer.
Another idea is "wide polarity range chromatography". Dry-load your extract on silica, and elute with hexane/ethylacetate gradient followed by ethyl acetate/methanol, followed by methanol/water. This can be run on silica.
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Wide%20Polarity%20Range%20Flash%20Purifications.pdf
You can dissolve that thin film in little water and if it dissolve re-precipitated it by adding four volume acetone. If u got the precipitate, you can sure that it is polysaccharidal nature comp. Go for hydrolytic analysis for identification of monosugar etc.
The thin appeared over the surface of solvent in case of leaves extract may be due to wax and leaf protein combination as some of leaves contain waxy material on upper and/or lower surface. You have not mention the name of plant and the specific phytochemical or group of phytochemals like lipids, phenolics, alkaloids, polysaccharides, etc, you intend to isolate. It would have been better to first extract the plant material with solvents like n-hexane, pet ether to remove oily or waxy materials, if present. Further, fraction of crude extract using solvents of eluotrope series will depend on the type of compounds to be isolated. Now, since you have already prepared methanol extract, you can suspend the extract in water and then partition through liquid-liquid partition chromatography with solvent of increasing polarity starting from n-hexane or pet ether followed by other solvents in order of increasing polarity. Different fractions so obtained should be to be distilled and dried. Then carry out chromatographic separation using techniques like silica column chromatography, flash column chromatography, etc for isolation of pure individual phytochemical constituents. You may get some pure compounds as well as mixture of 2 or more compounds; then you have to go for repeat chromatography.
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