I am trying to seperate and purify a steroid but I could not succeed through silica gel column. one more unknown compound (black spot on charring with anisaldehyde sulphuric acid reagent)also coming along with my steroid(violet colour after charring). please suggest me any possible method to get the pure steroid.
thank you
What solvent system did you use? Solvent systems which might work include hexane/ethyl acetate (you probably tried this one since that is a good starting point), hexane/dichloromethane, hexane/ether, hexane/toluene, and hexane/2-propanol. The second solvent in all of these are in different selectivity groups and so cause the compounds to elute in a different order. They are also miscible with hexane too.
The solvent systems can be evaluated by TLC. Note that the solvent system that gives the best TLC doesn't always mean the best column run If the compound runs 3/4 of the way up the plate (Rf=0.75) and the impurity has an Rf of 0.60, that mey not give a good separation. The first compound would elute, under isocratic conditions as used on the TLC plate, in 1.3 column volumes (CV = 1/Rf) and the second elutes in 1.7 CV. This gives a difference of 0.4 CV, which isn't much for a column. If you are familiar with HPLC, 1 CV corresponds roughly with the void volume. Using a weaker solvent system, where the compounds take longer to elute, will give a better purification on the column.
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Steroids can be extracted only by using neutral solutions, Ethyl acetate is the best solvent to extract steroid. I dont think in hexane fraction steriods would have come. If you want to confirm Run a chromatogram with your hexane fraction for steroids and spray with tetrazolium blue, heat the plate and look in for violet spots.
if you can separate both the component on TLC then you can use that mobile phase (or slightly less polar mobile phase) for column. your compound of interest should have Rf value less than 0.4 on TLC plate. separation will be better with increasing the column length and decreasing the sample load.
you can use HPLC in normal phase, using an isocratic mobil phase, try 50:50 iso-octane:Ethyl Ac. you have to use as a detector ELSD, because for this way, you can see all your impurities . once you know your retention time, you can take off the ElSD connection, not to waste your sample.
best wishes
For the TLC, switch to a C18 plate. Now the differences in the hydrophobic interactions will be more significant than the polar interactions (e.g.hydrogen bonding to the silica) If both compounds show up under UV, use the TLC plate with the phosphor. If not, you can consult the literature regarding detection reagents. Stay away from sulfuric acid as you will char your whole plate!! You can always try derivatization prior to elution but that's another headache
I think you can search in the Camag CBS data base, the possible mobile phase for separation of your steroids, after optimizing the system to get best separation (see comments from Jack above) you can apply to CC or HPLC or TLC preparative to isolate your steroids.
http://www.camag.com/en/tlc_hptlc/customer_magazine_cbs/ccbs_database.cfm
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