7.1: Paper Chromatography
The name of this method came from the Greek word khroma meaning colour. It was first used to separate colours, pigments and dyes. Nowadays, it can be used to separate colourless substances.
The principle involved depends upon the relative solubilities . In paper chromatography, the piece of paper used, like most paper, contains water loosely combined with the cellulose of the paper. If a dye is put in small spots, at the bottom of the paper, and another solvent is soaked up the paper, the solutes present in the dye dissolve to different extents. Some are more soluble in the mobile solvent moving up the paper. Other dissolve better in the water trapped in the paper and therefore do not travel very far up the paper. The difference in solubility allows the different pigments in the dye to be separated.
Chromatohraphy, although a simple technique, has many important applications besides identifying colour pigments. In medicine, proteins, which are complicated molcules, may be identified using chromatograms. Here, the building units of proteins are amino acids which, like pigments, travel different distances with solvents. Although amino acids are colourless, they can be sprayed with a liquid which reacts with them to make them visible. Other substances produced by the body, such as urine, also be analysed by chromatography. We call liquids, which, when sprayed onto chromatograms, make the separating visible, locating agents. A locating agent is a chemical which reacts with the substances to produce a visibly coloured product. One such liquid is ninhydrin spyray which can react with colourless amino acids to produce a purple stain.
7.3: Alternative Method
Paper chromatography can also be carried out with the solvent running down the paper. This descending method of paper chromatography works better for longer pieces of paper as the solvent does not have to move against gravity, and thus flows more quickly. This means that the solute which are separated can travel further and thus the separating between the spots is greater.
7.4: Rf Values
For any substance on a chromatogram,
Rf value = Distance moved by the substance/ distance moved by the solvent