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Personal Study: Basics of Chromatography

Chromatography is a separation technique that uses the size, shape, chemical properties or charge of molecules in a sample to separate the sample into its constituent components. It is often used to detect one, or a number of, components in a complex mixture.

High Performance Liquid Chromatograph (HPLC)

High Performance Liquid Chromatograph (HPLC)

For instance, a pharmaceutical manufacturer may use chromatography to test the concentration of an active ingredient in a formulated product, or a food manufacturer may use chromatography to test that the concentrations of harmful agents, such as pesticides, are at, or below, safe levels.

There are a number of chromatography techniques including:

  • Paper Chromatography
  • Thin Layer Chromatography (TLC)
  • Gel Permeation Chromatography
  • Ion Exchange Chromatography
  • High Performance Liquid Chromatography
  • Gas Chromatography (GC).

There are a range of modern instrumental techniques available, but thin layer chromatography (TLC) shall be used to demonstrate the principles of chromatography.

Thin Layer Chromatography
The TLC plate is a thin piece of aluminium coated on one side with the stationary phase (this phase does not move), an inert material that does not react chemically with the sample components.

A mobile phase, which consists of a solvent that moves through the stationary phase, is used to move the components of the sample up through the stationary phase.

A drop of the sample is placed near the bottom end of the TLC plate. The bottom end of the TLC plate is then dipped into the solvent (mobile phase). The solvent creeps slowly up the TLC plate.

The various components of the sample move along with the solvent at a rate that depends on how strongly they are bound to the stationary phase. Strongly bound substances hardly move at all, a very weakly bound substance may move at almost the same speed as the solvent. Thus the different components (or fractions) of the sample are separated into bands (or spots) along the length of the TLC plate.

This separation process is the basis of the chromatography method.

Press 'play' below to view an animation of the chromagraphy technique.Skip flash movie

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Stationary and mobile phases are used in all chromatography techniques as detailed in the table below.

Technique Stationary Phase Mobile Phase
Paper The paper Solvent
Thin layer Thin layer of inert material coated onto a plate Solvent
Gel Permeation Ion Exchange HPLC Material packed into a steel or plastic column Solvent
Gas Chromatography Coated as a thin layer on the inside of a column Gas

 

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