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Thin Layer Chromatography (TLC) is a simple and inexpensive technique used to judge the purity of a synthesized compound or to indicate the extent of progress of a chemical reaction. It is a solid-liquid form of chromatography where the stationary phase is normally a polar absorbent and the mobile phase is a single solvent or combination of solvents.

Stationary Phase

A wide range of commercially available adsorbents, such as silica gel, silicic acid, alumina, aluminum oxide, kieselguhr (diatomaceous earth), and micro crystalline cellulose can be used for TLC . Silica gel or silicic acid are most commonly used adsorbents.

Mobile Phase

The mobile phase in TLC is a solvent system composed of one or more miscible solvents (Figure 1). The solvent system competes with dissolved analyte for active sites on the sorbent and is carefully selected to achieve separation of individual components. The solvent systems are selected by considering equilibrium between solvent, solutes, and sorbent layer and other factors, such as cost, availability, quality, toxicity, volatility, and miscibility.

Figure 1. Solvent preparation for TLC analysis
Figure 1. Solvent preparation for TLC analysis

Sample application and Component Detection

The sample is applied to the plate by micropipette or syringe (Figure 2).

Figure 2. Microsyringes for sample injection in chromatography
Figure 2. Microsyringes for sample injection in chromatography

The sample spot is generally placed 2.0 to 2.5cm from edge of the plate (Figure 3). In preparative TLC, the sample is applied as band rather than a single spot.

Figure 3: TLC plate representing sample applications
Figure 3: TLC plate representing sample applications

An ideal visualization or location procedure for thin-layer chromatography should be able to reveal the microgram quantities of the separated substances. Colored substances are easy to spot as they give contrast between the visualized area and the background. For colorless compounds, techniques like iodination, ninhydrin, and fluorescence can be used for the components detection.

Figure 4: Flow diagram representing steps involved in the process of TLC
Figure 4: Flow diagram representing steps involved in the process of TLC

Applications of Thin Layer Chromatography

TLC is used for screening of organic reactions, analysis of phospholipids, and sphingolipids of nervous system, quantification of cerebral lipids, plasma neutral lipids and free fatty acids, determination of plant components, analyzing ceramides and fatty acids, for quantitative inorganic analysis, and in radiochemical studies, fingerprinting of licorice (Glycyrrhiza spp.), to determine the radiochemical purity of radiopharmaceuticals.