Thin Layer Chromatography (TLC) is a powerful technique to separate compounds, based upon their polarity and interaction with silica, and to assess the purifty of a sample. To perform TLC, a solution of a compound or mixture of compounds is applied to a TLC plate with a thin capillary tube. First, mark the origin by making thin pencil marks on the left and right sides of your TLC plate about one-quarter of an inch from one end. To apply the sample, dip the capillary tube into the solution of compound to be analyzed, and then touch the tube between the marks on your TLC plate. Carefully lower the plate into a developing chamber, which can be as simple as a beaker with some filter paper around the sides and an aluminum foil lid. The developing chamber should have some developing solvent in it, but the level of this solvent should not be above the pencil marks and spots on your plate. Allow the solvent to climb up the TLC plate, and remove the plate when the solvent nearly reaches the top. Quickly, before the solvent evaporates, mark the solvent front. Visualize your plate by first circling any spots you can visually see, then spots you can see with the aid of an ultraviolet light source, and then lastly, if necessary, by adding your plate to an iodine chamber or staining solution. Circle any spots you see. Calculate the Rf value of each spot. The Rf value is also a physical constant of the 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 chromotography procedure. The marks on the plate are drawn one centimeter from the bottom of the plate IN PENCIL. This is the origin. Samples are applied between the marks. (Ideally, labels should be placed at the top of the lanes, not across the bottom as done in this photo.) The lables from left to right read C, A, and U, represent caffine, asprin, and the unknown.
The chamber in which TLC takes place is filled to less than one centimeter of solvent, ethyl acetate and acetic acid in this particular example. It is extremely important that the solvent in the developing chamber be lower than the spotos on the plate, 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, the sample may simply dissolve in the developing solvent. A piece of filter paper that has been cut along the bottom edge 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, simply dip the tube into the solution you wish to apply to the plate. The sample will be drawn into the tube by capillary action. Spot a small amount of sample onto the TLC plate as pictured at the left. Note that the sample is applied between the marks. Avoid spotting too close to the edge of the TLC plate (~ 3 mm) as solvents travel differently up the extreme edges of the plate.

developingA 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, proceeding rapidly at first. When the solvent reaches approximately one centimeter below the top of the plate, remove it from the chamber. Do not move the developing chamber while a plate is being developed.

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, several methods can be used to visualize the location of the sample on the plate. You may be able to see the spots without assistance. You may need to use and ultraviolet lamp. When UV is used, the area of the plate surrounding the spots will fluoresce , while the spots do 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. If these measures fail, you may be asked to use a staining solution to help visualize the spots. In any case, use a pencil to outline the location of the sample(s) for later calculations, as noted with the bottom arrow. Sample calculations are shown below.

Rf is calculated as shown.

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