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Chapter: Biochemistry: Biochemical Techniques

Types of chromatography

Column chromatography: In this type, the stationary phase is packed into a glass or metal columns (wide tubes or cylinders).

Types of chromatography

Column chromatography: In this type, the stationary phase is packed into a glass or metal columns (wide tubes or cylinders). The mixture to be separated is layered on the top of the column in the form of a solution at particular concentration. After equilibration the components are eluted out of the column one by one using specific mobile phases (Fig.10.2).The solvent used to elute the separated components is known as eluant.

Thin layer chromatography: In this type, the stationary phase is thinly coated on to a glass, plastic or foil plates. The mixture to be separated is applied on the stationary phase at one end and kept vertical in the petridish containing the mobile phase. When the mobile phase reaches the other end of the plate the plate is removed from the petridish and the compounds separated are identified by using specific staining reagents

 Paper chromatography: In this type, the stationary column chromatography phase is supported by the cellulose fibres of a paper sheet. The mobile phase flows through the stationary phase and effects separation.

Each of these three types of chromatography have their specific advantages, applications and method of operation.

Column chromatography:

All the major types of chromatography are routinely carried out using column type (Fig. 10.3). The different types of column chromatography are

i. adsorption chromatography

ii. partition chromatography

iii. ion-exchange chromatography

iv. exclusion chromatography

v.    affinity chromatography

i. Adsorption chromatography


An adsorbent may be described as a solid which has the property of adsorbing molecules at its surface, particularly when it is porous and finely divided. Adsorption can be specific so that one solute may be adsorbed selectively from a mixture. Separation of components by the method depends upon differences both in their degree of adsorption by the adsorbent and solubility in the solvent used for separation. Adsorption chromatography can be carried out in both the column and thin layer modes.

ii.Partition chromatography Principle:

This technique is based on the partitioning of compounds between a liquid stationary phase and a liquid mobile phase. The liquid stationary phase can be held on any solid support like paper. This technique is otherwise known as liquid- liquid chromatography.

iii. Gas liquid chromatography

Principle: This technique is based upon the partitioning of compounds between a liquid stationary phase and a gas mobile phase . It is a widely used method for the qualitative and quantitative analysis of a large number of compounds (eg. fatty acids) because it has high sensitivity, reproducibility and speed of resolution. A stationary phase of liquid material such as a silicone grease is supported on an inert granular solid . This material is packed into a narrow coiled glass or steel column 1 to 3 meter long and 2 to 4mm internal diameter. Through this column an inert carrier gas (the mobile phase) such as nitrogen, helium or argon is passed. The column is maintained in an oven at an elevated temperature which volatilizes the compounds to be separated.

The basis for the separation is the difference in the partition coefficients of the volatilized compounds between the liquid and gas phases as the compounds are carried through the column by the carrier gas. As the compounds flow, they leave the column and pass through a detector which is connected to a recorder and record a peak. The area of the peak corresponds to the concentration of the compound separated.

iv. Ion exchange chromatography

Principle: The principle of this form of chromatography is the attraction between oppositely charged particles . Many biological materials, such as amino acids and proteins, have ionisable groups and the fact that may carry a net positive or negative charge can be utilized in separating mixtures of such compounds. The net charge carried by such compounds depend on their pKa and on the pH of the solution.

Ion exchange separations are mainly carried out in columns packed with an ion exchanger, which contain the core matrix molecule with exchangeable ionic groups on its surface. There are two types of ion exchangers, namely cation and anion exchangers. Cation exchangers posses negatively charged groups and they will attract positively charged molecules. Anion exchangers have positively charged groups which will attract negatively charged molecules. The actual ion exchange mechanism composed of four steps;

·           selective adsorption of the molecules to be separated by the ion exchange resins. B release of the exchangeable group from the matrix.

·           Elution of the absorbed molecule by specific eluants.

·           Regeneration of the matrix by recharging with the original exchangeable groups.

Cation exchanger

Some of the ion exchange materials used in this technique are Amberlite IRC 50, Bio- Rex, Dowex 50, Sephadex etc.

v. Molecular exclusion chromatography

This chromatography is otherwise known as gel permeation chromatography.

Princple: This technique is based on the separation of molecules on the basis of their molecular size and shape and the molecular sieve properties of a variety of porous materials which serve as the solid stationary phase.

A column of gel particles or porous glass granules is in equilibrium with a suitable solvent for the molecules to be separated. Large molecules which are completely excluded from the pores will pass through the interstitial spaces and smaller molecules will be distributed between the solvent inside and outside the molecular sieve and will then pass through the column at a lower rate . So the larger particles will come out of the column first followed by smaller particles (fig. 10.4).

The gel materials generally used for this technique are cross-linked dextrans, agarose, polyacrylamide, poly styrene etc.

vi. Affinity chromatography:

Principle: This technique is based on the specific biological interaction of the compounds to be separated with the special molecules attached on the stationary phase called as ligands. This technique requires that the material to be isolated is capable of reversibly binding to a specific ligand which is attached to an insoluble matrix (stationary phase).

Under suitable experimental conditions when a complex mixture containing the specific compound to be purified is added to the insolubilised ligand generally contained in a chromatography column, only that compound will bind to the ligand. All the other compounds can be washed away and the compound subsequently recovered by displacement from the ligand. The purification of an enzyme by this technique is shown diagrammatically in Fig. 10.5.

In practice, particles which are uniform, spherical and rigid are used as matrix materials such as polystyrene, cellulose, porous glass and silica etc.


Thin Layer chromatography

Principle: Partition, adsorption, exclusion chromatography can be carried out in a thin layer mode. In this technique the stationary phase is made in the form of a slurry and applied as a thin coating on the surface of a glass plate.After activating the plate, the sample to be separated is applied at one end of the plate . The plate is kept vertically in a chamber specially designed for this purpose( TLC chamber) and allowed the sample and the mobile phase to raise through the stationary phase by capillary action. The whole procedure consists of.

a. Thin layer preparation : A slurry of the stationary phase, generally applied to a glass, plastic or foil plate as a uniform thin layer by means of a plate spreader starting from one end of the plate and moving progressively to the other. Calcium phosphate is incorporated into the slurry in order to facilitate the adhesion of the adsorbent to the plate. The plate is heated in an oven at 1000 C to activate the adsorbent.

b. Sample application : The sample is applied to the plate by means of a micropipette or syringe as spot or as a band on the stationary phase.

c. Plate development : Separation takes place in a glass tank which contains mobile phase to a depth of about 1.5 cm. This is allowed to stand for atleast an hour with a lid over the top of the tank to ensure that the atmosphere within the tank becomes saturated with solvent vapour.

d. Component detection : The components separated are detected by (i) spraying the plate with 50% sulphuric acid or 25% sulphuric acid in ethanol and heating; (ii) examining the plate under ultraviolet light; (iii) spraying of plates with specific colour reagents, for example ninhydrin for amino acids.


Paper chromatography

Principle: The cellulose fibres of chromatography paper act as the supporting matrix for the stationary phase. The stationary phase may be water or a non-polar material such as liquid paraffin. The components get separated between the liquid stationary phase and the liquid mobile phase. The procedure consists of

a. Paper development: There are two techniques which may be employed for the development of paper, ascending and descending methods. In both cases, the solvent is placed in the base of a sealed tank or glass jar to allow the chamber to become saturated with the solvent paper. The sample spots should be in a position just above the surface of the solvent so that as the solvent moves vertically up the paper by capillary action, separation of the sample is achieved.

b. Component detection: The separated components can be detected by (i) examining the paper under ultraviolet light; (ii) spraying of papers with specific colour reagents, for example ninhydrin for amino acids and sulphuric acid for simple sugars.

The identification of a given compound may be made on the basis of its Rf value (retardation factor) which is the distance moved by the component during development divided by the distance moved by the solvent from the point of origin (Fig. 10.6).

The value of Rf is constant for a particular compound under standard conditions and closely reflects the distribution co-efficient for that compound.


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