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Thin Layer Chromatography

Thin layer chromatography principle

Thin layer chromatography (TLC) is a chromatography principle or technique which we used to separate or identify non-volatile mixtures on a small scale. The working method of thin layer chromatography is similar to column chromatography. In TLC technique, the adsorbent is coated on a glass plate which acts as a stationary phase. Usually, we used alumina or silica gel with little calcium sulfate for the production of thin layer chromatography plate. The solvent or mobile phase which we used in thin layer chromatography is a very impotent step. Compared to other chromatographic techniques, it is applicable for quantitative analysis of samples.

Thin layer chromatography principle, method of instrumentation and experiment of TCL on amino acids

Solvent used in thin layer chromatography

The choice of solvent system and composition of thin layer plate determined the principle of TLC chromatography. The solvent used in the TCL technique should be nonpolar and volatile. The eluting powers of the common solvents are,

Pentane < hexane < carbon tetrachloride < chloroform < diethyl ether < ethyl acetate < acetone < ethanol < methanol < water.

Thin layer chromatography method

Thin layer chromatography instrumentation method or procedure contains the following four main steps,

  • Preparation of thin layer chromatography (TLC) plate
  • Application of sample
  • Chromatogram
  • Identification of components

Thin layer chromatography plate

TLC plate is a glass plate that has three different sizes as 25 × 75 mm, 5 × 20 cm, and 20 × 20 cm. We used alumina or silica gel with little calcium sulfate for the coating of TLC plates. Cellulose powder and diatomaceous earth can also be used for coating TCL plates. Photodensitometry plates are also used for quantitative analysis. Such analysis can be carried out by UV, visible and IR, or fluorescence spectrophotometry. Two methods are generally used for the preparation of TCL plates.

  • A uniform slurry of absorbent neither too thick nor too thin is prepared by water. It is speared on the glass plate by a commercial spreading device. The glass plate is dried at 1200 °C for the production of TLC plates.
  • A uniform 1:1 slurry of adsorbent can be prepared by mixing the adsorbent with chloroform and methanol and stored in a well-stopped bottle. Two microscope slides can be coated by dipping them in the slurry. The solvent is evaporated. Two TLC plates are separated and activated by drying in an oven.

Sample

Generally, 1 to 2% solutions are applied by a capillary tube or a microsyringe at about 1.5 to 2 cm from the edge. The spot must be 1 cm apart from each other.

Chromatogram

When the spot becomes dry, the plate is put into the chamber which contains a suitable solvent with its saturated vapour. The absorbent layer must be under the solvent and spots must be above the solvent. When the solvent front travels at least three fourth of the length of the plate, a chromatogram is developed.

Identification of components

Components of colour compounds mixture are directly identified by colour spots in the chromatogram. Components of colorless compounds can be identified by UV light on the dried plate to obtain fluorescence or placing it into an iodine chamber or spring by the suitable reagent to give coloured spots.

Thin layer chromatography experiment

It is useful for the analysis or identification of amino acids, dyes, and ink. A new technique like high-pressure thin layer chromatography (HPTLC) is applied for pharmaceutical analysis.

Thin layer chromatography of amino acids

Identification and analysis of an individual amino acid from a mixture of amino acids can be done by thin layer chromatography experiment.

  • 10 mg of each known amino acid are dissolved in 10 ml of 95% ethanol. Solution of unknown amino acids mixture also prepared by the same method.
  • The spotted thin chromatography plate is put into the solvent chamber.
  • The TCL plate is taken out from the solvent chamber and the solvent front is marked quickly. The plate is dried in an air oven.
  • Detection of spots can be done by spraying ninhydrin solution and drying again. The purple colour spots appear for amino acids at different distances corresponding to their respective Rf values.

Thin layer chromatography applications

It is very useful for the analysis of a large number of compounds, Compounds that are not volatile or too labile for gas liquid chromatography can be analyzed by TCL technique.

Forensic chemists used the TCL technique for various separations. It is applicable for the analysis of amino acids, plasticizers, antioxidants, ink, dye, etc. It has extensive applications in inorganic analysis. A new method like high pressure thin layer chromatography (HPTLC) is used widely in pharmaceutical analysis.