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JoVE Lab Manual
Lab: Chemistry

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Thin-Layer Chromatography


Chromatography is a technique used in organic chemistry to separate compounds in a mixture based on their differences in solubility between two different phases. The concept is similar to liquid-liquid extraction, except in chromatography, the two phases consist of the stationary phase and the mobile phase. The stationary phase is a solid — typically a microscale hydrogel bead — while the mobile phase is a carrier solvent.

In traditional chromatography, the stationary phase is packed into a vertical column, and the solution mixture introduced at the top of the column. As the mixture flows through the stationary phase, the compounds partition between the stationary phase and the mobile phase based on their structures and polarities, forming discrete bands. Solutes that interact weakly with the stationary phase move more quickly through the column and exit — or elute — first. Solutes that interact more strongly with the stationary phase move slowly through the column and elute later. The bands can be collected individually in order to isolate and purify compounds in a mixture.

There are several types of chromatography, each harnessing a different chemical property to achieve separation. For example, in ion-exchange chromatography, the stationary phase beads can be positively or negatively charged, attracting molecules with opposite charge only. In size exclusion chromatography, the porous stationary phase is composed of beads such as agarose or dextran polymers. Smaller molecules can enter the pores more readily, while the larger molecules flow past the pores and elute faster.

Thin-Layer Chromatography

Thin-layer chromatography (TLC) is a type of chromatography technique that separates compounds based on their polarity. Like traditional chromatography, there are three components of a TLC system: the stationary phase, the mobile phase, and the solute. However, unlike traditional chromatography, the stationary phase is arranged in a thin layer on a plate rather than packed into a column. TLC most often uses polar silica gel, a form of silicon dioxide, as the stationary phase. The stationary phase forms hydrogen bonds due to the OH groups on its surface.

First, a starting line is drawn on the bottom of the TLC plate using a pencil. The compounds or mixture being analyzed are spotted on the starting line using a thin capillary. Then, 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, passing the solute spots and carrying some of each component with it.

As the solvent travels up the plate, the components partition to either the mobile phase or the stationary phase. If the component is polar, it interacts more with the polar stationary phase. It travels slowly and only moves a short distance on the TLC plate. If the component of the sample is less polar — and more soluble in the mobile phase than it is in the stationary phase — it interacts more with the mobile phase and travels farther on the TLC plate. The extent of the polarity of the component and the mobile phase are essential to understanding and predicting the separation.

TLC plates typically contain a UV-reactive fluorescent dye that will glow under a UV source of 254 nanometers. Therefore, TLC plates can be analyzed by observing them under UV light. Compounds within the TLC plate, such as the solutes of interest, will show up as dark spots compared to a green background. By circling the spots with a graphite pencil, the distance the compounds traveled relative to the solvent front can be measured. The spot of the organic compound, if not fluorescent itself, masks the fluorescence of the plate and shows up as a dark spot. Some organic compounds are UV-active and emit light when exposed to UV light. These are typically conjugated compounds, meaning those with alternating double and single bonds, and can be identified by the wavelength emitted.

Retardation Factor

By analyzing the retardation factor (Rf) of a component with a specific solvent, an unknown solute can be determined using TLC. The retardation factor is the ratio of the distance traveled by a component to the distance traveled by the mobile phase.

The distance traveled by the solute is measured from the starting line to the center point of the spot, and the distance traveled by the mobile phase is measured from the same starting line to the solvent front. The retardation factor of a compound is dependent on the mobile phase used. The retardation factor is large for compounds that are highly non-polar with a non-polar mobile phase. Low retardation factor values are seen for polar components with a non-polar mobile phase.

In the case of a highly non-polar mobile phase, some polar components may not move at all. This results in an extremely low retardation factor and an insufficient separation. A highly polar mobile phase causes the compound to move with the solvent and yields an extremely high retardation factor. This results in very little separation between the components.

For separation to be effective, the retardation factors of the components should be about 0.3 - 0.7 apart. To find the efficient mobile phase, trial and error is employed. Often, a mixture of two solvents proves most effective.


  1. Harris, D.C. (2015). Quantitative Chemical Analysis. New York, NY: W.H. Freeman and Company.

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