Biochemical Techniques and Instrumentation

2.1.1. Chromatographic separation

In chromatographic separation, compounds are separated based on their distribution between two immiscible phases: stationery phase and mobile phase. Two components in a mixture is introduced at time, t_o into a mobile phase that is in contact with stationery phase. As the compounds come in contact with stationery phase, they distribute or partition between the two phases depending on their relative affinity for the phases. Their relative affinity depends on their molecular structure and intermolecular forces. A compound with a high affinity for stationery phase will move through the system very slowly, whereas a compound with lower affinity will migrate more rapidly. This migration results in separation of compounds as they move through the system. Time for the distribution of solute molecules between phases must be rapid compared to the velocity of mobile phase.

Under these conditions, the system is characterized by a distribution or partition coefficient (K_d). Distribution coefficient is defined as ratio of the concentration of compounds in the mobile phase (Cm) to that of the concentration of compounds in the stationery phase (Cs).

           K_d = Cm/Cs = constant

Distribution coefficient of a compound is constant for any two immiscible phases ie. stationery and mobile phases at a given temperature. "K_d" is a characteristic physical property of a compound, which depends on the structure of the compound, nature of two phases and temperature.

Effective distribution coefficient is defined as the total amount of compound present in one phase (mobile phase) divided by the total amount present in the other phase (stationery phase). If the distribution coefficient of a compound between two phases A and B is 1, and if this compound is distributed between 10 cm³ of A and 1 cm³ of B, the concentration in the two phases will be the same, but the total amount of compound in phase A will be 10 times the amount in phase B.