I have done the flash column chromatography of the crude mixture. After the slow elution at 4% Ethyl acetate in petroleum ether, the polar impurity is separated and there is not signal of the impurity in flash chromatography. when the compound elution starts, the initial non-polar impurity still persists and comes along with the final product. I have tried washing with n-hexane and methanol. The final compound and impurity also goes into the non-polar solvent layer, and methanol layer also have both the compounds. I dont know how to separate this impurity from the final compound. Rf of impurity is 0.98 and Rf of compound is 0.65.
Hi,
indeed there are cases where one organic compound tends to associate with an admixture making the purification harder. Things You can try:
a) if accessible the most simple way should be reversed phase column chromatography
b) find a system where filtration through silica would work -> where the rf of one compound would be effectively 0.00 and the other would move in this case the seperation should occur.
In retrospect You could have overloaded the silica and used too strong of a system - it should be noted that removing gases and starting with an eluent in which the highest RF does not exceed 0.2 should be preferred.
Cheers,
Kaspars
Normally separation of two substances with such a big Rf difference by chromatography should not be a problem.
Make sure you do not overload the column (use more silica), you may also try to reduce polarity of the mobile phase slightly. Pay attention how you load your sample on the column - use non-polar solvent for this, and smallest possible volume (if your mixture is liquid, do not use any solvent at all); try to make a "band" on the column as thin as possible. Sometimes changing the mobile phase helps too.
Good luck !
I'm assuming you are using the same elution for the flash as is being used for the TLC. The Rf shown doesn't suggest the chromatography will be good. The non-polar compound has an Rf of (essentially) 1, and the product has an Rf of 0.65. Retention on a column is, in column volumes (CV)= 1/Rf. The column volume approximates the void volume on an HPLC. So the impurity comes out at 1 CV and the impurity comes out at ~1.5 CV. The column volume is merely the space taken by the mobile phase in the column and nothing can elute faster than 1 CV.
You would prefer the compound to come out at 2-5 CV, or an Rf of 0.2 through 0.5 and that will be difficult to achieve as you are running 4% EtOAc in pet ether now . I suggest trying a less polar mobile phase such pet ether/THF or pet ether/ether. One can also try pet ether/toluene. Pet ether/dichloromethane (DCM) could work but DCM is nearly as polar as EtOAc. Likewise, pet ether/acetone is similar in polarity to pet ether/EtOAc and so probably won't work well.
For TLC, make sure the chamber has time to saturate with vapor, and make sure vapor can't escape. A watch glass covering a beaker doesn't give the best results because the beaker spout allows vapor to escape. Use a proper TLC chamber or jar that can be completely closed.
Use solid load for best results (dry the sample onto silica)- see this app note for a comparison of loading techniques: http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Overview%20of%20Silica%20Column%20Sample%20Loading%20Techniques%20App%20Note.pdf
If you are running a flash column on an instrument with such a solvent system, make sure the equilibration is aet to 5-7 column volumes. The EtOAc adsorbs on the silica, and compounds often don't elute until this adsorption is complete. If packing columns, pack in at least 1-2% ethyl acetate of you are running a step gradient for the same reason. People often dry-pack columns and this may be one case where that can adversely affect the chromatography.
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