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Chromatography

Definition of chromatography

Chromatography is an analytical technique used to separate mixture of chemical substances into its individual compounds. Different types of chromatography are used in lab. e.g. column chromatography, thin-layer chromatography, gas chromatography etc.

By Klaas1978 (Own woBy Klaas1978 (Own work (image created with MS Paint)) [Public domain], <a href="https://commons.wikimedia.org/wiki/File%3AChromatography_column.PNG">via Wikimedia Commons</a>rk (image created with MS Paint)) [Public domain], via Wikimedia Commons
Fig: Column chromatography

Principles of chromatography

Chromatography consists of two phases: one mobile phase and one contiguous stationery phase. The stationery phase is liquid or solid and the mobile gas is gas or liquid. The compound mixture moves along with the mobile phase through stationery phase and separates depending on the different degree of adhesion (to the silica) of each component in the sample or the compound mixture.

Chromatography terms

  • A chromatography is a physical method of separation while chromatograph is an equipment to separates mixture of compounds into its components.
  • A mobile phase is the solvent that moves through the column.
  • A stationery phase is the solid (e.g. silica) that stays inside the column.
  • Eluent is the fluid that enter into the column.
  • Eluate is the fluid that leaves the column or collects into a flask.
  • Analyte/ sample is the compound mixture what needs to be separated by chromatography.

Process

Similar procedure used in all chromatography. Lets explain the process of column chromatography to understand the general procedure.

By quantockgoblin (Own work) [Public domain], via Wikimedia Commons

  • A column is firstly packed with silica gel, the stationery phase.
  • Then a solvent (mobile phase) is passed through the column.
  • The sample is poured slowly on the top of the silica.
  • A suitable solvent is poured on the sample to flow through the silica under the gravity pressure. It separates the components of the compound mixture depending on different degrees of adhesion to the silica.
  • The separated compounds are then collected in different flask or test tube for identification.

Types of chromatographpy

Column chromatography

This is also known as liquid column chromatography. In this type of chromatography silica or alumina is used as stationery phase and a suitable solvent is used as a mobile phase. The column is packed with the slurry of silica or alumina. Here the each compounds in the mixture or sample is separated depending on the polarity of the molecules.

Thin layer chromatography (TLC)

Thin layer chromatography is the most widely used method in chemical or biochemical lab. Here as a stationery phase a thin layer of silica is used on a glass, metal or rigid plastic. The sample needs to be separated is placed as a small drop or line on to the chromatography and placed into a sealed container with a swallow layer of  suitable solvent. The solvent rises up the chromatography taking each component of the sample with it. The components travel with the solvent depends on their polarity. The non polar components travel faster than the polar component. Thus separated bands of components are observed under UV-light.

By Tlc_sequence.png: Quantockgoblin derivative work: Shakiestone (This file was derived from  Tlc sequence.png:) [Public domain], via Wikimedia Commons
Fig: Thin layer chromatography

Paper chromatography

The similar technique is used on paper chromatography. Difference is, instead of using a thin layer of silica on metal, it uses a special type of chromatography paper as stationery phase. This paper is made of cellulose. Drops or line of reaction mixture is placed on to it and then placed into a sealed container of swallow solvent.  The non polar compound travels fast in this chromatography. Polar compound make bond with cellulose and thus does not go far.

Summary

  • Chromatography is an analytical technique used to separate mixture of chemical substances into its individual compounds.
  • Different types of chromatography are used in lab. e.g. column chromatography, thin-layer chromatography, gas chromatography etc.
  • Chromatography consists of two phases: one mobile phase and one contiguous stationery phase.
  • The mobile phase flows along with the each component through the stationery phase depending on the degree of adhesion of each component to the stationery phase. Thus they are separated.

 

 

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