Chemistry 1021 North Park University
Thin Layer Chromatography of Analgesics
Reference: Adapted from Identifying an Unknown Analgesic by Melting Point and Thin Layer
Chromatography, Organic Chemistry by Vernier, Vernier Software and Technology
Introduction
Thin-layer chromatography (TLC) is an important technique in organic chemistry. TLC uses the different
affinity a compound has between the mobile (liquid) and stationary (solid)phases to achieve separation of
mixtures of organic compounds. TLC can also be used to identify compounds by comparison with known
samples, check the relative purity of a compound, and monitor the progress of a reaction.
In TLC, the stationary phase is the adsorbent (usually silica or alumina) coated on a sheet of glass, metal,
or plastic. The sample is applied as a spot near the bottom of the plate. The TLC plate is then placed in a
developing chamber containing a shallow layer of mobile phase where the mobile phase (liquid solvent)
slowly rises up the solid sheet by capillary action. In our experiment, the chamber is a large beaker.
Under a given set of conditions, a specific compound will travel a fixed unique distance relative to the
solvent front. Different compounds generally move at different rates based on their attraction to the
absorbent on the plate versus their attraction to the solvent. As a result, if the sample is a mixture of
compounds it will separate into a series of spots at varying distances up the plate (see Figure 1). If the
sample is pure, then only one spot will result. Often the spots are not visible to our eyes in regular light.
A UV light source is used to visualize the spots on the TLC plate. Under UV light, the chemical deposits
will appear as dark spots against a bright background.
TLC separation results are expressed in terms of R f (retention factor) values. The R f is a ratio calculated
by dividing the distance traveled by the sample by the distance traveled by the solvent at the end of the
experiment.
Figure 1. Example TLC plate at the end of the experiment.In this experiment, TLC will be used to identify the active ingredients in an unknown over-the-counter
medicine tablet. The tablet you will test contains one or more of the following: acetylsalicylic acid
(aspirin), acetaminophen and caffeine. The structures of these compounds are shown in Figure 2.
Acetylsalicylic acid Acetaminophen Caffeine
(aspirin)
Figure 2. Structures of analgesics
Procedure
1. Obtain a pea sized amount of your unknown analgesic sample and transfer to a 50 mL beaker.
Add about 5 mL of ethyl acetate to your sample and use your glass stir rod to break up any
pieces. The sample will not fully dissolve.
2. Obtain one TLC plate. Handle the plate carefully and by the edges so that the adsorbent does not
flake off. Following directions a - e for the preparation of your TLC plate:
a. Using a pencil (NOT an ink pen) and a ruler, lightly draw a line across the plate, approximately 1-
2 cm from the bottom. Across this line, evenly mark four places indicating the location where the
sample will be spotted, making sure they are not too close to the edge of the plate (see Figure 1).
b. Under each mark, lightly label each spot starting left to right as Ac, As, C, and U for
acetaminophen (Ac), aspirin (As), caffeine (C) and the unknown (U).
c. Take a spotting tube and dip one end into the solution containing acetaminophen. Capillary action
will draw the liquid into the tube.
d. Lightly tap the tube on the line for acetaminophen (Ac) on the TLC plate. Only a small amount of
sample needs to be delivered. The spot should be 1-2 mm in diameter.
e. Repeat Steps d-e for the aspirin, caffeine, and unknown sample.
3. Prepare the TLC chamber by pouring enough 1:1 ethyl acetate/hexanes solution into a 250 mL
beaker to coat the bottom of the beaker. Add 1 drop of acetic acid to the beaker. Cover the top of
the beaker with a watch glass.
4. Place the TLC plates in the chamber and cover with the watch glass. The solvent level must not
be above the spots on the plate or your samples will dissolve into the solvent.
5. When the solvent has risen to within 1 cm from the top of the plates, remove the plates from the
chamber and with a pencil, gently draw a line to mark the position of the final solvent front.
6. After the plate has dried, observe the TLC plates under a UV lamp. Lightly outline the spots with
the pencil and use this to calculate R f for each plate.

OOH
O
CH3
O
N
N
O
O
CH3
CH3 N
N
CH3
OH
NH
O
CH3Chemistry 1021 North Park University
Thin Layer Chromatography of Analgesics Report
Name:
Lab Partner(s):
Lab Time (circle): 8:00 am 9:50 am 12:40 pm 2:30 pm
Experimental Data
Part I Thin-Layer Chromatography
Your TLC plate
Plate 1: Calculated R f
Another group’s TLC plate
Plate 2: Calculated R f
Acetaminophen
acetylsalicylic acid
Caffeine
Unknown (R f for each spot!)
Data Analysis and Discussion
1. Draw a picture of your TLC plate.
2. Compare your plate (Plate 1 in your data table) with a group near you. Record their R f values in
your data table above (Plate 2). Did both plates have the same number of spots? If you had
different results for your two TLC plates, discuss what may have happened.3. Based on the R f values of the unknown, identify your unknown analgesic. Be sure to use
evidence from your known samples to back up your conclusion (compare values of the standards
to that of the unknown).
4. Why did the components in your unknown separate the way they did? Which of your standards
travelled the furthest and why? Which travelled the least distance and why?