1 2 3 4 5

1. Anacin2. Bufferin3. Excedrin4. Tylenol5. Reference Solution

1 2 3 4 5

1. Acetaminophen2. Aspirin3. Caffeine4. Salicylamide5. Reference Solution

1

Kristyn MurphyOrganic 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

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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

1 2 3 4 5

1. Acetaminophen2. Aspirin3. Caffeine4. Salicylamide5. Reference Solution

1 2 3 4 5

1. Anacin2. Bufferin3. Excedrin4. Tylenol5. Reference Solution

3

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 FormulaMW

(g/mol)Amount

(g)Moles State

Density(g/ml)

M.P.(˚C)

B.P.(˚C)

Theo. Yield

Hazards

acetaminophen C8H9NO2 151.16 16.6155*

10-3 s - 168-170 - - respiratory, skin, and eye irritant

aspirin C9H8O4 180.16 15.5506*

10-3 s - 134-136 - -respiratory, skin, and eye irritant;

toxic if swallowed

caffeine C8H10N4O2 194.19 15.1495*

10-3 s - 232 - -respiratory, skin, and eye irritant;

toxic if swallowed

salicylamide C7H7NO2 137.14 17.2918*

10-3 s - 140-144 - - respiratory, skin, and eye irritant

methylene chloride CH2Cl2 84.93 13.25 .1560 l 1.325 - 39.8-40 -respiratory, skin, and eye irritant;

toxic if swallowed

ethanol C2H6O 46.07 7.89 .17126 l .789 - 78 - respiratory, skin, and eye irritant

iodine I2 253.81 - - s/g - 113.5 - -respiratory, skin, and eye irritant; harmful to mucous membranes

ethyl acetate C4H8O2 88.11 - - l - -76.5-77.5

-respiratory, skin, and eye irritant;

may cause drowsiness

glacial acetic acid C2H4O2 60.05 - - l - - 117-118 -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 X’s were ones that developed the brownish color after exposure to the iodine.

Figure 6. Developed TLC Plates

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The Rf values were calculated based on these observations. They are shown in Table 2.

Table 2. Rf Values

NameDistance from Base to Center of Spot

(cm)÷

Distance from Base to Sovlent

Front (cm)= Rf Value

acetaminophen 4.0 ÷ 5.5 = .73

aspirin 4.7 ÷ 5.5 = .85

caffeine 2.4 ÷ 5.5 = .44

salicylamide 4.6 ÷ 5.5 = .84

Anacin 2.6 ÷ 5.8 = .45

Bufferin 5.0 ÷ 5.8 = .86

Excedrin 2.6 ÷ 5.8 = .45

4.3 ÷ 5.8 = .74

Tylenol 4.3 ÷ 5.8 = .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

5

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.

5

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 weren’t 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 chloride—just 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.