Thin-Layer Chromatography Analysis of

Analgesics
I. Introduction
A. Objective

The purpose of this experiment is to identify an unknown proprietary drug using thinlayer chromatography. The unknowns 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

b.p.
C

Density g/mL

m.p. C

Solubility

Poten

Aspirin

C9H8O4

180.160

140

1.40

138140

H2O

Irritan
exces

Caffeine

C8H10N4O2

194.0956

178

1.23

237

H2O

Irritan
exces

Acetaminophen

C8H9NO2

151.17

1.263

168-172

H2O

Irritan
exces

Ibuprofen

C13H18O2

206.27

157

75 78

H2O

Irritan
exces

C. Experimental Procedure

First, five 13100 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
A. Data

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.
Substance

Rf

Aspirin

0.76

Acetaminophen

0.52

Caffeine

0.22

Ibuprofen

0.91

Unknown Spot #1

0.24

Unknown Spot #2

0.58

III. Conclusions
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.

Thin Layer Chromatography

Introduction
(Adapted from Mohrig, 1st ed., pp. 151-162.)
Chromatography is a sophisticated method of separating mixtures of two or more compounds. The separation is
accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving.
Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two
phases between which they are to be partitioned.

Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a solid (stationary phase) and a
liquid (moving phase). Solids most commonly used in chromatography are silica gel (SiO 2 x H2O) and alumina (Al2O3 x
H2O). Both of these adsorbents are polar, but alumina is more so. Silica is also acidic. Alumina is available in neutral,
basic, or acidic forms. Thin Layer Chromatography (TLC) is a sensitive, fast, simple and inexpensive analytical technique.
It is a micro technique; as little as 10-9g of material can be detected, although the sample size is from 1 to 100x10 -6 g. TLC
involves spotting the sample to be analyzed near one end of a sheet of glass or plastic that is coated with a thin layer of
an adsorbent. The sheet, which can be the size of a microscope slide, is placed on end in a covered jar containing a
shallow layer of solvent. As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs
between the components of the mixture dissolved in the solvent the stationary adsorbent phase. The more strongly a
given component of a mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and
the more slowly it will migrate up the plate. The following are some common uses of Thin-Layer Chromatography:

1.

To determine the number of components in a mixture.

2.

To determine the identity of two substances.

3.

To monitor the progress of a reaction.

4.

To determine the effectiveness of a purification.

5.

To determine the appropriate conditions for a column chromatographic separation.

6.

To monitor column chromatography.

In this experiment, you will use TLC to identify unknown analgesic painkillers using the table of analgesics and their
components in the experimental section of this experiment.

Apparatus

Experimental Procedure for TLC Analysis of Analgesic Drugs

(adapted from Fieser & Williamson, pp. 128-129)
Obtain 2 TLC plates. Draw a light pencil line about 1 cm from the end of each chromatographic plate. Spot one plate with
your 4 known standards (Acetaminophen, Aspirin, Caffeine, and Ibuprofen) and the other plate with the 5 unknown
commercial painkillers. Both plates should also have a Reference spot that contains all 4 standards. Use a separate
capillary tube for each standard and unknown solution. Make each spot as small as possible, preferably no more than 2-3
mm in diameter. Examine the plate under the ultraviolet (UV) light to see that enough of each compound has been
applied; if not, add more.
The standards and commercial painkillers will be dissolved in a 50/50 Ethanol/Ethyl Acetate solution.

Prepare a developing chamber as indicated in the picture using a large beaker as the chamber, a half-piece of filter paper
inside, and foil or plastic wrap to cover. Pour the eluting solvent, a 99/1 mixture of Ethyl Acetate/Glacial Acetic Acid, into
the beaker to a depth of approximately 1 cm. Place the prepared TLC plates in the developing chamber.
After the solvent has risen to near the top of the plate (about 1 cm from the top), remove the plate and mark the solvent
front with a pencil. Keep the plates in the hood until the majority of the eluting solvent has evaporated from the plates.
Examine the plate under UV light to see the components as dark spots against a bright green-blue background.

Outline the spots with a pencil and note anything distinctive about any of the compounds. The spots should also be
visualized by putting the plate in an iodine chamber. The iodine chamber is pre-made and contains a few crystals of iodine
in the bottom of a capped jar. More than 2 plates can be placed in the iodine chamber at one time. Remove the plates
when a definite change in appearence takes place on your plates. Note which compounds stained with iodine and to what
intensity. The iodine stains will dissipate over time.
Wrap your TLC plates in plastic wrap and scan them into your e-lab.
Calculate the Rf values for each spot. Unknowns can be identified using Rf values, fluorescence in UV light, changes due
to iodine exposure, the reference spot and Table 1.