One of the tasks of biochemists is to identify, separate and purify one or more biological components in a mixture of such compounds in a biological sample. One of the most important convenient methods for achieving such separation is the use of chromatographic techniques.
The term chromatorgraphy was originally applied by Micheal Tswett, a Russian Botanist, in 1906 to a procedure where a mixture of different coloured pigments (chlorophylls and xanthophylls) was separated from each other.
Principle : The basis of all forms of chromatography is the partition or distribution co-efficient which describes the way in which a compound distributes itself between two immiscible phases. For a compound distributing itself between equal volumes of two immiscible solvents A and B (Fig. 10.1), the value of distribution co-efficient is a constant at a given temperature and is given by the expression
Basically all chromatographic systems consists of two phases. One is the stationary phase which may be a solid, liquid or a solid liquid mixture which is immobilized. The mobile phase may be a liquid or a gas and flows over or through the stationary phase.
Separation starts to occur when a compound to be separated is held more firmly by the stationary phase than the other which tends to move on slower in the mobile phase. Thus, the underlying principle of chromatorgraphy is to adsorb the components of the mixture on an insoluble material and then to differentially remove or elute these components one by one with suitable solvents.
The term effective distribution co-efficient is defined as the total amount as distinct from the concentration of substance present in one phase divided by the total amount present in the other phase. Thus, a distribution co-efficient of a substance between alumina (stationary phase) and butanol (mobile phase) might be 0.25 which means that the concentration of the substance in butanol is four times that in the alumina.The choice of stationary or mobile phases is made so that the compounds to be separated have different distribution co-efficient.
In practice separations may be achieved by using different types of chromatographic techniques