Thin Layer Chromatography Principle

Chromatography is a method of separation in which the components to be separated are distributed between the two different phases, one is the stationary phase and the other one is a mobile phase that moves in a definite direction. There are different types of chromatographic techniques such as column chromatography, paper chromatography, partition chromatography, thin-layer chromatography, gas-liquid chromatography, and ion-exchange chromatography.

In this article, we will study thin layer chromatography in detail.

Thin Layer Chromatography (TLC)

Thin-layer chromatography is the simple, fast, easiest, and least expensive of all the chromatographic techniques which are used in quantitative and qualitative analysis of separation of organic compounds, and to test the purity of compounds.

TLC is a form of liquid chromatography that consists of two phases given as follows-

1. A mobile phase (solvent)

2. A stationary phase (glass plate coated with silica gel) 

The analysis is done under atmospheric pressure and room temperature.

Thin Layer Chromatography Principle

Thin-layer chromatography is a method of separation, or identification of a mixture of components by using finely divided adsorbent solid/ liquid over a glass plate, and liquid as a mobile phase. 

The distinction depends on the relative affinity between the stationary and mobile phases of the compounds. The compounds move over the surface of the stationary phase under the influence of the mobile phase (driven by capillary action). The compounds with greater affinity for the stationary phase travel slowly during this movement while the others travel faster. The isolation of components in the mixture is thus accomplished. The individual components are visualized as spots at a different stage of travel on the plate once separation happens. 

Thin Layer Chromatography Process

The process includes the following steps-

• Adsorption of substances on the stationary phase.

• Separation of adsorbed substances by the mobile phase.

• Separated substances are recovered by the mobile phase through elution.

• Qualitative and quantitative analysis of eluted substances.

In a solid phase, the adsorbent is coated onto a solid support such as a sheet of glass, aluminium, or plastic as a thin layer of about 0.25mm thick. 

The mixture that needs to be separated is dissolved in a solvent to form a solution. Thus, this solution is applied at the base of the thin-layer plate. The solution is allowed to move in an upward direction under the influence of capillary action. The solid will absorb some fraction of each component of the mixture and the remaining will be left in the solution. Anyone molecule will remain attached to the solid surface while the other components continue moving up the plate with solvent. 

Separation of mixtures on a flat surface by the movement of solvent due to differences in solubility, migrating rates, size, and charge. Elution is stopped when the solvent reaches the opposite side of the silica-coated glass.

Advantages and Disadvantages of Chromatographic Techniques

Advantages of Thin-Layer Chromatography

1. A simple method of component separation. 

2. Fewer types of equipment are used in this technique. 

3. As the components elute rapidly, the separation is achieved in a very short time. 

4. It is possible to visualize all elements of UV light. 

5. By this process, the non-volatile compounds can be isolated. 

6. It is also possible to separate the microlitre volume of the sample through TLC. 

7. Easy isolation and recovery of the components of complex mixtures.

Disadvantages of Thin-Layer Chromatography

1. It is difficult to reproduce the findings obtained from the experiment. 

2. Applicable for components of soluble mixtures only. 

3. Qualitative analysis, not the analysis in quantitative terms. 

4. It is not an automatic mechanism. 

5. A thin layer of chromatography operates in an open system, and humidity and temperature can influence the outcomes. 

6. As the plate length is limited, the separation process takes place up to a certain length.

Applications of Thin Layer Chromatography

1. Pharmaceuticals and Drugs

Identification, purity testing, and determination of active substances and preservatives in drugs and in drug preparations.

2. Clinical, Forensic and Biochemistry

Determination of active substances and their metabolites in biological matrices, diagnosis of metabolic disorders like phenylketonuria, cystinuria, and maple syrup disease in babies.

3. Cosmetology

Dye raw materials and end products, preservatives, surfactants, fatty acids, constituents of perfumes.

4. Food Analysis

Determination of pesticides and insecticides in drinking water, residues in vegetables, salads, and meat, and vitamins in soft drinks.

5. Environmental Analysis

Groundwater analysis, determination of pollutants from soils and surface water.

6. Analysis of inorganic Chemistry 

Determination of metals.

Did You Know?

The retardation factor (R) in chromatography is the fraction of an analyte in a chromatographic system's mobile phase. The retardation factor is the ratio of the distance travelled by the centre of a spot to the distance travelled by the solvent front.

For the study and comparison of various substances, the retardation factor, Rf, is widely used in paper chromatography and thin-layer chromatography. It can be defined by the following ratio mathematically:

Rf= migration distance of substance/migration distance of solvent front.