It depends a lot on what do you want to get out of the extract. There are three main technologies you can use:

-Solid liquid separation: Using a resin you can adsorb some of the compounds from the extract and using a solvent/mixture of solvents, you can elute them. In this way you can separate different fractions with different compounds.

-Distillation: Adding heat you can vaporize certain compounds and retrieve them by condensing the vapor. If you have lots of volatiles or compounds sensitive to heat (proteins, etc.) this is not a good idea.

-Liquid-liquid extraction: By adding a solvent that doesn't mix with the extract, you can separate all the compounds that have more affinity for that solvent.

Do you have a methanolic extract?, in this case add a little volumen of water and try a liquid partition with less polar solvent to sepárate according compounds' polarity and each extract shoul be separated by chromatographic column,

in other case you can run directly  a chromatographic column starting with hexane  followed by dicholomethane or ethyl acetate and then methanol again. Each portion should be run by chromatographic techniques again,

Assuming you don't know what causes the biological activity....

Do a literature search on your plant and see what may have been isolated already. Keep these compounds in mind.

If doing antibacterial/antifungal screens, run thin layer chromatography (silica, alumina, diol, C18) with a variety of solvent systems and do bio-autograms which will allow you to see that the biological activity moves, and if it is resolved from other compounds.

If doing other biology, I like to run a "column screen" with "wide polarity range chromatography". The "wide polarity range chromatography" is as Olga described, except I like to continue on beyond methanol and use water as well. I use this solvent system as part of a column screen which includes silica, alumina, and diol columns. For reverse phase, I like to run C18 and run a gradient of water -> methanol -> dichloromethane. I also like to include ion exchange columns in my screen (see links below).

Collect all fractions and submit for biological assay. Run active fractions through an LC-MS to see if the molecular weights match known compounds, be aware of fragments and adducts.

The column screen provides information on a purification strategy and information about the polarity of your compound. It can provide enough material for LC-MS and possibly initial purification. Although I used an automated system, it can be run with glass columns as well.

More information on column screens:

http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20Poster.pdf#search=column%20screening

http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20II%20Poster.pdf#search=column%20screening

Column Chromatography will give separate fractions. Elute the samples with different polarity solvents, use non-polar to polar solvents in the column. 

I suggest column chromatography and even then you should have an idea of the nature of compounds suspected or known to be present in your extract. Are these compounds polar or relatively non polar. Consider also the state of your extracts e. g. solid extracts can be used in column chromatography. 

If your sample is crude then you can go for successive extraction  from high polar solvent to least polar solvents so that we can obtain maximum number of compounds. Then you can go for TLC followed by column chromatography . With simple and low cost we can separate more number of compounds.

Based on the compound of interest you can proceed further with HPLC , HPTLC etc

Please find our developed method to extract and fractionate flavonoids HPLC based.

Thesis Chapter 2-TO INVESTIGATE THE ANTIOXIDANT PROPERTIES OF DIETA...

Thanks everyone for all the extremely valuable insights that you have provided.

All answers you get here indicate the same: there is no general method you can use in all situations. In fact, Your extract maybe of essential oils. These are in general volatile compounds. But your extract may be of lipophilic or, on the contrary, hydrophilic compounds. The same techniques cannt be apply in both situations. Thus if your want us to give you an adequate methodology for your particular case, please be more detailed on what kind of extract you need to handle. 

Dear Prof.Alphonse,

Thank you for your contribution to this query. I wish to prepare 50% ethanol/methanol extracts of whole plant material, and because  I will be using whole plant material(leaves,roots,bark,stem...etc), am expecting a wide range of phytocompounds as well as interfering compounds to be present in the prepared extracts.In view of this, I am inquiring on the most efficient method that would enable me obtain as many pure fractions as possible for screening using bioassay methodologies of my choosing.

Regards,

Mitchel Otieno Okumu 

 Hello everyone. Rather than using individual plant materials, I wish to extract whole plant material and therefore I am expecting a myriad of phytocompounds each with their own distinct/related character. To this end I am hoping to choose the most appropriate extracting solvent/solvents to make crude extracts, then go on to use an optimum but cost-effective method for fractionating the crude extracts to obtain multiple fractions which would all be subjected to bioassay.Kindly advise.

Regards,

Mitchel Otieno Okumu.