I an trying to elute secondary metabolites from plant extract via column chromatography ( silica slurry method).But unfortunately, I got uneven extract distribution in column which is not moving further. It is stucked. Hexane: ethyl acetate is using as a mobile phase . I packed column very efficiently but don't know what is the matter?
How do you know the compound should elute with hexane and ethyl acetate?
Are you trying to purify a known compound? If so, try to follow the procedure previously used for this material. Verify that a standard will run on TLC. Did you run a step gradient to 100% ethyl acetate?
One problem with natural products is that we may not know which compounds are active, so we don't know how to purify them- we can only try procedure that maximize our chance to purify to active compounds.
If you are working with antibacterials or antifungals, you can run TLC plates and do bioautograms. For other activity, try running your silica column with hexane to 100% ethyl acetate, 100% ethyl acetate to 100% methanol, and finally 100% ethyl acetate to 100% water. This gives all the compounds a chance to elute from the column.
Plant extracts contains several components that could reduce the efficiency of your separation. Firstly, you have to ensure removing pigments, sugars, protein by using physical methods or liquid-liquid extraction so that you have fractions that contain less secondary metabolite to simplify the separation otherwise you would have to do a lot of sub-columns. Also from I can see your loaded sample might be too much for the packed column, there is golden role in separation that for easy to moderate separation for each gram you should have to use 25-50 gm silica for packing while in complex separation you should use upto100 gm silica this could be determined by the behavior of the compounds in TLC. pay attention for the flow rate to avoid diffusion of the bands if it is too slow or too fast. good luck
I performed TLC before conducting column with different ratios of hexane and ethyl acetate. I picked best one among them which showed some sort of bands separation. But unfortunately, in column it was not so as on TLC.
Is it possible you mixed the solvents improperly? I sometimes reverse the solvent lineswhen I run automated flash chromatography. Try the step gradient with increasing concentrations of ethyl acetate to elite the compound.
Hi Aisha,
You can change the eluent. Alternatively, remove the stuck silica gel column and dissolve it again, followed by recrystallization or other purification operations.
I tried Hexane;Ethyl acetate (9:1), then pure hexane after column stuck, but it didn't work, then I elute my column with pure Ethyl acetate little bit fractions. Finally by adding acetone, I eluted my whole plant extract efficiently. It was a difficult column. Thanks for your cooperation.
For another column, I want to isolate and purify compounds from chloroform extract of Moringa oliefera leaves. What do you suggest for successful separation?
I have approximately 34 grams of Moringa oliefera leaves extract. Crude extract extracted in chloroform solvent.
I agree with Abdullah A. Elgazar
I think the weight of the silica used is not enough and I recommend using suitable column size to obtain the optimum separation.
Furthermore I think using liquid- liquid extraction followed by VLC especially if you are targeting defined compounds which could be traced by spraying reagent on TLC would shorten the time of separation process since you will focus only on the targeted fractions.
finally i think these tips and tricks would help you
10.1002/chemv.201200074
10.1002/chemv.201200084
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