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Chromatography is a recent technique and is now widely used for the purification of organic compounds. It can also be used for the identification of compounds. This method is used in various forms, such as column chromatography, thin layer chromatography (TLC), gas chromatography, paper chromatography, etc. .

Different Types of Techniques of Chromatography

Type of chromatography

Mobile phase

Fixed phase

Principle involved

Main use

Column chromatography

solvent or mixture of solvents

Silica gel or alumina


purification of compounds

Thin layer chromatography (TLC)

solvent or mixture of solvents

Silica gel or alumina



Gas liquid chromatography (GLC)

inert gas like N2 or He

paraffin oil or silicon grease




mixture of solvents

water supported over cellulose of filter paper



Column Chromatography

Column chromatography is a simple chromatographic technique. It consists of a long tube of glass fitted with a stopcock at one of its ends. This tube is filled with a suitable solid adsorbent such as silica gel or alumina (Al2O3). This solid adsorbent is also called the fixed phase.

The mixture of compounds that is to be purified is dissolved in a suitable solvent and then poured over the adsorbent. The compounds present in the mixture get adsorbed to different extents over the adsorbent, depending upon their chemical nature. A solvent or a mixture of solvents is then allowed to percolate (pass) through the column. Such a solvent is called the mobile phase. The compound that is least adsorbed over the adsorbent gets deadsorbed and begins to move along with the solvent. This process is called elution. A compound that is strongly adsorbed moves much slower than the less strongly adsorbed ones. Thus, different compounds occupy different positions in the column. If the solvent is allowed to flow in the column for a long time, then the compound that is least adsorbed comes out of the column through the stopcock. Other compounds come out later. Thus the solution of the compound is collected as a separate fraction. The compound is obtained by evaporating the solvent.

Fig 1.6 Separation by column Chromotography
(i) starting stage (ii) intermediate stage and (iii) Final stage
Thin Layer Chromatography (TLC)

Thin Layer Chromatography

Thin layer chromatography (TLC) is another type adsorption chromatography, which involves separation of the substance of a mixture over a thin layer of an adsorbent. A thin layer (about 0.2mm thick) of an adsorbent like silica gel or alumina is spread over a glass plate of suitable size. The plate is known as thin layer chromatography plate. The solution of the mixture to be separated is applied as a small spot about 2 cm above one end of the TLC plate. The glass plate is then placed in a closed jar containing the solvent. As the solvent in jar moves up the plate, the components of the mixture move up along the plate to different distances depending on their degree of adsorption and separation takes place. The relative adsorption of each component of the mixture is expressed in terms of its retention factor i.e. Rf value.
Rf = Distance moved by the substance from base line (x)
       Distance moved by the solvent from base line (y)  

The spots of coloured compounds which are invisible to the eye but which fluoresce can be detected by putting the plate under ultraviolet light.

Partition Chromatography

Partition chromatography is based on continuous differential partitioning of components of a mixture between stationary and mobile phases. Paper chromatography is a type of partition chromatography. In paper chromatography, a special quality paper known as chromatography paper is used. Chromatography paper contains water trapped in it, which acts as the stationary phase.

A strip of chromatography paper spotted at the base with the solution of the mixture is suspended in a suitable solvent or a mixture of solvents. This solvent acts as the mobile phase. The solvent rises up the paper by capillary action and flows over the spot. The paper selectively retains different components according to their differing partition in the two phases. The paper strip so developed is known as a chromatogram. The spots of the separated colored compounds are visible at different heights from the position of initial spot on the chromatogram. The spots of the separated colourless compounds may be observed either under ultraviolet light or by the use of an appropriate spray reagent as discussed under thin layer chromatography.

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