I recently tried to isolate a compound which apparently looked like a single spot in the TLC of the reaction mixture but actually was 2 spots when I was doing TLC when running Column Cromatography. I later repeated the experiment and found out that when the TLC is run few times repeatedly in the same mobile phase it separates out.The number of repeatations can be lowered or even cut down to one if a longer TLC plate is cut.I tried various solvent combinations to get the most suitable mobile phase to increase the separation between the spots but in vain.Is there a logical way to determine the most appropriate combination of solvents to narrow in on a mobile phase?I have used the method of trial and error and am looking for a logical approach which will atleast reduce, if not eliminate the method of trial and error.
Advice on combination of solvents may be sufficient.I can find out the proportion of each by trial and error.
1. Increase or decrease the elution strength by changing the quantitative composition of your eluent until your spot(s) has an Rf value of about 0,5 after one separation.
2. Change the qualitative composition of your eluent without changing its elutions strength -> chromatographic selectivity changes.
See recommended publication for details.
Article The "PRISMA" Mobile Phase Optimization Model in Thin-Layer C...
1. Increase or decrease the elution strength by changing the quantitative composition of your eluent until your spot(s) has an Rf value of about 0,5 after one separation.
2. Change the qualitative composition of your eluent without changing its elutions strength -> chromatographic selectivity changes.
See recommended publication for details.
Article The "PRISMA" Mobile Phase Optimization Model in Thin-Layer C...
Being that the solvent system you are using for column chromatography resolves the spots, this suggests the solvent system is suitable for your compounds on TLC as well. Are you running a gradient on your column, or isocratic?
I'm assuming that the column chromatography and the TLC are both silica, since it is possible to, for example, run C18 column chromatography while using silica TLC. The two media would have different "selectivity", or retention for the compounds.
Occasionally, the moisture content and pH of the TLC plate silica and the column silica are different leading to differences in resolution and selectivity. I once saw a case where the elution order was opposite on TLC compared to the column chromatography.
change the proportions of the mobile phase the ratio of polar to non-polar solvent from 10% to 90%.
Try to decrease the flow rate if not working you have to change your system
Do you know the nature of the two spots, acidic,neutral or alkaline. Try to match the solvent pH with your target spot to get maximum separation.
you can modify you organic solvents system with pure formic acid or ammonia to enable you target analytes to exist in their free base or free acid form, not the ionic form.
It depends on the nature of your compounds
try to use different systems with TLC first until you can see good speration between them after that apply that system with HPLC, put in your consideration the flow rate as well
Try to change the proportion of your solvent proportion based on polarity.
While I have used TLC in the past, I prefer HPLC for this reason. Use the resolution equation in HPLC. But the compounds, because of a similar solvency and thus polarity (they are probably structurally similar like Vitamin A Alcohol and Vitamin A Acetate) can only be separated by HPLC (an enhanced form of Liquid Chromatography). HPLC uses a smaller particle size and can perform gradient elution.
One determine the most suitable mobile phase to separate 2 very close spots in TLC either by decreasing the polarity of by flash chromatography.
I am with @Alaa and @Bruce. HPLC is superior for so many reasons. The fact that after extensive chromatography, one begins to resolve another spot on TLC makes me suspicious that all the manipulation and exposure to oxygen and the acidic surface of silica gel may in fact be degrading your analyte into a closely-eluting artifact that you are detecting.
Try to change the proportion of your solvent proportion based on polarity.
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