Thin-layer chromatography (TLC) is a chromatography technique that separates compounds based on their polarity. TLC typically uses polar silica gel, a form of silicon dioxide, as the stationary phase. The silica gel contains hydroxyl (OH) groups on its surface, which form hydrogen bonds with polar compounds, influencing their adhesion to the stationary phase.

To begin the analysis, a mixture of compounds is spotted on the starting line on the TLC plate using a thin capillary. The bottom of the plate is immersed in the mobile phase, which is usually an organic solvent that is less polar than the stationary phase. The solvent travels up the plate by capillary action, carrying the solute spot. As the solvent travels up the plate, the components of the mixture partition between the mobile phase or the stationary phase. The polar components interact more strongly with the polar stationary phase, which results in them traveling slowly and only moving a short distance on the TLC plate. The less polar components of the sample are more soluble in the mobile phase and tend to interact more with the mobile phase, allowing them to travel farther up the TLC plate.

Understanding the polarity of the components and the mobile phase is essential for predicting separation outcomes. The mobile phase (solvent) should be polar enough to move the most polar solute effectively while ensuring good compound separation. The retardation factor, R_f, is defined as the ratio of the distance traveled by a component to the distance traveled by the mobile phase. Understanding this concept is crucial in TLC analysis. The R_f of a compound is dependent on the mobile phase used. The R_f is large for nonpolar compounds with a nonpolar mobile phase. Low R_f values are seen for polar components with a nonpolar mobile phase.