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Kristyn Murphy Organic Chemistry I Laboratory

TLC Analysis of Analgesic Drugs Introduction In this experiment, thin-layer chromatography (TLC) was used to determine the composition of various over-the-counter (OTC) analgesics: Anacin, Bufferin, Excedrin, and Tylenol. The TLC plates were first viewed under ultraviolet (UV) light and then treated with iodine vapor in order to visualize the spotting. Experiment Scheme 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 1. Acetaminophen 2. Aspirin 3. Caffeine 4. Salicylamide 5. Reference Solution
1 2 3 4 5 1 2 3 4 5

1. Anacin 2. Bufferin 3. Excedrin 4. Tylenol 5. Reference Solution

Figure 2. Acetaminophen

Figure 3. Aspirin

Figure 4. Caffeine

Figure 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 Table 1. Chemical Table1
Name acetaminophen aspirin caffeine salicylamide methylene chloride ethanol iodine ethyl acetate glacial acetic acid Formula C8H9NO2 C9H8O4 C8H10N4O2 C7H7NO2 CH2Cl2 C2H6O I2 C4H8O2 C2H4O2 MW (g/mol) 151.16 180.16 194.19 137.14 84.93 46.07 253.81 88.11 60.05 Amount (g) 1 1 1 1 13.25 7.89 Moles 6.6155* 10-3 5.5506* 10-3 5.1495* 10-3 7.2918* 10-3 .1560 .17126 State s s s s l l s/g l l Density (g/ml) 1.325 .789 M.P. (C) 168-170 134-136 232 140-144 113.5 B.P. (C) 39.8-40 78 76.577.5 117-118 Theo. Yield Hazards respiratory, skin, and eye irritant respiratory, skin, and eye irritant; toxic if swallowed respiratory, skin, and eye irritant; toxic if swallowed respiratory, skin, and eye irritant respiratory, skin, and eye irritant; toxic if swallowed respiratory, skin, and eye irritant respiratory, skin, and eye irritant; harmful to mucous membranes respiratory, skin, and eye irritant; may cause drowsiness respiratory, skin, and eye irritant; harmful to mucous membranes

After the plates were removed from the development container, they were dried and then observed. They were first viewed under UV light, where any spots were circled with a pencil. They were then placed in a jar with iodine where some spots developed a brownish color. Figure 6 shows approximately what each plate looked like after it was viewed. Any circles with Xs were ones that developed the brownish color after exposure to the iodine. Figure 6. Developed TLC Plates 1. Acetaminophen 2. Aspirin 3. Caffeine 4. Salicylamide 5. Reference Solution
1 2 3 4 5 Solvent Fronts 1 2 3 4 5

1. Anacin 2. Bufferin 3. Excedrin 4. Tylenol 5. Reference Solution

The Rf values were calculated based on these observations. They are shown in Table 2. Table 2. Rf Values
Name acetaminophen aspirin caffeine salicylamide Anacin Bufferin Excedrin Distance from Base to Center of Spot (cm) 4.0 4.7 2.4 4.6 2.6 5.0 2.6 4.3 Tylenol 4.3 Distance from Base to Sovlent Front (cm) 5.5 5.5 5.5 5.5 5.8 5.8 5.8 5.8 5.8 = = = = = = = = = = Rf Value .73 .85 .44 .84 .45 .86 .45 .74 .74

Discussion This experiment was designed to determine which analgesic compounds common OTC drugs contained. According to research done after the experiment, the testing successfully determined which compounds the sample drugs contained. The results showed that Tylenol contains acetaminophen, Excedrin contains caffeine and acetaminophen, Anacin contains caffeine, and Bufferin most likely contains aspirin but could have salicylamide instead or could have both aspirin and salicylamide. According to the research, Bufferin is only aspirin. Excedrin and Anacin contain aspirin as well, but this was not shown by the data. One problem that arose in this experiment was that the reference plate gave very similar Rf values for aspirin and salicylamide and neither of them turned color after exposure to the iodine. This made it difficult to tell which compound was in Bufferin. Salicylamide and aspirin are similar compounds2, so it would make sense for our tests to show them to have similar properties.

The other problem encountered with the results was that aspirin did not spot in Excedrin or Anacin. There were two spots on the top left and right side of the sample plate and some coloration in between. These things were all ignored and considered erroneous because they werent in line with the other spots. The two larger spots were in the approximate location of where the plate was handled, so this could be what created those spots. It could be that the aspirin was in the coloring between the two spots. If too much of the sample was applied, it could cause the aspirin to tail and not create defined spots. It could also be that too little sample was applied and no spots were created for the aspirin. The problem of not applying the correct amount of solvent could be resolved by using better capillary micropipets. The ones used in this experiment were crudely made, and it was difficult to judge exactly how much sample had been applied. It would have been interesting to see if other methods of visualization showed other spots on the TLC plates. While we only used UV light and iodine, some other methods we could have used involve silver nitrate, sulfuric acid charring, and ferric chloridejust to name a few. In conclusion, the experiment was mostly successful because it correctly determined the composition of the sample drugs with the exception of the problems with aspirin. Questions 1. Too much sample will cause the spots to tail and overlap. 4. The ink from a pen would rise up the TLC plate along with the development solvent and the chemicals being tested. This would make it very difficult to determine which spots belonged to the sample being tested. References 1. Sigma Aldrich. http://www.sigmaaldrich.com/safety-center.html Date accessed: August 31, 2009. 2. Miriam Webster Online Dictionary. http://dictionary.reference.com/browse/salicylamide Date accessed: September 5, 2009.