I am trying to isolate active compounds from bacteria. I have used an ODS HPLC column but the active compound did not elute within this column. Can anybody give me any suggestions? My compound is MeOH-soluble and it does not bind to HP20 resin or wa 30 resin. It's an oil-like substance.
By "did not elute" do you mean it is stuck on the column? WA 30 is an anion exchange resin so you compound may not bind to it. Or do you mean the compound just shot through the column? What was your solvent system for the reverse phase? Are you running a gradient?
Assuming the compound was stuck on the column, try non-aqueous reverse phase. don't use HP20 for this, you need a silica-based C18. Polymeric reverse phase (such as HP20) will swell in the solvents. For examples, see: http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Facile_Isolation_of_Carotenoid_Antioxidants.pdf and also http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN58_Non-aqueous_Reverse_Phase_with_RediSep_Rf_Gold_C18.pdf
Assuming the compound does not bind to the column, it may because you are dissolving it in too strong a solvent. The compounds elute with the "slug" of strong solvent and fail to interact with the packing. Consider using a weaker solvent to load the compound or dry loading it. I'd expect a methanol soluble compound to interact with reverse phase, unless it is extremely polar. For a comparison of reverse phase loading techniques, see: http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN88_C18_Flash_Column_Loading.pdf
Finally, the compound may be so polar that it fails to interact with reverse phase. Normal phase HILIC may be the purification technique you need:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN78_HILIC_Purification_Strategies_for_Flash_Chromatography.pdf
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Strategies_for_the_Flash_Purification_of_Highly_Polar_Compounds.pdf
Thank Dr. Jack Silver for your nice suggestions and references. I will try following your suggestions. My compound may be more polar and I used firstly 100% water later 100% MeOH as a mobile phase in HPLC. I used Inersil-3 ODS column. My compound is MeOH soluable. I used only water to HPLC sample preparation. My compound is also not bind in SPE cartridge C18.
Depending on the C18, the column may have been subject to "phase collapse" at 100 % aqueous solutions. The mobile phase is so polar that the C18 "strands" interact with each other rather than the solvent system and you compound.
You can try starting at 5% methanol, or wash the column with methanol, followed by a very short equilibration (one or 2 column volumes) and load your compound as quickly as possible.
One can also try a column designed for highly aqueous purification too. (see http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN76_RediSep_Rf_Gold_C18A_for_Highly_Aqueous_Mobile_Phases.pdf )
However, I suspect HILIC or another technique may work better for you since your compound failed to bind on an SPE cartridge.
Thank Dr. Jack Silver for an important suggestion. I should follow your suggestions and inform you in this regard.
Try to use 0.1% Formic acid, or 0.1 % Acetic acid, along with THF or even isopropanol as mobile phase. Retention may improve.
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