High Performance Liquid Chromatography : Module 1

General Introduction to Chromatography

       “Tell me and I’ll forget :  show me and I may remember; involve me and I’ll understand”

                                                                                                                    —- Chinese Proverb 

High Performance Liquid Chromatography Diagram

Chromatography is the most widely used laboratory technique for separation, identification and quantification of components of liquid and gaseous mixtures. Solid mixtures are also analyzed by first converting them to a liquid or gaseous state, using suitable sample preparation techniques. Differential affinity of components between the carrier and stationary phases forms the basis of separation. Components retained by stationary phase have slower migration rates than unretained or partially retained components.

You can think of the molecular mixture as a family passing by a candy store which is the stationary phase. The children will tend to get retained because of their affinity for candy, while the parents will keep on moving like un-retained molecules leading to a separation between children and parents!

Various techniques have been adapted to identify and quantify the components that migrate through the chromatographic system in a sequence depending on operating parameters. We will be discussing these detectors in-depth in module number 6.

In earlier days liquid mobile phase was commonly used in paper, thin layer and column chromatography .In paper and thin layer chromatography separation of components takes place as the solvent moves along the filter paper or coated plates by capillary action. In column chromatography an empty glass tube is packed with finely powdered stationary phase and a small volume of liquid mixture is applied to top of column. Mobile phase liquid is continuously added which elutes sample components sequentially based on affinity with stationary phase. The individual components can be detected visually or with a detection system.

Gas Chromatography Diagram

In Gas chromatography same principle is used but carrier is a gas instead of a liquid. The stationary phase is an immobilized liquid bound to an inert support or simply applied to the inner surface of a column. Gas chromatography is effectively used for analysis of gas mixtures or liquids having low boiling points. On the other hand liquid chromatography is applied for separation of thermally labile liquid mixtures or those having high boiling points.

Size exclusion chromatography is based on separation of molecules on the basis of their size .Stationary phases are selected on basis of their pore size and selective retention takes place depending on the  pore size of stationary phase .No chemical interaction takes place between the stationary phase and the eluting species.

Liquid Chromatography has played a key  role in development of HPLC. The next module takes you through the  evolution of Liquid Chromatography  to its present day status.

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P.S

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