high performance liquid chromatography

What is Thin Layer Chromatography?Thin-layer chromatography is a technique in which the analytes of a mixture are separated by differential migration through the stationary phase. TLC is performed on glass, aluminum or plastic sheets. It is coated with a thin layer of adsorbent material, typically made using silica gel, aluminum oxide, or cellulose and is known as a stationary phase. After applying the sample to the TLC plate, a mobile phase (solvent mixture) is drawn up to the plate by capillary action. Since each analyte has a different affinity to the TLC plate; hence separation is achieved at different rates.

Thin Layer Chromatography Principle:TLC is a separation technique where the molecules of mixtures separate using differential migration through a stationary phase, the solvent mixture flowing through the virtue of capillary forces. After chromatography is complete, solutes are detected on the surface of a TLC plate by visualizing the reagents and using a UV cabinet. TLC is a type of planar chromatography, such as paper chromatography, but the stationary phase in TLC is a thin-split Sorbent, which spreads as a thin layer of aluminum, glass, or plastic supporting flat plate.

Thin Layer Chromatography Diagram:

Principle and Procedure of Thin Layer Chromatography

Thin Layer Chromatography Procedure:Before beginning a TLC experiment, we must recognize the various components essential to perform the process.

Thin Layer Chromatography Chamber: To develop the plates, the TLC chamber is used. This maintains a stable environment and prevents solvent evaporation, which helps spots develop.

Thin Layer Chromatography Plates: The separation occurs on the TLC plate, in which the stationary phase is applied as a thin layer on its surface. Nowadays, ready plates are available in the market. They are chemically inert, stable and of high quality.

Thin Layer Chromatography Mobile phase:A solvent or mixture of solvents with suitable polarity is used as the mobile phase to achieve the required separation.

Experimental Procedure of TLC:

  1. The adequate mobile phase is poured into a TLC chamber to give liquid about 0.5 cm deep. Subsequently, cover the chamber for a few minutes to allow the atmosphere to become saturated with solvent vapor.
  2. The small amount of liquid sample is “spotted” on the TLC plate with close to an edge and parallel with an edge. The plate is then placed in contact with the mobile phase in the chamber and is covered.
  3. The mobile phase is drawn from the edge of the TLC plate through the Sorbent bed, mainly by capillary action, Allow the development process until the mobile phase is approximately half a centimeter below the top of the TLC plate. The mobile phase and sample are displaced in parallel passages in the direction of flow, and the components separate according to their affinity.
  4. Remove the plate from the chamber and mark it immediately in front of the solvent. 
  5. Allow the plate to dry with warm air or room temperature.
  6. Circle the visual spot lightly with a pencil. Most analytes are not colored they are to be visualized by spraying chromogenic reagent or in UV cabinet. Mark the sample spot and calculate the Rf value. The retention factor (Rf value) will always be in the range 0 to 1.

Thin Layer Chromatography Applications:

  • It is used to separate and identify the number of components present in a sample mixture.
  • TLC can be used to select the appropriate mobile phase system in column chromatography.
  • It is used for the separation and identification of sweetening agents, preservatives, dyes, and different cosmetic products.
  • It is used for the separation and identification of food products, pharmaceutical formulations, and natural products.
  • Thin-layer chromatography can be used to monitor the progress and the rate of reactions at particular intervals.

The Advantages of Thin Layer Chromatography are as follows.

  • TLC is a very easy and sensitive mode of separating analytes.
  • The molecules are separated in a short period, as they are quickly eluted.
  • Very small amounts of the sample need to be analyzed.
  • A complex mixture of sample solution can be easily separated and recovered.
  • This can be automated using high-performance thin-layer chromatography (HPTLC).
  • This requires very few types of apparatus.

The Disadvantages of Thin Layer Chromatography are as follows.

  • Only non-volatile compounds are separated by this method.
  • It is difficult to reproduce to obtain from TLC.
  • The separation length in TCL is insufficient, as it does not have a long stationary phase.
  • Humidity and Temperature can affect TLC results.
  • Another disadvantage of TLC is that it only analyzes soluble analytes.

Commonly asked questions on thin layer chromatography are as follows.

What is the principle of TLC?TLC comprises a stationary and mobile phase, in which a thin layer of alumina or silica is coated on glass, metal, or plastic that is a stationary phase and a solvent or mixture of solvents is a mobile phase. The sample mixture travels through the stationary phase with the mobile phase and separates according to the degree of their adhesion.


What type of chromatography is TLC?Thin-layer chromatography is a type of planar chromatography.

What is the TLC plate made of?Generally, TLC plates are made using silica gel, cellulose, aluminum oxide or similar material. A thin layer of special granular ground matrix is ​​coated as on a glass plate, aluminum or a plastic film.

What does TLC Rf value mean?It is the proportion of separation traveled by an analyte to distance traveled by a mobile phase front. It is used to identify analytes due to their specificity for each compound.

What is the major advantage of thin-layer chromatography?The major advantage of TLC is that many samples can be analyzed simultaneously over rapid time.

What is the major difference between TLC and HPTLC?The major difference between TLC and HPTLC is that the particle size of the absorbing material. An HPTLC plate contains very small particles of adsorbent material while the TLC contains larger particles.

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