I did column chromatography to separate the compounds present in plant extract using sephadex LH 20 as stationary phase and 100 % as the solvent system. Now I need to find out whether the elute has single compound or multiple compound. So, can anyone tell me how I can find it? I tried TLC but am failing to standardize the solvent system.
Kindly help me in this regard.
have u try reverse phase TLC with different water and methanol combination system
Use TLC as said above and multiple bond gives yellow color spot with Iodine. After running TLC yellow spots will evolved during Iodine chamber.
Rajesh,
TLC is a good option if you don´t have HPLC. First try with pure solvents, run in a saturated chamber (put a filter paper inside the chamber), dry and visualize the spots under UV lamp (254 or 366 nm) or with reagents like iodine, sulfuric acid, sulfuric acid and vanillin, ninhydrin, 2,4-dinitrophenylhydrazine, etc. The best is to use a non polar solvent like hexane, toluene, etc in greater proportion with a better eluant (more polar solvent like acetonitrile, alcohols, etc) in a minor proportion, depending on the compounds polarity/solubility. When you can see that the initial spot moves forward in the plate, change the mixture composition until the Rf values are in between 0,2 and 0,8. If you are not sure if a spot in TLC is a single compound, you can change the mixture until compounds separate, or you can run a square plate once in a solvent mixture and then again in another solvent mixture, turning the plate 90 degrees for the second time, so you can get a 2D TLC.
Best regards
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