Sangeetha Pulapaka

Chromatography is usually introduced as a technique for separating and/or identifying the components in a mixture. The basic principle is that components in a mixture have different tendencies to adsorb onto a surface or dissolve in a solvent. It is a powerful method in industry, where it is used on a large scale to separate and purify the intermediates and products in various syntheses.

There are several different types of chromatography currently in use – ie paper chromatography; thin layer chromatography (TLC); gas chromatography (GC); liquid chromatography (LC); high performance liquid chromatography (HPLC); ion exchange chromatography; and gel permeation or gel filtration chromatography. Basic principles

All chromatographic methods require one static part (the stationary phase) and one moving part (the mobile phase). The techniques rely on one of the following phenomena: adsorption; partition; ion exchange; or molecular exclusion.

Paper chromatography

This is probably the first, and the simplest, type of chromatography that people meet. A drop of a solution of a mixture of dyes or inks is placed on a piece of chromatography paper and allowed to dry. The mixture separates as the solvent front advances past the mixture. Filter paper and blotting paper are frequently substituted for chromatography paper if precision is not required. Separation is most efficient if the atmosphere is saturated in the solvent vapour

As each solute distributes itself (equilibrates) between the stationary and the mobile phase, the distance a solute moves is always the same fraction of the distance moved by the solvent. This fraction is variously called the retardation factor or the retention ratio, and is given the symbol R or Rf

So as long as the correct solvent and type of chromatography paper are used, a component can be identified from its retention ratio.

It is possible that two solutes have the same Rf values using one solvent, but different values using another solvent (eg this occurs with some amino acids). This means that if a multi component system is not efficiently separated by one solvent the chromatogram can be dried, turned through 90 degrees., and run again using a second solvent,