Thin Layer Chromatography

By Sushmita Baniya •  Updated: 06/06/22 •  10 min read

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

On a flat surface like glass or plastic plate, a thin layer of stationary phase is formed. 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. The rate of transfer of analysis is determined by their distribution coefficient, Kd.

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. The movement of the analytes is expressed by their retention value.

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 which is 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 silica which is hydrophilic and it is again coated by wax or paraffin making it hydrophobic.
  4. Ion exchange TLC: The exchange of ions takes place 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 which is 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 the separation of 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: It 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 is used for the separation of 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 generally 20 x 20 cm and 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: Most commonly used method is to obtain a thin and uniform layer. The slurry is placed in an applicator. This is either moved over the stationary plate or it is held stationary and the plate is pushed or pulled through. In one method the adsorbent reservoir containing the slurry 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 needs to 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 the 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 the ultraviolet (UV) light. Eg: fumaric acid, malic acid. 
  3. Reflected light or transmitted light: If the substances are in form of colored spots, they are visible under reflected light.

Chemical methods

It employs the use of different chemical reagents when the compounds are invisible under ordinary light, they can be visualized by using certain 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. For eg: 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

Quantitative evaluation of chromatogram can be carried out by 2 methods

  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. In this method, the spot containing the separated compound is scrapped and scrapings are 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 even the trace amount can be detected by TLC.
  • 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.
  • In a pathological lab for the analysis of urine and blood, TLC can be used.
  • For the identification of organic compounds, TLC is used. E.g. acids: citric acid and tartaric acid have been separated on layers of silica gel G by using a butyl acetate, acetic acid, and water mixture.
  • TLC is used for the isolation of the proteins from urine by using the silica gel plate. Vitamins, antibiotics, and food products have been separated by TLC on a thin layer of silica gel G by using 2 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 by using 10 % citric acid, butanol, and methanol as a solvent system.
  • 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 and hence separation takes place only up to a certain length.
  • The separation takes place in an open system, hence there are chances that 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/.
  • Coming up next “A Complete Note on Instrumentation”. Upendrats.blogspot.com. (2022). Retrieved 18 May 2022, from https://upendrats.blogspot.com/2016/11/coming-up-next-complete-note-on.html.

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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