A solution of the mixture to be separated is “spotted”, usually from a micropipet, near one edge of a piece of filter paper, and the solvent is evaporated. Usually several sample and standard spots are placed along the edge. Then the chromatogram is “developed” by immersing that edge of the paper in a solvent that migrates through the paper as the mobile phase. The solvent often has two, three or four components, one of which is usually water. Development is normally done in a chamber that is saturated with solvent vapor.
The water from the solvent, in particular, is adsorbed and tightly held on the paper fibers, so the sample components partition between the migrating mobile phase and the tightly-held water. If the bottom edge of the paper is immersed in the solvent, the process is “ascending” chromatography. Alternatively, the upper edge can be immersed in a trough of solvent and hung down for “descending” chromatography. Usually the process is stopped before the solvent front reaches the opposite edge of the paper, and the solvent is evaporated.
The ratio of the distance moved by a particular component to the distance moved by the solvent front is the “Rf”, or “retardation factor”, which is characteristic of that component under the conditions used. After the separation, any strongly colored spots are visible on the “chromatogram”. Colorless materials can be visualized by heating the paper in an oven so that substances (but not the paper) char and leave black spots. Sometimes the paper is first sprayed with a solution of sulfuric acid for better charring.
Fluorescent materials can be visualized with ultraviolet light. Reagents specific for certain components may be sprayed on to make colored spots. Radioactive spots can be located with a detector, or the chromatogram can be pressed against X-ray film for minutes or hours to expose the film. Sample spots can be tentatively identified if they have the same Rf values as known standard spots. Sometimes spots, once located, are cut out so that the material in the spot can be recovered.
There are also instruments that (more or less accurately) quantitate the material in the spot by measuring light absorbance. Variant: Circular chromatography: the sample is applied to the center of a circle of filter paper. Then the mobile phase is slowly dripped onto the spot and the chromatogram develops as a circle. Variant: Electrochromatography: as the mobile phase flows downward, an electrical field is applied transversely (electrophoresis) to get a two-dimensional separation of ionized molecules.
Variant: Two-dimensional chromatography: One spot is applied near a corner of a sheet of paper, and is developed with one solvent system. Then a second solvent system is applied from another edge so that mixed spots that did not separate with the first system may separate with the second one. These sheets may be as large as 75 cm x 75 cm. Lab reports or procedures will usually specify the solvent mixture (e. g. , methyl isobutyl ketone:isopropyl alcohol:ethyl acetate:water:ammonium hydroxide (50:45:35:18:3), the paper used (often Whatman #3) and the means by which the spots are visualized.