The purpose of this experiment is to identify an unknown proprietary drug using thin-layer chromatography. The unknown’s behavior in thin-layer chromatography will be compared with that of its possible component analgesics. The possible unknowns and their analgesic ingredients will be Anacin (aspirin, caffeine), Excedrin (acetaminophen, caffeine, aspirin), Motrin (ibuprofen), and Tylenol (acetaminophen).
B. Materials and Safety
|Chemical Name||Molecular Formula||Molecular Weight (g/mol)||Liquid||Solid||Solubility||Potential Hazards|
|b.p. ºC||Density g/mL||m.p. ºC|
|Aspirin||C9H8O4||180.160||140||1.40||138–140||H2O||Irritant, harmful if excess swallowed|
|Caffeine||C8H10N4O2||194.0956||178||1.23||237||H2O||Irritant, harmful if excess swallowed|
|Acetaminophen||C8H9NO2||151.17||—||1.263||168-172||H2O||Irritant, harmful if excess swallowed|
|Ibuprofen||C13H18O2||206.27||157||—||75 – 78||H2O||Irritant, harmful if excess swallowed|
C. Experimental Procedure
First, five 13×100 test tubes will be labeled 1-asp, 2-ace, 3-unk, 4-caf, and 5-ibu. Then
About 20-30 mg of each of the four known compounds will be added to the appropriate test tube, and 20-30 mg of the unknown will be added to the test tube marked 3-unk. Next, 0.5 mL of methanol will be added to the four test tubes with known compounds and they will be swirled until all the solid has dissolved. The test tube with unknown will receive 1.0 mL of methanol and a glass stirring rod will be used to mix the sample. Any insoluble material will be allowed to settle.
Next, two 3” x 1” sheets of fluorescent TLC silica gel plates will be obtained. Capillary tubes will be used to spot the samples of 1-asp, 2-ace, and 3-unk on the coated side of one plate. The spots are to be evenly spaced, 0.5-1.0 mm in diameter, 1 cm from the bottom of the plate, and at least 0.5 cm from the side of the plate. The plate will be viewed under UV light to make sure that enough of each sample has been applied. Capillary tubes will also be used to spot 3-unk, 4-caf, and 5-ibu on the other plate in the same manner. Again, UV light will be used to make sure enough of each sample has been applied.
Then, each TLC plate will be placed spotted end down into a developing jar containing a pool of ethyl acetate that is about 0.5 cm deep. The jar will be capped and when the ethyl acetate rises within 1-2 cm of the top of the TLC plate, the TLC plate will be removed from the jar and allowed to dry. Once dry, the TLC plate will be analyzed under the UV light and the appearance of the spots will be drawn in a laboratory notebook.
II. Experiment and Results
First, five clean test tubes were labeled 1-asp, 2-ace, 3-unk, 4-caf, and 5-ibu. Twenty to thirty mg of each of the four known compounds were added to the appropriately labeled test tube, and all of the unknown in our given vial, labeled “K”, was added to the vial labeled 3-unk. Next, 0.5 mL of methanol was dispensed into the four test tube containing known compounds, and the test tubes were swirled until the solid had dissolved. Then 1.0 mL of methanol was dispensed into the test tube with the unknown, and it was also swirled until the solid was dissolved. A spatula was used to help mix any solids that were having trouble dissolving.
Next, two fluorescent TLC silica gel plates measuring about 3” x 1” were obtained. Micro capillary tubes were used to spot 1-asp, 2-ace, and 3-unk on the coated side of one plate. The spots were evenly spaced and about 1 cm from the bottom of the plate and 0.5 cm from the side of the plate. On the second plate, 3-unk, 4-caf, and 5-ibu were plated in the same manner. The plates were then placed spotted side down in a developing jar that contained a 0.5 cm high pool of ethyl acetate. The lid of the jar was screwed on and the plates were allowed to sit in the jar until the ethyl acetate rose to about 2 cm from the top of the plate. The plates were then removed from the jar and then analyzed under an ultraviolet light. The appearance and measurements of the spots were recorded in a lab notebook.
|Unknown Spot #1||0.24|
|Unknown Spot #2||0.58|
It appears that the unknown contained caffeine and acetaminophen judging by the Rf numbers. The average Rf for unknown spot #1 was 0.24, which is very close to the Rf of caffeine (0.22). The average Rf for unknown spot #2 was 0.58, which is fairly close to the Rf of acetaminophen (0.52). However, none of the unknown drugs contain only caffeine and acetaminophen. Excedrin contains caffeine, acetaminophen, and aspirin, which leads me to believe that we may have misinterpreted our gel when looking at it. It was hard to tell if there some of the streaks actually had spots in them or not, so we may have missed on what should have been a third spot above the acetaminophen spot on our gel plate. This third spot would have been aspirin. I do not remember even seeing a streak above our second spot on the gel plate for the unknown, so this means we may not have spotted enough of the solution on the plate. It could also mean that there is a very low concentration of aspirin in Excedrin, which would account for why we could not see it on the gel plate.