Categories: General

Thin Layer Chromatography; Principle, Procedure and Diagram

Thin Layer chromatography (TLC)

The chromatography is used to distinguish mixtures of materials into their components. All types of chromatography effort on a similar principle. They all have a stationary phase which is a solid, or a liquid maintained on a solid and a mobile phase which is a liquid or a gas. The mobile phase runs through the stationary phase and brings the components of the mixture with it. Different components travel at different rates. We will see the aspect of the reasons for this further down the page. The Thin layer chromatography is done precisely as it says ‘using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic’. The silica gel or the alumina is the stationary phase. The stationary phase for thin layer chromatography also often covers a substance which fluoresces in UV light – for details you will see later. The mobile phase is an appropriate liquid solvent or mixture of solvents.

Principle

  • Based on the principle of adsorption chromatography and type planar chromatography.
  • An analyte migrates up the layer of the stationary phase under the influence of the mobile phase which moves through the stationary phase by capillary action. The distance moves by the analyte are determined by its relative affinity for S.P V/S M.P.
  • Oldest technique
  • It is used for the separation of a minute amount of sample.

Instrumentation of Thin Layer Chromatography

TLC Plate

  • The plate should be 5cm long and 2cm wide.
  • Absorbents are usually silica or alumina.
  • First, we made slurry by mixing silica in water and a thick paste is formed called slurry.
  • Then this slurry is applied on the TLC plate usually by four methods.

Spreading

Glass tube having a plastic end is used to spread slurry usually slurry is 250nm. We can change the thickness of slurry by using glass rods of different plastic endings.

Pouring

It is the most used method. This slurry is applied on the plate and spread by rotating the plate.

Dipping

In this method, the plate is dipped in slurry and dried.

Spraying

In this method, the slurry is sprayed and is not used usually.

Marking of TLC plate

  • After drying of TLC plate a line of 1.5cm long is marked on TLC plate
  • After marking sample is placed on that marks
  • The line is standard or guideline
  • Two spots are marked

Activating thin layer chromatography plate

  • The marked plate is kept in the oven for 15mins at 50-60 C.
  • It is kept because water molecules in slurry should be dried.

Spotting of thin layer chromatography plate

  • The sample should be in liquid form.
  • A capillary tube is dipped in a sample and placed on spots made on marking of slurry.
  • After spotting it is called an origin.

Developing of TLC plate

Choice of solvent is necessary for having the following characteristics

  • It should be non-reactive
  • It should be cheap
  • It should be easily available
  • It should be pure
  • It should be inert

Visualization

  • By UV light (Observed under UV lamp)
  • By staining
  • Iodination in which iodine gives a brown color
  • Ninhydrin for amino acids it gives purple spots

Advantages of Thin Layer Chromatography

  • Better separation
  • Better resolution
  • Economical
  • The short duration of time
  • Simplest technique
  • Manually operated

Applications of Thin Layer Chromatography

  • Qualitative analysis
  • Quantitative analysis
  • Microanalytically technique for the small number of samples
  • Isolation of biological compounds
  • Purification of biological compounds
  • Separation of the complex mixture
  • Calculation of Rf value (0-1)
  • Identification of drugs
  • Qualification and quantification of the complex pharmaceutical dosage form.
  • In forensic labs (700 drugs are identified using thin-layer chromatography).

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