Introduction to Chromatography:
A technique for the preparation of components of a become of the distribution of the components between two phases, is called chromatography. This separation is due to the difference in solubilities of the component between these phases.
The phase over which mobile phase flows in chromatography is called the stationary phase.
- It may be solid or liquid supported on solid or a gel.
- It may be packed in a column.
- It may be spread as a layer or distributed as a film.
- Chromatographic had is a term used to denote any of the different forms in which the stationary phase is used.
- Examples: Silica gel, alumina, filter paper, etc. are some important stationary phases.
- It is also called eluant.
- It competes with the stationary phase for components of the mixture.
- Examples: Water, ethanol, acetic acid, acetone or gas, etc, are some important mobile phases.
Principle of chromatography:
There are possibilities for the value of KD.
- A component having a smaller value of KD mostly remains in the stationary phase as the mobile phase flows over it.
- A component having a higher value of KD remains largely dissolved in the mobile phase and it quickly passes over the stationary phase.
Classification of chromatography
- On the basis of shape
- On the basis of phase
- On the basis of the mechanism
On the basis of shape:
Chromatography is classified into various types depending upon the way in which it is carried out.
Examples: Column chromatography, thin layer chromatography (T.L.C), paper chromatography, ascending, and descending paper chromatography.
On the basis of phase:
It is also classified on the basis of phases. As described before the mobile phase can be gas or liquid while the stationary phase can be liquid or solid. Thus we have four combinations:
- Liquid-liquid chromatography
- Liquid – solid chromatography
- Gas-liquid chromatography
- Gas – solid chromatography
On the basis of mechanism:
Different ways of paper chromatography:
- Ascending paper chromatography
- Descending paper chromatography
- Radial/circular paper chromatography
Ascending paper chromatography:
Then spot a point with a narrow capillary tube on the line with the sample mixture solution. To make identification of components easy, spot the points of known compounds on the same line. All spots should be pointed using separate capillary tubes Fig (2.6) B and C are two pure components. A is a mixture of components B and C.
Uses of chromatography:
Although it is a simple technique yet it has many important applications:
- It is very useful in the separation and identification of colored pigments.
- Medicines and proteins, which are complicated molecules can be identified.
- Amino acids travel different distances with solvents and hence can be identified.
- Substances produced by a body like urine can also be analysed by chromatography.
- The purity of the compounds was checked by chromatography. Pure substance gives one spot only.
- It separates substances from complete mixtures of closely related substances which cannot be separated by ordinary chemical methods
- It separates a mixture of very small quantities at very low concentrations.
- It is equally important in qualitative and quantitative analysis.
- It is very useful in organic synthesis for separation, purification, and identification of products.