Question

What if any would you have to change in terms of the mobile phase if you wanted toactually perform the column chromatography in order to separate the pigments of spinach compare to the one used for the TLC Experiment? Explain

Answer 1

Say you have run a TLC in 10% ethylacetate in hexane mobile phase. The Rf value are 0.1, 0.3 and 0.6. Now when we move to column chromatography of the same mixture separation we would first use only Hexane as the mobile phase. Once the first componenet comes off, we would increase the polarity slightly say 2% ethylacetate in hexane to elute the second compound off the column. Finally we can use 5-6% ethylacetate in hexane to slute the last compound of the mixture. So as we have shown, in TLC we have used a mixture of solvent to determine the polarity values in terms of Rf of the compounds in the mixture. In column chromatography we always start from less polar solvent and then go on increasing the polarity of the mobile phase to separate the compounds.