Column Chromatography

This is the most commonly used mode of chromatography. It can be defined as a separation process involving the uniform percolation of a liquid through a column packed with finely divided material. The stationary phase attached to a matrix (inert insoluble support) is packed in a glass or metal column and mobile phase is passed through it either by gravitational flow or with help of a pump. The selected stationary phase retards the movement of certain components of mobile phase which leads to effective separation of different components. This retardation in movement may be either due to direct interaction of solute component with stationary phase or indirect adsorption of solute component on the surface of stationary phase.

A typical column chromatographic system uses liquid mobile phase, consist of a column, a mobile phase reservoir and delivery system (pump), a detector for identification of separated analytes as they emerge in the effluent from the column, a recorder to maintain the record of analytes in combination with detector and a fraction collector to collect each analyte separately (Fig.1).

The pictorial representation of detector response as a function of elution time or volume is known as chromatogram which consists of a series of peaks, representing the individual analytes. After application on the column the time require to elute by an analyte is known as retention time (t_R ) of that analyte. The retention time of any analyte has two components. The first one is time taken by that analyte to cross the column through free spaces between matrixes. This volume is referred as void volume (V₀ ) and the time taken is called dead time (t_M ). The value of dead time (t_M ) will be same for all analytes and can be measured by the analyte which does not show any interaction with stationary phase. The second component is the time the analyte retained by the stationary phase and known as adjusted retention time (t_R'). This is the characteristic of analyte and can be given by;

t _R' = t _R - t _M

The additional time taken by an analyte to elute from the column relative to an excluded analyte that does not interact with the stationary phase is known as capacity factor (k').

Thus, Capacity factor (k') = t _R− t_M / t _M = t _R '/ t_M (capacity factor has no units)