I tried 2 separate 2 spots which are not so close in a 4cm long TLC plate but everytime it used to come together.I packed a long column in 230-400 Silica, took minimum amount of slurry and did gradient elution but 2 spots always used to come together.What can be the possible reason?They started coming relatively quickly.
if your two spots elute together on every chromatography TLC or Open column, then it is concluded that that your mobile phase or combination of solvent system is not accurate. the polarity of solvent is high. u start with minimum possible amount of polar solvent and increase the polarity of solvent gradually by observing your result.
you can visit my research paper. my whole work is based on chromatography. i think my paper would help you to sort out your problem.
I think you should try the different mobile phase combinations with increasing the order of their polarity or vice versa.
There could be several things going on.
Let's start with TLC.
Having spots "which are not so close" isn't quite enough. Although there is a great difference in retention factor (Rf), there may not be a large difference in column volumes (CV). The Rf =(distance spot moved)/(distance solvent front moved). From this, we can predict the elution on a matching silica column in CV with the equation CV=1/Rf. So if a compound runs at the solvent front on TLC, it will elute at 1 CV from the column (the void volume). So if you have well resolved spots @ Rf= 0.8 and Rf= 0.6, they will elute at 1.25 CV and 1.6 CV respectively. You really want an Rf of the desired compound to be between 0.5 and 0.2 (2 to 5 CV) for fast and good chromatography. This will appear that the spots aren't moving fast enough on TLC, but will make for good chromatography. Also note that you can estimate the difference in CV the spots will elute. You may be overloading the column if the difference in CV is small.
There needs to be proper TLC technique. Make sure you run the TLC in a sealed container (jar with a screw lid or a TLC chamber with a lid) to prevent the solvent vapors from escaping. Also let the solvent/vapor equilibrate inside the chamber before running the TLC. If the vapor can escape (for example, using a beaker with a watch glass cover, the fumes escape via the spout), you never achieve equilibration. without a stable, saturated, chamber, the solvent with the lower boiling-point evaporates form the TLC plate during the run, affecting the results.
The column has technique as well. It needs to be well packed.
How are you loading the sample? If you are loading dissolving the sample in a "strong" solvent, it can "carry" the sample down the column. Dry-loading is usually better. This app note compares different loading techniques:
http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Overview%20of%20Silica%20Column%20Sample%20Loading%20Techniques%20App%20Note.pdf
if your two spots elute together on every chromatography TLC or Open column, then it is concluded that that your mobile phase or combination of solvent system is not accurate. the polarity of solvent is high. u start with minimum possible amount of polar solvent and increase the polarity of solvent gradually by observing your result.
you can visit my research paper. my whole work is based on chromatography. i think my paper would help you to sort out your problem.
The spots separate well in TLC but it comes together while doing Column.I loaded the sample by performing dry loading.I have also tried switching from Hexane/Ethyl Acetate to Hexane/DCM but it doesn't help. For Aluminium Oxide, it is recommended to check TLC in Alumina plates instead of Silica TLC as the polarity will vary and hence the solvent system required for elution of the same spot.We don't have Alumina plates
you should try changing stationary phase as earlier mentioned by Pierre-Antoine Nocquet, try C18.
Can you show a picture of your TLC?
If it resolves on TLC, it should also resolve on your column. What is the Rf of your spots, and what is the solvent system?
Solvent system is DCM/Hexane..I think it is due to the nature of a particular compound.I am seeing this pattern in 2 reactions where one component is same and the other has been changed.The component that is same comes at a much lower polarity than determined from TLC even after packing a long column with 230-400 Silica and using dry packing with minimum amount of silica.
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