Do you want to know the possible compounds in your extract? In my opinion the best way is by using LC- QTofMS, or you can do it by GC MS. Or you can purify first using CC, HPLC preparative or TLC preparative than determine the chemical structure of the pure isolate using spectroscopy methods.

in this case i will recomend to measure a NMR of the crude to try to identify the mayor type of molecules you have in that crude. Another option is make a liquid-liquid partition with solvents of different polarities.  You will separate the molecules of the crude according their polarity and the measure again NMR and LCMS. 

Also would be a good idea to monitor every fraction against the crude in a control TLC plate, it could be developed with Dichloromethane: methanol 95:5, and try to localize fraction or partitions with the mayor compounds and also you wil have an idea of the polarity of the molecules in each partition. 

You may try acid or base. Means try to add acid and base separately in your aqueous layer and fractionate it with organic solvent. Then check your activity with both extract you obtained.

Is your question about extracting water soluble compounds that can't be extracted by EtOAc? Or how to identify the nature of compounds in EtOAc crude extract?

If you mean the second one.. you can inject about 50 microgram of your crude extract in HPLC-MS so you can predict the nature of your compounds from UV and molecular weight.

on the other hand, I'm extracting my bacterial cultures with EtOAc and I think it can extract most classes of compounds 

@Dr. Gunawan, For QTOF & HPLC i could not select the solvent for elution since i am not sure what kind of compounds are present.

@Quirós, i tried liquid-liquid partition with chloroform, ethanol and butanol separately but the extracts did not show activity.

@Neeraj Sethiya, Do you have any suggestions of acid or base? 

@Osama, i wanted to know how about extracting water soluble compounds that can't be extracted by EtOAc.                                                                                                   I agree EtOAc can extract different compounds but i could not find my active fraction in it. 

@Quirós, i tried running TLC of aqueous EtOAc extract with Chloroform:methanol:water(65:25:4), n-Hexane: EtOAc(1:1) and (7:3) but the spot did not move. As you suggest, i will try Dichloromethane:methanol(95:5) and get back to you.

Try sulphuric acid and ammonia.

As many metabolites are basic in nature. As you add acid these converted in salt, which is freely soluble in water means in your aqueous layer. After you add ammonia again to basify your aqueous layer, then salt break down and active metabolites again converted to basic form, which is highly soluble in medium polar solvent. i.e. ethyl acetate.

In case of plant metabolites it happen, but can not say about bacteria.

Excellent, is better always start with this kind of mixture to see kind of the whole picture and then modify the composition of solvent, is not normal start with complex mixtures if you do not know the kind of components.. another think you can try if the spots do not move is Butanol saturated with water, this is usefull for sugars... maybe your extract could have some because is aqueous...

Another stuff, is not necesary to know the kind of compunds to chose the solvent for HPLC.. you always have to start with a preview usign a liner gradient of aqueous methanon or acetonitrile in a run of 35-50 min.. then you modify the gradient, step, isocratics, etc to get a better resolution in a shorter time...

@neeraj.. Will try and let you know if it is same with bacteria.

@ Quiros, would you suggest i run HPLC of supernatant directly with methanol and Acetonitrile as solvent?

Dear Aravind,

I think you can do first simple TLC analysis of your extracts (it will be nice if you use the same stationary phase of TLC (maybe RP18), and column that will be used for HPLC or LC QTof MS; after you get good separation by TLC, you can used the mobile phase for HPLC and or LC QTof MS.

For TLC separation, please search in this data base (maybe someone has done):

http://www.camag.com/en/tlc_hptlc/customer_magazine_cbs/ccbs_database.cfm?method=query

Dear Arivind

I do not have that much of relevant information about bacteria. But, it may work as most of organism consist of secondary metabolites. Which must work on same principle.

basicaly you can make two things... normally what i do for the chemical profile in plants, honeys, etc is normally start as i suggest before with a linear gradient in a long time and then try to improve it in time and resolution. But as you may know the most common solvent systems for HPLC are Methanol-Water and Acetonitrile-Water. so you can chose one and try....

Or the second thing is the last answer... make a profile in a reverse phase TLC (there you just can use water:methanol or acetonitrile-Water mixtures), once you get a good resolution you have a starting point in HPLC..

When you say supernatand is the Ethyl acecate extract of the growing media of the bacteria?

Dr. Gunawan Indrayanto, thanks for the link.. seems useful. i will refer to it.

@Neeraj. thank you.

@ Quiros. I have tried HPLC both with methanol-water N aetonitril- water run for 60mins. i couldnt see any peak. , But on TLC with Dichloromethane:methanol (95:5) i could see three spots close together eluted very near to base. Upon changing the propotion with gradient (starting from 50:50 - 90:10) the spots were found to smear!!