I have collected one active fraction from Si-gel CC using Hexane:EtOAc gradient. The active fraction eluted with 3:2 ratio. Now its time to purify the active compounds from this fraction. TLC analysis shows the presence of many spots, that's why now which method I need to follow to purify the compounds?
Expert suggestion would highly be acknowledged.
Run more column chromatography to separate the spots. If you can see the spots on the TLC, it is likely to be separable by varying the solvent ratio and column size.
If the compound is UV active and you have a prep HPLC that will help a lot. Prep TLC is also possible.
When I run TLC the spot is easily visible, no need to put under UV lamp. But the spot are very closer and I have decided to do Si-Gel CC again. In this case which eluent can I use?
How do you know the visible spot is the bio-active compound? The active compound might be co-eluting with the spots that you see and may not be visible.
As for solvents to try to resolve the spots better, try a different "selectivity" group. Run TLC with hexane/dichloromethane, hexane/toluene, and hexane/ethanol. Each of these solvents has a different selectivity. Alternatively, try a different stationary phase. Look at alumina, or CHP-20 (a reverse phase resin). By changing the stationary phase, you are also changing the selectivity.
It is difficult to say for sure without seeing the actual plate.
But as a rough guide I will start with 7/3 Hex/EA and increase the EA %. You can also try ether in place of EA, and pentane in place of Hex.
I doubt you will be able to separate everything in 1 or 2 runs. For example, if you have 3 spots the aim should be to get at least one or two pure fractions of each spot for further testing. At this point it is really trial and error without more knowledge of the compounds
what is the weight of your fraction ? if you are limited by the weight do not run it in Silica CC. Since the recovery is around 70% of what you load on the column. If the weight is 200 mg or below you should try RP-Semi prep HPLC. after developing a method on analytical level. If sample weigh around 300-500mg you can try MPLC or CC. centrifugal TLC (chromatotron) could be another decent option for isolation of pure midpolar compounds.
Since you are having different spots on the TLC plate, it means that the fraction is not yet pure. Hence, run another column chromatography (maybe with smaller column) and observe any colour separation along the column, which should be collected differently. If there is no colour separation, collect fractions at interval of say, 5-10ml in small beakers. Collect as many fractions as possible. You can as well vary the polarity of the solvent system after about 100-200ml have been collected. Solvent polarity can be varied. Then run TLC on each of the fractions. Combine fractions with same Rf value(s). Run the TLC of the combined fractions by varying the polarity of the eluent.
If there are still more than one spot after TLC, repeat the above procedure until you get a fraction having just one spot on TLC plate, even with different polarity of the eluent and always remember to view the plate under UV lamp or iodine vapour in case of any hidden spot(s).
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