Thin Layer Chromatography (TLC) is a powerful technique to separate compounds based upon their polarity and interaction with silica and to assess the purify of a sample. To perform TLC, a solution of a compound or mixture of compounds is applied to a TLC plate by using a thin capillary tube. First, make a thin pencil mark on your TLC plate about one-quarter of an inch up from one end. Dip the capillary tube into the solution of compound and then touch the tube onto the line on your TLC plate. Now place they plate into a developing chamber (can be a simple beaker with some filter paper and aluminum foil cover). The developing chamber should have some developing solvent in it but the level of this solvent should not be above the pencil mark on your plate. Allow the solvent to move up the TLC plate and remove the plate when the solvent nearly reaches the top. Mark the distance as to where the solvent traveled. Now visualize your plate by first noting any spots you can visually see, then spots you can see with the aid of an ultraviolet light source, and then lastly by adding your plate to an iodine chamber. Once your spots have been visualized you can calculate the Rf value of each, the Rf value is also a physical constant of an organic molecule.

This procedure is shown below.

Pictured at the left is a TLC plate. The white matte surface pictured is the solid phase of this chromatography procedure. The solid line on the plate is drawn one centimeter from the bottom of the plate IN PENCIL. This is the line on which the the sample is applied. The labels from left to right read C, A, and U, represent caffeine, aspirin, and the unknown.

The chamber in which TLC takes place is filled to less than one centimeter of solvent (ethyl acetate and acetic acid). It is extremely important that the solvent in the developing chamber be filled to LESS THAN ONE CM because the solvent must be drawn upward through the sample in order to draw the sample along with it. If the sample is dipped in to the solvent, it will damage the results. A piece of filter paper is placed in the chamber to draw the solvent into the top of the chamber. Finally it is important to cover the chamber to be sure that the solvent does not evaporate.


The samples are applied to the TLC plate with a capillary tube. To draw sample into the tube, heat the one end of the tube on a hot plate and then dip the cool end into the sample. This will draw the sample up into the tube. Then spot a small amount of sample onto the TLC plate as pictured at the left. Note that the sample is applied on the 1cm line.

After all samples are loaded, the plate can be placed in to the chamber. Be sure that the solvent is below the line on which the samples were applied and that the plate is not touching the filter paper. Also, keep an eye on the plate. It only takes a few minutes for the solvent to travel up the plate. When the solvent reaches approximately one centimeter below the end of the plate, remove it from the chamber. Be sure that the plate does not touch the filter paper.



As soon as the plate is removed from the chamber, mark the solvent front for later calculations (top arrow). Allow the plate to dry. After the plate is dried, two methods can be used to visualize the location of the sample on the plate. Ultraviolet light can be used. When UV is used, the area of the plate surrounding the solvent will appear fluorescent, while the solvent does not. Another way to visualize the sample is using an iodine chamber. Placing a few pieces of iodine in a covered container and then adding the plate will turn some samples brown. In either case, using a pencil outline the location of the sample for later calculations (bottom arrow). These calculations are shown below.



Rf is calculated as shown.

Return to the skills page.


Chemistry Department, Wake Forest University, Salem Hall, Box 7486
Winston-Salem, NC 27109. Phone: (336) 758-5325 FAX: (336) 758-4656
Campus Map
Web page created and maintained by Tommy Murphy