Biochemical Techniques and Instrumentation

2.6.1 Principle

The different compounds in the sample mixture migrate at different rates due to the differences in their attraction to the stationary phase, and differences in solubility in the mobile phase. The separation of compounds is therefore based on the competition of the compound and mobile phase for the binding sites on the stationary phase.

Silica gel is used as the stationary phase is polar. So, the more polar compound has a stronger interaction with the silica gel and it dispels the mobile phase from the binding sites. Consequently, the less polar compound moves higher up the plate. If the mobile phase is changed to a more polar solvent or solvent mixture, it dispels the compounds from the binding sites and all compounds on the plate will move higher up the plate. Strong solvents move the compounds up the plate, while weak solvents do not move them.

The order of strength/weakness depends on the stationary phase. In silica gel coated TLC plates, the solvent strength increases in the following order: perfluoroalkane, hexane, pentane, carbon tetrachloride, benzene/toluene, dichloromethane, diethyl ether, ethylacetate, acetonitrile, acetone, 2-propanol/n-butanol, water, methanol, triethylamine, acetic acid, formic acid. In C-18 coated TLC plates, the order is reverse. An eluotropic series is mainly used as a guide in the selection of the mobile phase.