TLC thin layer chromatography: Principle, Procedure

TLC chromatography or Thin layer chromatography

is a type of planar chromatography. TLC is routinely used by researcher in the field of phyto-chemicals, biochemistry etc. to identify the components in a compound mixture like alkaloids, phospholipids, amino acids etc..

It is a semi quantitative method of analysis and its sophisticated version or highly precise quantitative version is High performance thin layer chromatography (HPTLC).

Thin layer chromatography principle:

Similar to other chromatographic methods TLC is also based on the principle of separation. The separation depends on the relative affinity of compounds towards stationary and mobile phase. The compounds under the influence of mobile phase (driven by capillary action) travel over the surface of stationary phase. During this movement the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus separation of components in the mixture is achieved.

Once separation occurs individual components are visualized as spots at respective level of travel on the plate. Their nature or character are identified by means of suitable detection techniques.

TLC System consists of

a) TLC plates preferably ready made with stationary phase: These are stable and chemically inert plates on to whose surface a thin layer of stationary phase is applied. The stationary phase on the plates is of uniform thickness and consists of fine particle size.

b) TLC chamber: This is used for the development of TLC plate. The chamber maintains uniform environment inside for proper development of spots. It also prevents the evaporation of solvents and keep the process dust free.

c) Mobile phase: This comprises of a solvent or solvent mixture recommended for the purpose. The mobile phase used should be particulate free and of highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, stationary phase.

d) A filter paper moistened in the mobile phase, to be placed inside the chamber. This helps uniform rise in mobile phase over the length stationary phase.

The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize. But now a days ready made plates are preferred.

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

Then samples solutions are applied on the spots marked on the line at equal distances.

The mobile phase is poured into the TLC chamber to a level few centimeters above the chamber bottom. A filter paper moistened in mobile phase is placed on the inner wall of the chamber to maintain equal humidity in the entire chamber and there by avoid edge effect.

Then the plate prepared with sample spotting is placed in TLC chamber such that the side of the plate with sample line is towards the mobile phase. Then the chamber is closed with a lid.

The plate is immersed such that sample spots are well above the level of mobile phase but not immersed in the solvent as shown in the picture for development.

Allow sufficient time for development of spots. Then the plates are removed and allowed to dry. The sample spots are visualized in suitable UV light chamber or any other methods as recommended for the said sample.

The Thin layer chromatography advantages include:

It is simple process with short development time.

It helps in visualization of separated compound spots easily.

The method helps to identify the individual compounds.

It helps in isolation of most of the compounds.

The separation process is faster and the selectivity for compounds is higher (even small differences in chemistry is enough for clear separation.

The purity standards of the given sample can be assessed easily.

It is a cheaper chromatographic technique.

1. To check purity of given samples.

2. Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics etc.

3. To evaluate reaction process by assessment of intermediates, reaction course etc.

4. To purify samples i.e for purification process.

5. To keep a check on the performance of other separation processes.

Being a semi quantitative technique, tlc chromatography is used for rapid qualitative measurements than for quantitative purposes. But due its rapidity of results, easy handling and inexpensive procedure, it finds its application as one of the most widely used chromatography techniques