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.
Please join StudyMode to read the full document