Chromatography is a technique to separate different molecules of organic origin from each other; every organic molecule is different from other in its physical properties, and owing to this difference they tend to get separated from each other while migrating in the form of solution over a solid surface, every molecule get separated from each other at a specific distance over the solid surface at specific distance and after specific time. This phenomenon is applied over wide range of laboratory techniques of quantification (assay) and qualitative analysis (Identification) every molecule has its own characteristics with respect to…
1) Number of Carbon atoms
2) Number of electron rich moieties over it.
3) Presence of loan pair of electrons over just like amines -N: C=0 and hydrogen atoms, which can form polar bonds easily in polar solvent.
4) Polarity of entire molecule
5) Molecular weight of molecule.
6) Spacial arrangement of molecule
7) Chiral carbon atoms in molecule.
8) Structural isomerism
All above characteristics of molecule impart specifically different characteristics to a molecule, when different molecules are dissolved in solvent; these molecules (solute) are solvated in solvent by different forces like hydrogen bonding with solvent molecule. Polar molecules tend to get solubilised with ease with polar solvents. Non polar molecules tend to get solubilised with ease with no polar solvents.
When a mixture of molecules is dissolved in solvent different molecules have different degree of solvation. The mixture will be termed as solution. When a small drop of this solution is placed over a highly porous surface like, porous silica, the dissolved solute travels with different speed through the solid surface, solution migrates through porous surface (stationary phase) by capillary action, which is the only force applied in thin layer chromatography, in case of HPLC solution (mobile phase) is pumped through a column consisting a stationary phase.
There are many applications and use of chromatography , by making changes in stationary phase and mobile phase ,different types molecules are separated from each other , on the basis of , its size, special orientation, nature of molecule, even stereoisomer’s are separated from each other by chromatographic technique.
Following are the types of chromatography
1) Column chromatography :
Here stationary phase which is made up of silica, bentonite ect , is packed inside a column through which a solution consisting solute (mobile phase) is passed through, different types of molecule are eluted at different time, and thus retention time over solid phase is different for different molecule, it is specific for a particular molecule.
2) Paper chromatography:
Here a typical paper is used in place of solid stationary phase, paper is of uniform thickness throughout, and uniform in weight at any given portion over it. It is placed in solution contained in a chamber and covered. Solvent migrate through paper along with solute, different soutes are separated and concentrated over different spots over paper. It is used in identification tests.
3) Thin layer chromatography: TLC: here a thin layer of stationary phase is spread over the surface of a plate. And it is widely used in identification tests in pharmaceutical analysis, TLC can also be used in quantitative estimation of a compound.
Liquid chromatography : high performance liquid chromatography
Affinity chromatography : Supercritical fluid chromatography
Expanded Bed Adsorption (EBA) Chromatographic Separation
Simulated moving-bed chromatography
Pyrolysis gas chromatography
Fast protein liquid chromatography