Thin Layer Chromatography (TLC): Principle, Procedure, and Applications

Thin-layer chromatography (TLC) is a method for chromatographic analysis on a thin layer of absorbent held on a glass plate. It is also known as open column chromatography, drop strip, spread layer, or surface chromatography.

Principle

A thin layer of stationary phase is formed on a flat surface like glass or plastic plate. Due to the thin layer, rapid movement of the mobile phase takes place by capillary action. As the mobile phase move across the layer from one end to the opposite, it transfers any analytes (sample) placed on the layer. Their distribution coefficient, Kd, determines the transfer rate.

Kd = Concentration of solute in stationary phase/concentration of solute in the mobile phase

The movements of analytes stop when the mobile phase reaches the end of the layer, and capillary action flows stop when the plate is removed from the mobile phase. Their retention value expresses the movement of the analytes.

Rf= distance traveled by solute/distance traveled by the solvent

Types of TLC 

  1. Partition TLC: Both the stationary and mobile phases are in the liquid. The stationary phase is liquid held on a suitable solid such as cellulose.
  2. Adsorption TLC: In this phase, the stationary phase is solid, and the mobile phase is liquid. Silica gel and alumina are used in the stationary phase. The mobile phase is an organic solvent.
  3. Reversed-phase partition TLC: Plate is coated by hydrophilic silica and again coated by wax or paraffin, making it hydrophobic.
  4. Ion exchange TLC: The exchange of ions occurs between the ions in a liquid phase and a solid, insoluble substance containing an ionic state (ion exchange resins).
Thin layer chromatography
Thin layer chromatography

Adsorbents used in Thin Layer Chromatography

Silica gel, alumina or aluminum powders, cellulose powder, and kieselguhr (natural adsorbent) are used as adsorbents in TLC. In general, silica gel and alumina are used for adsorption TLC, and silica gel, cellulose, and kieselguhr are used for partition TLC.

  1. Silica gel: It is slightly acidic. Gypsum salt, a binding agent, is added with the silica gel to hold the adsorbent firmly on the plate. Silica gel is the active absorbent used to separate all classes of compounds.
  2. Alumina: They are slightly basic and used in separating bases and steroids.
  3. Kieselguhr (diatomaceous earth): It is a natural adsorbent. It is weakly absorbing in nature as compared with silica gel and alumina. It is an inactive type of adsorbent. It can be added to silica gel to give a less active mixed adsorbent. It has been used for the separation of sugars.
  4. Cellulose is used only as support for the stationary liquid phase in partition TLC and in the same way as in the sheet of paper chromatography. Modified cellulose separates phospholipids, glycolipids, amino acids, and different pigments.

Steps of Thin Layer Chromatography

Preparation of plates

In TLC, the adsorbent supports are usually glass plates of square or rectangular plates, and the size of the plates is generally 20 x 20 cm, and a thickness of 0.4 cm is used. Generally, glass plates are used. Plates can be of synthetic polymers or metallic tails, or plastic plates. It should be cleaned with detergents and ethanol.

Preparation of slurry

The slurry is prepared in water. The required amount of adsorbent powder is weighed out and mixed in water. To prepare a slurry, 0.02M sodium acetate can be used instead of water. It avoids cracking the absorbent layer. A slurry of silica gel can be prepared by mixing silica gel with 0.02 M sodium acetate in a ratio of 1:2. Proper mixing is required during slurry preparation to obtain a homogenous mixture. The slurry should not be too thick or too thin. If it is a too thin layer and if it is too thick, spreading produces lines along with the plate.

Preparation of thin layer on a plate

Thin layers of absorbent slurry can be spread on a glass plate in several ways:

  • Dipping method: In this method, a slurry of adsorbent is prepared then glass plates are dipped in the slurry (Adsorbent). After about 20 sec, the plates are carefully withdrawn and allowed to dry. This method is not much used.
  • Spraying method: In this method, a slurry of adsorbent is prepared and then sprayed onto the glass plates with the help of an ordinary spray.
  • Spreading method: The most commonly used method is obtaining a thin and uniform layer. The slurry is placed in an applicator. This is either moved over the stationary plate or is held stationary, and the plate is pushed or pulled through. In one method, the slurry’s adsorbent reservoir is held stationary, and the plate to be coated is moved underneath. In another method, the reservoir is moved over the plate.

Activation of adsorbent

After making thin layers on the plate, the liquid associated with the layer must be removed as completely as possible.  It can be done by drying the thin layer plate for 30 min in the air and then in an oven at 110°C for another 30 minutes, and this drying makes the adsorbent layer active. Since water or other polar solvents greatly affect the development by adsorption chromatography. They should be removed from the chromatoplates and this is known as activation.

Sample application

The sample is loaded on chromatographic paper using a micropipette or micro-syringe. In small spots above 2 cm from the edge and at least 1.5 cm apart, the sample is then dried with the help of a dryer. The sample can be applied in different amounts ranging from 0.5 µl to 500  µl.  The amount of sample that can be applied depends on the thickness of the plate and the principle of chromatography employed.

Choice of solvent

Mixtures of two or more solvents of different polarities often give better separation than chemically homogeneous solvents. The choice of solvents depends on two factors;

  1. Nature of substance to be separated
  2. Material on which separation is to be carried out

Development of chromatogram

Development of chromatogram is carried out by ascending method in which the solvent is allowed to rise to the height of about 10-15 cm above the origin line. The plates are removed from the tank, and the solvent front is marked with a pencil. The plates are then dried in a hot air oven at 110°C for 10-15 minutes.

Location of compounds in chromatographic plate

The compound can be located by using physical and chemical methods.

Physical methods

  1. By radioactive method: If the substance is radiolabelled, it can be detected by counter devices.
  2. Fluorescence method: Some compounds which aren’t visible under ordinary light, fluorescence when held in ultraviolet (UV) light. Eg: fumaric acid, malic acid. 
  3. Reflected light or transmitted light: If the substances are in the form of colored spots, they are visible under reflected light.

Chemical methods

It employs different chemical reagents when the compounds are invisible under ordinary light, they can be visualized using specific chemical agents. The chemical phenomenon occurs between reagents and separated compounds. The chemicals are sprayed with the help of a customizer in a TLC plate from the left to the right direction. E.g., For the lipid, the reagent will be iodine vapor.

Calculation of Rf value

After the spots are visualized, Rf values are calculated immediately because of the possibility of fading. It can be used for qualitative analysis. The Rf value is always specific for a compound. It is used to compare with standard compounds.

Quantitative analysis

Two methods can carry out a quantitative evaluation of chromatogram

  1. Direct method: Comparison with visible spots. The photodensitometry method and fluorimetry method are used in the direct method.
  2. Indirect method: This method is also known as the elution method. This method scrapped the spot containing the separated compound, and scrapings were transferred into a test tube with a suitable solvent. After a few minutes, the optical density of the colored solution is measured with the help of a colorimeter or spectrophotometer at an appropriate wavelength.

Advantages of Thin Layer Chromatography

  • Use of simple equipment. A simple technique with low cost.
  • Can be used for both organic and inorganic compounds.
  • Separation can be done at rapid speed within 15-20 mins.
  • A wide choice of stationary phases can be used. The method may be employed for adsorption, partition, or ionic exchange chromatography.
  • Sensitivity is 10-100 times more than the paper chromatography, due to which TLC can detect even the trace amount.
  • TLC gives a sharper distinct spot. The diffusion of spots in TLC is low.
  • It is possible to remove the powdery coating of a plate by scraping it with a knife.
  • Easy visualization of separated components.
  • Non-fluorescence but UV absorbent, a substance can be obtained on a thin layer containing fluorescent indicator.
  • Corrosive reagent and acid can be spread on thin layer chromatography without adverse effects.
  • Capacity of thin layers of an adsorbent is higher than that of paper chromatography.

Application of Thin Layer Chromatography

  • TLC is used for qualitative and quantitative analysis.
  • In organic chemistry, it is used as an analytical tool. As compared to paper chromatography, it has a high speed.
  • TLC can be used as a purification process. TLC has 10-100 times more sensitivity in comparison to paper chromatography.
  • TLC can be used in a pathological lab for the analysis of urine and blood.
  • For the identification of organic compounds, TLC is used. E.g., citric acid and tartaric acid have been separated on layers of silica gel G using a butyl acetate, acetic acid, and water mixture.
  • TLC is used to isolate the proteins from urine by using the silica gel plate. TLC has separated vitamins, antibiotics, and food products on a thin layer of silica gel G using two solvent systems acetone and methanol or benzene and formamide. Erythromycin has been separated on silica gel G using methyl chloride, methanol, benzene, and formamide. Tetracycline uses 10 % citric acid, butanol, and methanol as solvent systems.
  • Vitamins A, D, and E can be separated on silica gel G using 80 % cyclohexane and 20 % diethyl ether.
  • TLC can be used to separate inorganic ions, cations, anions, and organometallic compounds. 

Limitation of Thin Layer Chromatography

  • It can only be used for small-scale production.
  • The plate length is limited; hence, separation takes place only up to a certain length.
  • The separation takes place in an open system; hence, samples may be affected by humidity and temperatures.

References

  • Thin Layer Chromatography (TLC) – Principle, procedure, Applications on BYJU’S. BYJUS. (2022). Retrieved 18 May 2022, from https://byjus.com/chemistry/thin-layer-chromatography/.

Sushmita Baniya

Hello, I am Sushmita Baniya from Nepal. I am a postgraduate student of M.Sc Medical Microbiology. I am interested in Genetics and Molecular Biology.

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