Have you done the phytochemical profiling of the extract? Which type of constituents you are expecting from the plant.? Is there any previous literature for your plant regarding isolation and chemical characterization? On the basis of chemical tests, you should firstly find out the categories of constituents present. Then only you can proceed for isolation of individual components.

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First of all you have to decide that what constituent/class you want to isolate. In ethanol most of the constituents may extract out including higher glycosides. You can isolate the constituent of your interest. Suppose, you are trying to isolate a flavanoid, you should assure that the extract having the flavanoids which can be confirmed by various chemical tests like Shinoda test. After that, you can run a silica gel column by chloroform with increasing polarity of methanol.

HI Bhupendra

As your mentioned that your extract contains flavonoids and glycosides, you should run the TLC with Chloroform:methanol in varying proportions from 9:1 to 5:5. I beieve you will get the best resolution in any of these proportions. Then you should target the major compound for isolation. You can either perform column chromatography or a preparative TLC depending on the amount of extract you have. Once you isolate the desired spot, leave it in methanol in a refridgerator for crystallization. You should get the compound by repeated crystallization. Then you can purify using acetone to get the isolated compound. Remember you have to simultaneously run the TLC at each step to ascertain the presence of that single compound only.

Gud Luck

Regards