Initially, sixteen capillary micropipets were created in order to spot the TLC plates. Two TLC plates were then obtained and marked with pencil for spotting. A line was drawn 1 cm from the bottom of each plate, and five small, evenly spaced marks were made along those lines (see Figure 1). Each mark indicated where a substance would be spotted.
All compounds used were in solutions of 1g of each dissolved in 20 ml of a 50:50 mixture of methylene chloride and ethanol. The first plate made was the reference plate. Capillary micropipets were used to spot the first four marks with acetaminophen, aspirin, caffeine, and salicylamide (in that order). (See figures 2-5 for chemical structures.)
The last mark was spotted with a reference solution of all four chemicals. The second plate made was the sample plate. The first four marks were spotted with Anacin, Bufferin, Excedrin, and Tylenol. The fifth mark was spotted with a reference solution of all four drugs. Figure 1. Prepared TLC plates
Figure 2. AcetaminophenFigure 3. Aspirin
Figure 4. CaffeineFigure 5. Salicylamide
A development container was created with a wide-mouthed screwcap jar. It was filled with the development solvent, which was .5% glacial acetic acid in ethyl acetate, so that the solvent was approximately .
5 cm deep.The first TLC plate was then carefully placed into the development container. Great care was taken to ensure that the plate went in evenly so that the solvent could rise evenly up the plate. Once the solvent front had reached approximately 1cm from the top of the plate, the plate was removed, the solvent front was marked with a pencil, and the plate was allowed to dry.
The second plate was then placed in the development chamber in the same manner as the first. Once the solvent front reached approximately 1cm from the top of the plate, the plate was removed, the solvent front was marked with a pencil, and the plate was allowed to dry. Each plate was then viewed under the UV light.
Any spots that were seen were lightly circled with a pencil, and their color was noted. The orders of elution (Rf values) were calculated by dividing the distance from the baseline to the center of the spot by the distance from the baseline to the solvent front. After all observations and calculations were made, the plates were placed in a jar containing iodine.
The jar was warmed with hands so that the iodine vaporized. The plates were then removed from the jar and observed. The reference and sample plates were then compared to determine which compounds the drugs on the sample plate contained. Data