Thin-Layer Chromatography

Thin-layer chromatography, or TLC, is a technique used to separate organic compounds based on differences in their polarities. There are three components of a TLC system: the stationary phase, the solute, and the development solvent.

The stationary phase is the surface on which the compounds will be separated, which is usually composed of extremely polar silica gel. The compounds or mixtures being analyzed are the solutes. Dilute solutions of the solutes are spotted on one edge of the plate using thin capillaries.

The bottom of the plate is then immersed in the development solvent, which is usually an organic solvent that is less polar than the stationary phase. The solvent travels up the plate by capillary action. As the solvent passes through the solute spots, it carries some of each solute with it. This solute-solvent mixture is the mobile phase.

The top of the mobile phase is called the solvent front. As the mobile phase travels up the plate, solute and solvent molecules temporarily stick to the stationary phase based on their polarity.

More polar solutes are strongly attracted to the stationary phase, so they stick more often and stay stuck for a while. Less polar solutes stick less often and don't stay stuck for long. This means that less polar solutes spend more time in the mobile phase than more polar solutes do. In other words, less polar solutes travel faster than more polar solutes.

To achieve good separation between compounds, the solvent should be just polar enough to move the most polar solute. Here are some common solvents in order of increasing polarity. Sometimes, two solvents are combined to achieve the desired polarity.

Because most organic compounds are white or colorless, TLC plates usually have a fluorescent compound in them. Under UV light, the organic compounds will appear as dark spots on the glowing plate.

Once the compounds are visualized, you can measure how far they traveled and calculate the retardation factor, or R_f, for each one. This value is the distance traveled by the solute divided by the total distance traveled by the mobile phase.

If the solvent is chosen properly, each solute will have a distinctly different R_f. This allows us to identify the component compounds in mixtures by matching the R_f values to known compounds.

In this lab, you will use TLC to determine the R_f of several known compounds, and then you will use those R_f values to determine the identity of an unknown compound.