Thin Layer Chromatography

What Is Thin Layer Chromatography?

Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The experiment is conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent material. The material usually used is aluminium oxide, cellulose, or silica gel.

On completion of the separation, each component appears as spots separated vertically. Each spot has a retention factor (Rf) expressed as:

Rf = dist. travelled by sample / dist. travelled by solvent

The factors affecting retardation factor are the solvent system, amount of material spotted, absorbent and temperature. TLC is one of the fastest, least expensive, simplest and easiest chromatography technique.

Thin Layer Chromatography Principle

Like other chromatographic techniques, thin layer chromatography (TLC) depends on the separation principle. The separation relies on the relative affinity of compounds towards both the phases. The compounds in the mobile phase move over the surface of the stationary phase. The movement occurs in such a way that the compounds which have a higher affinity to the stationary phase move slowly while the other compounds travel fast. Therefore, the separation of the mixture is attained. On completion of the separation process, the individual components from the mixture appear as spots at respective levels on the plates. Their character and nature are identified by suitable detection techniques.

Thin Layer Chromatography Diagram

Diagram of Thin Layer Chromatography

Diagram of Thin Layer Chromatography

Thin Layer Chromatography Procedure

Before starting with the Thin Layer Chromatography Experiment let us understand the different components required to conduct the procedure along with the phases involved.

  1. Thin Layer Chromatography Plates – ready-made plates are used which are chemically inert and stable. The stationary phase is applied on its surface in the form of a thin layer. The stationary phase on the plate has a fine particle size and also has a uniform thickness.
  2. Thin Layer Chromatography Chamber – Chamber is used to develop plates. It is responsible to keep a steady environment inside which will help in developing spots. Also, it prevents the solvent evaporation and keeps the entire process dust-free.
  3. Thin Layer Chromatography Mobile phase – Mobile phase is the one that moves and consists of a solvent mixture or a solvent. This phase should be particulate-free. The higher the quality of purity the development of spots is better.
  4. Thin Layer Chromatography Filter Paper – It has to be placed inside the chamber. It is moistened in the mobile phase.

Thin Layer Chromatography Experiment

The stationary phase that is applied to the plate is made to dry and stabilize.

  • To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil.
  • Apply sample solutions to the marked spots.
  • Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened filter paper in the mobile phase.
  • Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample faces the mobile phase.
  • Immerse the plate for development. Remember to keep the sample spots well above the level of the mobile phase. Do not immerse it in the solvent.
  • Wait till the development of spots. Once the spots are developed, take out the plates and dry them. The sample spots can be observed under a UV light chamber.

Thin Layer Chromatography Applications

  • The qualitative testing of Various medicines such as sedatives, local anaesthetics, anticonvulsant tranquilisers, analgesics, antihistamines, steroids, hypnotics is done by TLC.
  • TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical metabolites from its blood plasma, urine, body fluids, serum, etc.
  • Thin layer chromatography can be used to identify natural products like essential oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc
  • It is widely used in separating multicomponent pharmaceutical formulations.
  • It is used to purify of any sample and direct comparison is done between the sample and the authentic sample
  • It is used in the food industry, to separate and identify colours, sweetening agent, and preservatives
  • It is used in the cosmetic industry.
  • It is used to study if a reaction is complete.

Disadvantages Of Thin Layer Chromatography:

  1. Thin Layer Chromatography plates do not have longer stationary phase.
  2. When compared to other chromatographic techniques the length of separation is limited.
  3. The results generated from TLC are difficult to reproduce.
  4. Since TLC operates as an open system, some factors such as humidity and temperature can be consequences to the final outcome of the chromatogram.
  5. The detection limit is high and therefore if you want a lower detection limit, you cannot use TLC.
  6. It is only a qualitative analysis technique and not quantitative.

Frequently Asked Questions on Thin Layer Chromatography

What is the principle of TLC?

TCL is based on the principle of separation through adsorption type. The separation relies on the relative empathy of compounds towards the mobile phase and stationary phase.

Who discovered thin layer chromatography?

It was developed by Izmailov in the year 1938 considering Mikhail Tswett’s description on column chromatography.

What does TLC Rf value mean?

It is the ratio of distance travelled by a substance to distance travelled by a solvent front. Higher the Rf value lesser the polarity of the substance. Lower the Rf value higher is the polarity of the substance.

What is the major application of thin layer chromatography?

Separating multi-component pharmaceutical formulations is one of the major uses of TLC.

What is the major difference between Thin layer chromatography and paper chromatography?

The stationary phase used in paper chromatography Cellulose filter paper containing water in its pore whereas in TLC the stationary phase used is Glass plate coated with silica gel.

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