“Chroma” means colour and “graphy” means graphical representation. Separation of components of a mixture on the basis of colour and then identifying the components via graph is the simplest meaning of chromatography.
History of chromatography:
- Mikhail Tswett in 1903 separated plant pigments using chromatography. He gave the name chromatography because he separated coloured components.
- Later Martin and Synge discovered various types of partition chromatography like paper chromatography, gas chromatography etc.
Concept of partition coefficient:
Suppose you have two phases, which cannot mix with each other. If you have to dissolve a analyte (substance or compound whose analysis has to be done with chromatography) ‘x’ in each of these liquids, then the solubility of the substance ‘x’ may be different in the two liquids.
Kd = (concentration of analyte in first phase / concentration of analyte in second phase.
2 phases of chromatography:
- Stationary phase: This phase may liquid, gel, solid-liquid mixture or gas.
- Mobile phase: This constitutes the movable part of the chromatography. Mobile phase moves over the stationary phase, for the purpose of separation of substances.
This phase may be liquid or gas.
Types of chromatography
Two types of chromatography are there basically:
- Planar chromatography: In planar chromatography, stationary phase is coated on the surface of a glass or plastic plate. Sample or analyte is applied as a thin spot on the stationary phase. Via capillary action, the mobile phase moves over the stationary phase. During movement it carries the analyte along with it. Rf value of various components of analyte present in the sample are calculated by differential migration of different components. Various types of planar chromatography are:
- Paper chromatography
- Thin layer chromatography
- Column chromatography
In this type of chromatography, the stationary phase is packed into a glass column, forming a sieve like structure. Then the analyte is applied on the stationary phase; finally mobile phase is applied. The mobile phase carries the analyte along the pores of stationary phase. Movement occurs either by gravity, or by applying pressure. Nowadays, most chromatographic techniques used in laboratories are modifications of column chromatography.
Various modifications of column chromatography are:
- Gel-filtration chromatography
- Ion-exchange chromatography
- Affinity chromatography
- Gas chromatography
- High performance liquid chromatography
Details of each and every technique are provided on the website separately, as practical. Any bioanalytical technique, I believe, can be understood only by performing and actually doing it in the laboratory.