Chromatography- introduction, definition, types, uses

Introduction

Chromatography is a separation technique used to separate and analyze different components of a mixture based on their physical and chemical properties. It involves the separation of a mixture into its individual components by passing it through a stationary phase, such as a solid or a liquid, and a mobile phase, such as a gas or a liquid.

Definition

It is a technique of separation in which components of a mixture are separated on the basis of their relative affinities for stationary and mobile phases.

Meaning

The word chromatography originates from the Greek word “chromatic” meaning “color writing”. This is because the earliest chromatographic methods were confined to the separation of colored substances from their mixture.

Phases in chromatography

Chromatography involves the distribution of a solute between two phases, a stationary phase, and a mobile phase.

Stationary Phase

The phase in which the mobile phase travel is called the stationary phase. It may be a solid or a liquid supported as a thin film on the surface of an inert solid.

Examples: Alumina, silica gel, charcoal, Whatman’s filter paper, acetone, water, ethyl alcohol, etc.

Mobile Phase

The phase which is travel on the stationary phase is called the mobile phase. It may be liquid or gas. It flows over the surface of the stationary phase. While passing over the stationary phase, it competes with it for the components of the mixture.

Examples: Acetone, water, ethyl alcohol, benzene, H₂ gas, N₂ etc.

Types of Chromatography

Adsorption Chromatography

The chromatography in which the stationary phase is solid is called adsorption chromatography.

In this type, a substance leaves the mobile phase to become adsorbed on the surface of the solid stationary phase.

Examples

• Column chromatography
• Thin layer chromatography
• Gas solid chromatography

Partition Chromatography

The chromatography in which the stationary phase is a liquid is called partition chromatography.

In this type, the separated substances are distributed throughout the stationary and mobile phases.

Examples  

• Paper chromatography
• Gas-liquid chromatography

Paper Chromatography

It is a technique of partition chromatography.

Here the stationary phase is a liquid adsorbed on paper. The adsorbed water behaves as immiscible liquids towards the mobile phase, which passes over the paper.

The mobile phase is usually an organic liquid.

Ways of Paper Chromatography

There are three common ways of carrying out paper chromatography namely

(i) Ascending      (ii) Descending   (iii) Radial/Circular

Ascending Paper Chromatography

• In this technique, the solvent is a pool at the bottom of a vessel in which the paper is supported and the solvent travels upwards by capillary action. This vessel is called a chromatographic tank.

• A solvent mixture, specially composed in accordance with the sample to be separated, is poured into the chromatographic tank. Cover the tank to homogenize its inner atmosphere.

• Take about 20 cm strip of Whatmann’s Chromatographic paper No.1 and draw on it a thin pencil line about 2.5 cm from one end.

• Spot a point, on the pencil line, with the sample mixture solution. To facilitate the identification of the components of the mixture, spots of the known compounds may also be placed alongside.

• When the spots have dried, suspend the paper with clips so that the impregnated end dips into the solvent mixture to a 5-6 mm depth.

• Cover the tank. As the solvent front passes the spots, the solutes begin to move upward.

• The rate at which they move depends on their distribution coefficients. When the solvent front has risen to about 3/4th of the length of the paper, remove the strip, mark the solvent with a pencil, and allow the strip to dry.

Chromatogram

The finished dried pattern with the color bands of various components of a mixture obtained in the chromatographic experiment is called a chromatogram.

The components of the mixture, if colored e.g. mixture of red, blue, and green inks can visually be identified.

Development of Chromatogram

If the components of a mixture are colorless, then the chromatogram has to be developed by two methods:

1. Chemical Methods: By using locating agents such as H₂S, Ninhydrin, Rubenic acid, etc.
2. Physical Methods: We can identify the spots by heating or throwing ultraviolet radiation.

Uses of Chromatography

Chromatography is a separation technique used to separate components of a mixture based on their differential partitioning between a stationary phase and a mobile phase. The technique finds various applications in different fields such as:

1. Analytical chemistry: Chromatography is used for the qualitative and quantitative analysis of compounds in a mixture. It can be used for the identification and determination of impurities, purity, and potency of pharmaceuticals, food products, and environmental samples.
2. Forensic science: Chromatography is used for the detection and identification of drugs, poisons, explosives, and other illicit substances in forensic samples.
3. Biotechnology: Chromatography is used for the purification of proteins, enzymes, nucleic acids, and other biomolecules from complex biological mixtures. It is also used for the analysis of biological fluids and tissues.
4. Environmental science: Chromatography is used for the monitoring and analysis of pollutants in air, water, soil, and other environmental samples.
5. Petrochemical industry: Chromatography is used for the analysis and purification of petroleum and its products.
6. Food industry: Chromatography is used for the analysis and separation of food additives, flavors, and fragrances.
7. Pharmaceutical industry: Chromatography is used for the purification and separation of active pharmaceutical ingredients and drug formulations.
8. Quality control: Chromatography is used for the quality control of various products, such as foods, pharmaceuticals, and chemicals.
9. Research and development: Chromatography is used for the isolation, purification, and characterization of new compounds and materials in research and development activities.