Chromatography is based on the principle where molecules in a mixture are applied onto the surface or into the solid, and fluid stationary phase (stable phase) separates from each other while moving with the aid of a mobile phase.
The factors effective in this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights.
Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster.
Based on this approach three components form the basis of the chromatography technique.
- Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface a solid support”.
- Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
- Separated molecules
The type of interaction between the stationary phase, mobile phase, and substances contained in the mixture is the basic component effective in the separation of molecules from each other.
Chromatography methods based on partition are very effective in the separation, and identification of small molecules such as amino acids, carbohydrates, and fatty acids.
However, affinity chromatographies (ie. ion-exchange chromatography) are more effective in the separation of macromolecules as nucleic acids, and proteins.
Paper chromatography is used in the separation of proteins, and studies related to protein synthesis; gas-liquid chromatography is utilized in the separation of alcohol, Esther, lipid, and amino groups, and observation of enzymatic interactions, while molecular-sieve chromatography is employed especially for the determination of molecular weights of proteins.
Agarose-gel chromatography is used for the purification of RNA, DNA particles, and viruses.
A stationary phase in chromatography is a solid phase or a liquid phase coated on the surface of a solid phase.
The mobile phase flowing over the stationary phase is gaseous or liquid. If the mobile phase is liquid it is termed liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC).
Gas chromatography is applied for gases, mixtures of volatile liquids, and solid materials. Liquid chromatography is used especially for thermal unstable, and non-volatile samples.
The purpose of applying chromatography which is used as a method of quantitative analysis apart from its separation, is to achieve a satisfactory separation within a suitable time interval. Various chromatography methods have been developed to that end.
Some of them include column chromatography, thin-layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, high-pressure liquid chromatography, and affinity chromatography.