Thin Layer Chromatography

Chromatography is a method to separate mixtures of substances or compounds into their components. It was developed by Izmailov in 1938. It is generally performed with aluminum foil, a sheet of plastic, or glass coated with a thin layer of adsorbent material such as alumina, silica gel, and cellulose. It is an analytical tool widely used because of its simplicity, relatively low cost, high sensitivity, and speed of separation. TLC is mostly used by researchers to identify the components in a compound mixture, like alkaloids, phospholipids, and amino acids. Now we will discuss the thin layer chromatography principle.

Thin Layer Chromatography Principle

Just like other chromatographic methods like Partition chromatography or Paper chromatography, the thin layer chromatography principle is also based on the principle of separation.

• The separation depends on the relative affinity of compounds towards the stationary phase and the mobile phase.

• The compounds of the mobile phase travel over the surface of the stationary phase. During the movement, the compounds with lower affinity to stationary phase travel faster while the others travel slower. Thus, the separation of components in the mixture can be achieved. The stationary phase can be prepared by coating a thin layer of either alumina or silica on metal, plastic, or glass.

• Once the separation process is done, the individual components are visualized as spots at a level of travel on the plate. Their nature or character is identified using suitable detection techniques.

Let’s have a look at the thin layer chromatography diagram.

Thin Layer Chromatography Diagram

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Components Required

TLC Plates: 

The plates used are chemically inert and stable, where a thin layer of stationary phase is applied on its surface layer. The stationary phase on the plates is of fine particle size and is of uniform thickness.

TLC Chamber: 

This chamber is used in the development of the TLC plate. The chamber maintains a normal environment inside so that proper development of spots can take place. This chamber prevents the solvents from further evaporation and keeps the process dust-free.

Mobile Phase: 

This phase consists of a solvent or a solvent mixture. The mobile phase must be particulate-free and should be pure for the proper development of TLC spots. The solvents used should be chemically inert with the sample, a stationary phase.

A Filter Paper: 

This is moistened in the mobile phase so that it can be placed inside the chamber. This helps to develop a uniform rise in the mobile phase.

Thin Layer Chromatography Theory

Working of chromatography is based on the fact that different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned as shown in the Thin layer chromatography diagram. TLC is a solid-liquid technique in which the two phases are a solid phase (stationary) and a liquid phase (moving). Solids used in chromatography are silica gel and alumina. Both of them are polar. Silica is also acidic in nature and Alumina is available in neutral, basic, or acidic forms. TLC is an analytical technique that is simple, fast, sensitive, and inexpensive.

Thin Layer Chromatography Procedure

A thin mark is made at the bottom of the plate with the help of a pencil in order to apply the sample spots.

Now, sample solutions are applied to the spots marked on the line at equal distances.

The mobile phase is poured into the TLC chamber to a few centimeters level in order to maintain equal humidity, after that place a moistened filter paper in the mobile phase.

Now, place the plate in the TLC chamber and close it with a lid. It should be kept in such a way that the sample faces the mobile phase and close the chamber with a lid.

The plate is then immersed, in such a way that sample spots are well above the level of the mobile phase for development.

Wait for some time for the development of spots. After spots, remove the plates and allow them to dry. The sample spots can now be easily observed in a UV light chamber or any other methods for the said sample.

Applications

It is used in the purification process and to check the purity of the given samples.

• It is used in the cosmetic industry.

• It is used in pharmaceuticals for separating multi-component.

• It is used in the food industry, to separate and identify colors, sweetening agents, and food preservatives.

• To monitor column chromatography, few types of equipment are used in this technique. The separation is done in a very short time as the components are removed rapidly.

Disadvantages

• This technique is a qualitative analysis and not quantitative.

• The results generated from TLC are not easily reproducible.

• TLC plates do not have a longer stationary phase.

• The detection limit is high and if we want a lower detection limit, we cannot use TLC.

• As TLC operates as an open system, some factors including humidity and temperature can create a problem in the final outcome of the chromatogram.

Did you know?

What does the R_f value mean?

Answer: It is the ratio of distance traveled by a substance to distance traveled by a solvent front. Higher the R_f value lesser the polarity of the substance and vice versa.

R_f = \[\frac{\text{Distance travelled by Component}}{\text{Distance travelled by Solvent}}\]

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Eg: In the given figure, If the red component traveled 1.8 cm from the baseline while the solvent had traveled 8.0 cm, then the R_f value for the red dye is:

R_f = \[\frac{1.8}{8}\] = 0.225