Biochemical Techniques and Instrumentation

2.1.3. Equilibrations

Assume the distribution coefficient of a compound is 1. Equilibrations occur continuously in a column since the mobile phase is continuously added. Then, after five equilibrations, the compound is distributed throughout the whole column but is maximally concentrated at the center of the column.

If the distribution coefficient is <1, more than 50% of the compound would be left on stationery phase after each equilibration and the concentration peak is above the center of the column and vice versa. The greater the number of equilibrations, the greater becomes the concentration of compound on a certain part of the column.

Two important factors that influence the pattern of separation or resolution are effective distribution coefficient and sharpness of band on the column. This sharpness depends on the number of equilibrations. The number of equilibrations which takes place is termed as “Theoretical plates” which describes the efficiency of the column. The greater the number of theoretical plates, the column is more efficient. The distribution of compound with effective distribution coefficient is affected by the number of theorticial plates(n).