Tech Overview – Thin Layer Chromatography

By Tristan Willis

June 5, 2018 -- This time in The Science Advisory Board’s chromatography series, we will cover thin layer chromatography (TLC). This method was introduced in the 1950’s as a fast, easy, and inexpensive method for qualitative analysis. This technique can be employed on the analytical scale or preparative. Like all other forms of chromatography, TLC follows a similar principle in that it contains a mobile and stationary phase.

Advion Plate Express – TLC/CMS

The mobile phase will be drawn through the stationary phase by means of capillary action. TLC uses a glass, plastic or metal surface, which is coated with a thin layer of particles of silica gel or alumina. The thickness of the layers and the particle sizes determines if it is either traditional TLC or high performance TLC (HPTLC). The stationary phase in HPTLC plates uses finer particle sizes than traditional TLC plates. This allows for higher resolution in less time, but it will significantly reduce sample capacity. It is important to note that TLC plates can separate a sample by normal or reverse-phase partitioning.

A critical step in TLC is the application of the sample to the plate. In order to “spot” the plate properly, samples are applied a few centimeters from the edge of the plate. This is generally done using a capillary tube containing the sample. Researchers may also opt to use an automated sample applicators to apply a precise volume in a specific position on to the plate. In order to develop the plate, one must immerse the edge in the developing solvent and the solvent will carry the spotted sample through the stationary phase. As the sample travels through the stationary phase the mixture will separate into individual analytes based on its physical properties.

CAMAG TLC Visualizer 2

Once the solvent has travelled two-thirds of the plate, it can be set aside to dry. There are multiple ways to visualize the analytes. If the mixture is organic, the plate is sprayed with a solution of iodine or sulfuric acid. This will react with the organic compounds to yield dark colored spots. Another method of detection includes incorporation fluorescent material into the stationary phase. The dried plate can be viewed under a UV light with will expose the location of the sample components.

The Retention Factor is an important component to TLC. After separation, the individual compounds can be visualized as spots on the plate. Each component has a retention factor (Rf) equivalent to the distance migrated over the total distance covered by the solvent. This value can be a tool to identify compounds or compare different compounds under identical conditions. It is important to note that Rf can be affected in a number of ways such as temperature, moisture on TLC plate, and saturation. Scientists interested in determining each compounds position relative to that of another substance, x, may employ the relative retention value, or Rx. This is equivalent to the distance of compound from the origin over the distance of compound x from the origin.

Thin layer chromatography can be an excellent technique to meet your chromatography needs. Sample purity can be analyzed and compared with a standard to quickly detect impurities and produce useful chromatograms for research.

What do you think the pros and cons are of TLC? Join the discussion below!

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