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Chapter: 11th Biochemistry : Biochemical Techniques

Principle of chromatography

Chromatography involves a stationary and a mobile phase.

Principle of chromatography

 

Chromatography is defined as the method of separation of different substances based on their partition coefficients in two immiscible phases. Chromatography involves a stationary and a mobile phase. The sample to be analysed, when placed in the stationary phase (solid, bonded coating) will gradually move along in the same direction as the mobile phase (liquid or gas). A component that is soluble in the stationery phase takes a longer time to travel as compared to the one that is insoluble. Therefore, the differences in their mobility can be ascribed to the interaction of the sample with the mobile and stationary phase. For a compound distributing itself between equal volumes of two immiscible solvents A & B, the distribution or partition co-efficient (Kd) is given as,





As shown in Fig.10.1, substances present in X will tend to cross the boundary between the two liquid layers, to form a dynamic equilibrium. 


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