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Paper chromatography is an analytical technique for separating and identifying mixtures that are or can be colored, especially pigments. 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 spot of solution that contains the sample is applied to a strip of chromatography paper about 1 cm from the base. 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, 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 and to one another. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent. 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 into multiple spots, showing the presence of more than one pigment. As before, the value is calculated, and the two pigments are identified.

After development, the spots corresponding to different compounds may be located by their color, ultraviolet light, ninhydrin (Triketohydrindane hydrate)or by treatment with iodine vapors. The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes using the R_f value.

As in most other forms of chromatography, paper chromatography uses R_f values to help identify compounds. R_f values are calculated by dividing the distance the pigment travels up the paper by the distance the solvent travels (the solvent front). Because R_f values are standard for a given compound, known R_f values can be used to aid in the identification of an unknown substance in an experiment.