Chemistry - Chromatographic techniques

Chromatographic techniques : 

Chromatography is a technique used to separate components of a mixture, and also purify compounds. The name of the technique

comes from the Greek word Chroma meaning

Colour. In 1903, Tswett discovered this technique for separating the coloured components found in plants. The principle of separation of substances in this technique is similar to solvent extraction i. e. distribution of the solutes in two phases. In chromatography we use two phases for separation. (a) Stationary phase and (b) Mobile phase. This technique is based on the difference in rates at which components in the mixture move through the stationary phase under the influence of the mobile phase. First the mixture of components is loaded at one end of the stationary phase and then the mobile phase, which is a pure solvent or a mixture of solvents, is allowed to move over the stationary phase. Depending on the relative affinity of the components toward the stationary phase and mobile phase they remain on the surface of the stationary phase

or move along with the mobile phase, and gradually get separated. The stationary phase can be a solid or a liquid. Depending on the stationary phase, chromatography is classified into Adsorption Chromatography and Partition

Chromatography.

Adsorption Chromatography : 

This type of Chromatography is based on the

principle of Differential Adsorption. Different

solutes are adsorbed to different extent on the

stationary phase. Adsorption Chromatography

is of the following two types.

i. Column Chromatography : This type involves the separation of components over a column of stationary phase. The stationary phase material can be Alumina, Silica gel. A slurry of the stationary phase material is filled in a long glass tube provided with a stopcock at the bottom and a glass wool plug at the lower end. The mixture to be separated is dissolved in a small amount of appropriate solvent and is then loaded on top of the adsorbent column. A suitable mobile phase which could be a single solvent or a mixture of solvents is then poured over the adsorbent column. The mixture along with the mobile phase slowly moves down the column.

Column Chromatography

The solutes get adsorbed on the stationary phase and depending on the degree to which they are adsorbed, the solutes get separated from each other. The most strongly adsorbed component is retained on the column and others move down the column to various distances forming bands as seen in Figure. The component which is less strongly adsorbed is desorbed first and leaves the column first, while the strongly adsorbed component is eluted later. The solutions of these components are collected separately. On evaporating the solvent the solutes can be recovered.

ii. Thin Layer Chromatography : A thin layer (0.2 mm thick)of adsorbent silica gel or alumina spread over a glass plate acts as the stationary phase. The plate is called the TLC plate or chromplate. The mixture of solutes is applied on the Chromplate as a small spot about 2 cm from one end of the plate as shownin Figure.

(a) Stages in the thin layer Chromatography

The plate is then placed in a closed jar containing the mobile phase such that the spot is well above the mobile phase. As the mobile phase rises up the components of the mixture move along with it. They move upto different distances depending upon their degree of adsorption and thus get separated.

(b) Developed chromatogram

If the components are colored they appear as separated colored spots on the plate. If the components are not colored but have property

of fluorescence they can be visualised under UV light, or the plate can be kept in a chamber

containing a few iodine crystals. The Iodine vapors are adsorbed by the components and

the spots appear brown. Amino acids are visualised by spraying the plate with a solution of ninhydrin. This is known as spraying agent.

Partition Chromatography : 

In this type of chromatography the stationary phase and mobile phase both are liquids. Separation of components takes place by continuous differential partitioning of the components between the stationary and mobile phases.

For example, Paper Chromatography : In this

technique a special quality paper, Whatmann

paper number 1, is used. The water trapped in

the fibres of the paper acts as the stationary

phase.

Stages in paper chromatography

The solution of mixture is spotted on the strip of the Chromatography paper at the about 2 cm from one end of the paper using a glass capillary. The paper is then suspended in a chamber containing the mobile phase taking

care that the spot does not dip in the mobile

phase. The mobile phase rises up by capillary

action and flows over the spot. Partitioning of

the components takes place between stationary phase (water) and the mobile phase. Different solutes are retained differently on the paper depending on their selective partitioning between the two phases. This developed paper strip is the chromatogram. Similar to TLC (Thin layer chromatography) the colored components are visible as colored spots and the colourless components are observed under UV light or using a spraying agent.

Retention factor : Migration of the solute

relative to the solvent front gives an idea about the relative retention of the solutes on the

stationary phase.

Retention factor

This is termed as the  retention factor of the solute.