Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/Paper chromatography

Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be coloured, especially pigments. This can also be used in secondary or primary colours in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.

Technique of Paper Chromatography
A small concentrated spot of solution that contains the sample of the solute is applied to a strip of chromatography paper about two centimetres away from the base of the plate, usually using a capillary tube for maximum precision. This sample is absorbed onto the paper and may form interactions with it. Any substance that reacts or bonds with the paper cannot be measured using this technique. The paper is then dipped into a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container.

The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper; this can also be explained as differential adsorption of the solute components into the solvent. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibres in the paper. The more soluble the component the further it goes. Paper chromatography takes anywhere from several minutes to several hours.

In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same values in a particular solvent. In these cases, two-way chromatography is used to separate the multiple-pigment spots.

Ascending chromatography
In this method, the solvent is in pool at the bottom of the vessel in which the paper is supported

Descending chromatography
In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper. The liquid moves down by capillary action as well as by the gravitational force, thus this method is also known as the gravitational method. In this case, the flow is more rapid as compared to the ascending method, and the chromatography is completed more quickly. The apparatus needed for this case is more sophisticated. The developing solvent is placed in a trough at the top which is usually made up of an inert material. The paper is then suspended in the solvent. Substances that cannot be separated by ascending method can sometimes be separated by the descending method.

Chromatography as art
Chromatography can be used as an art alternative because of certain colours that it creates, but paper chromatography rarely produces decipherable colours if executed incorrectly. Sometimes, chromatographic results may start with undesirable "leafy" colours, but if done correctly and completely, magnificent hues can be produced.

Rƒ value
Rƒ value may be defined as the ratio of the distance travelled by the substance to the distance travelled by the solvent. Rƒ values are usually expressed as a fraction of two decimal places but it was suggested by Smith that a percentage figure should be used instead. If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front.