Paper chromatography is an analytical technique for separating and identifying mixtures that are or can be coloured, especially pigments. This can also be used in secondary or primary schools 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.
A small concentrated spot of solution that contains the sample is applied to a strip of chromatography paper about 2 cm 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 in to 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,also this can be explained as differential absorption
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 fibers in the paper. 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. The chromatogram is turned by ninety degrees, and placed in a different solvent in the same way as before; some spots separate in the presence of more than one pigment. As before, the value is calculated, and the two pigments are identified. Paper chromatography was invented by two British biochemists, Archer John Porter Martin
and Richard Laurence Millington Synge
After development, the spots corresponding to different compounds may be located by their color, ultraviolet
(Triketohydrindane hydrate) or by treatment with iodine
vapors. The paper remaining after the experiment is known as the Chromatogram
.The components which have been separated differ in their retention factor
i.e Ratio of distance traveled from the spot
by the solute component to that of the distance traveled from the spot
by the solvent. Retention Factor can never be greater than one. To calculate Rf
, use the following:
Distance traveled by sample/ distance traveled by solvent.
The final chromatogram can be compared with other known mixture
chromatograms to identify sample mixes using the Rf
value in an experiment. The retention values found can be compared to known values, and from that conclusions can be drawn.