Name: ____________________

Section: ___________________


Chemistry 115
TLC ANALYSIS OF ANALGESIC DRUGS

INTRODUCTION
In this experiment, thin-layer chromatography (TLC) will be used to determine the composition
of various over-the-counter analgesics. Chromatography is a technique used to separate and
identify individual components in a mixture. Chromatographic techniques make use of the fact
that components of a mixture tend to move at different speeds along a coated tube or flat surface.
The different rates of movement are the results of differing attractions of the components to the
coating material compared to the tendency of the components to remain in the moving fluid.

Thin-layer chromatography is one of the easiest of the many chromatographic techniques. A thin
layer of a suitable solid substance is coated on a sheet of glass or plastic. A very small sample of
the mixture to be analyzed is spotted onto the sheet. By immersing one edge of the sheet in an
appropriate liquid developing solvent, the solvent is drawn up the sheet by capillary action, and
the compounds of interest are carried along at different rates, effectively separating the
components. This is commonly called developing the plate. After the plate has been
developed, it is examined under ultraviolet (UV) light, which allows you to note the location of
the spots.

Experimental conditions in TLC include the solvent system used to dissolve the compounds, the
adsorbent coated onto the TLC plate, the thickness of the adsorbent layer, and the relative
amount of the material spotted onto the plate. Under an established set of experimental
conditions, a given compound always travels a fixed distance relative to the distance the solvent
front travels. This ratio of the distance the compound travels to the distance the solvent front
travels is called the R
f
value. The symbol R
f
stands for retardation factor, or ratio-to-front,
and it is expressed as a decimal fraction. The equation is shown below:

R
f
=
distance traveledbythe substance
distance traveled by the solvent front


When the conditions of measurement are completely specified, the R
f
value is constant for any
given compound, and it corresponds to a physical property of that compound.

In this experiment, each pair will be given two commercially prepared TLC plates with a flexible
backing and a silica gel coating with a fluorescent indicator. On these TLC plates, you will spot
solutions prepared from commercial analgesic tablets. The crushed tablets will be dissolved in an
ethyl acetate-ethanol-acetic acid solvent mixture (25 parts ethyl acetate-1 part ethanol-1 part
acetic acid). On each plate, you will also spot a standard reference mixture, which contains four
standard compounds often used in analgesic formulations dissolved in the same solvent mixture.
The standard compounds are:
Acetaminophen (Ac) Caffeine (Caf)
Aspirin (Asp) Salicylamide (Sal)
Revised 11/21/2005
1
TLC Analysis of Analgesic Drugs
In some analgesics, you may find ingredients besides those mentioned above. Some include an
antihistamine and some a mild sedative. For instance, Midol contains N-cinnamylephedrine
(cinnamedrine), an antihistamine, while Excedrin PM contains the sedative methapyrilene
hydrochloride. Cope contains the related sedative methapyrilene fumarate.

There is a choice of several developing solvents for this experiment, but ethyl acetate with 0.5%
glacial acetic acid is preferred. The small amount of glacial acetic acid suppresses ionization of
the aspirin, allowing it to travel upward on the plates. Without the acid, this compound does not
move.

After developing, you will observe your plates and a reference plate under illumination from a
short-wavelength UV lamp. See the example reference plate on page 7. The reference plate has
been spotted with the standard reference mixture along with solutions of each of the four
standard compounds dissolved individually in the ethyl acetate-ethanol-acetic acid solvent
mixture. The purpose of the reference plate is to observe the order of elution (R
f
values) of the
known substances and to index the standard reference mixture. The structural formulas of each
of these substances are shown below.
C
C
C
C
C
C
O
H
H
H
N
H
H
H
C
C
O
H
H
H
C
C
C
C
C
C
H
H
H
C
O
H
O
C C
O
H
H
H
O H
Acetaminophen
Aspirin
C
C
C
C
C
C
H
H
H
C
O
H
O
N H
H
H
Salicylamide
N
C
N
C
C
C
N
C
N
O
O
C H
H
H
C
H
H H
C
H
H
H
H Caffeine


You will use the UV lamp in a viewing chamber in the prep room. When using the UV lamp, you
must be careful not to operate the lamp outside of the viewing chamber, and always wear the
safety glasses provided. Under these conditions, some of the spots will appear as dark areas on
the plate, while others will fluoresce brightly. This difference in appearance under UV
illumination will help distinguish the substances from one another. On your plates, you will find
it convenient to outline very lightly in pencil the spots observed and to place a small x inside
those spots that fluoresce.

When observing the spots, take special care to notice that the salicylamide spot has a different
appearance when viewed under UV illumination. Aspirin presents some special problems
because it is present in a large amount in many of the analgesics and because it hydrolyzes
easily. For these reasons, the aspirin spots often show excessive tailing.
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TLC Analysis of Analgesic Drugs
Dispose of all waste in the container provided in the Waste Hood. The micropipettes used for
spotting the solutions should be placed in the container in the Waste Hood labeled for that
purpose. The TLC plates should be stapled to your lab report.

SAFETY PRECAUTIONS
Due to the use of organic solvents, gloves are required for this experiment. When using the UV
lamps, be sure to wear the UV safety glasses provided and only use the lamps inside the viewing
chambers. Wear departmentally approved eye protection at all times in the laboratory. Follow all
additional laboratory rules and regulations provided by your instructor. Know the location and
proper use of all laboratory safety equipment (safety showers, eye washers, fire extinguishers,
etc.). Dispose of all chemicals in the proper waste containers located in the Waste Hood.

A material safety data sheet (MSDS) for each chemical used in this experiment is located in a
binder in the lab. You should be familiar with the hazards associated with each chemical, as well
as the instructions on safe handling and appropriate disposal. Your instructor will be available to
assist you in interpreting this information.


EXPERIMENTAL PROCEDURE
Preparing the TLC Plates:
Obtain two 10 cm x 6.6 cm TLC plates from your instructor. These plates have a shiny flexible
backing but should not be bent excessively. Handle them carefully, and only by the edges, or the
adsorbent may flake off. Using a pencil (not a pen), on the non-shiny side, lightly draw a line
across the plate about 1 cm from the bottom (see figure below). When marking the plate, be sure
not to disrupt the adsorbent with your pencil. Using a centimeter ruler, start 1.5 cm from the edge
and lightly mark off three points 1.5 cm apart on the line. These are the points at which the
samples will be spotted.




Commercial Tablets
Sample Plates:













1 2 REF 3 4 REF
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TLC Analysis of Analgesic Drugs
Preparation of Analgesics:
Obtain half a tablet of four different analgesics. The experiment will be most interesting if you
make your choices to give a wide spectrum of results. Try to pick at least one analgesic each
containing aspirin, acetaminophen, and, if available, salicylamide. If you have a favorite
analgesic, you may wish to include it among your samples.

Take each analgesic half-tablet, place it on a smooth piece of notebook paper, and crush it well
with the curved edge of the padlock from your lab drawer. Transfer each crushed half-tablet to a
DRY, labeled 50-mL beaker. Using a graduated cylinder, add 5 mL of the Solution Solvent,
which is an ethyl acetate-ethanol-acetic acid mixture, to each of the crushed half tablets, and
swirl to mix. Work in your bench-top hood when working with the solvent mixture. Then heat
each of them gently for a few minutes on a hot plate set at low. Not all of the tablet will dissolve
because the analgesics usually contain an insoluble binder. In addition many contain inorganic
buffering agents or coatings that are insoluble in this solvent mixture. After heating the samples,
swirl again to mix and then allow the insoluble particles to settle.

Spotting the TLC Plates:
Obtain four capillary micropipettes to spot the plates with the four solutions of analgesic you
prepared above. The correct method of spotting a TLC plate will be demonstrated by your
instructor. It is important that the spots be spaced properly as described above and that they be
made as small as possible by briefly touching the tip of the micropipette very lightly to the plate.
If these cautions are disregarded, the spots will tail and will overlap one another after
development. The applied spot should be about 1-2 mm (1/16 in) in diameter. If scrap pieces are
available, it would be a good idea to practice spotting on these before preparing the actual
sample plates.

After the insoluble material has settled from your analgesic solutions, spot the clear liquid
extracts from two analgesic tablets on each of your TLC plates using a separate micropipette for
each solution. At the third position on each plate, spot the standard reference mixture using the
micropipette provided at the station in the prep room. The standard reference mixture contains
acetaminophen, aspirin, caffeine and salicylamide, and is provided as a solution of 1 gram of
each (0.5 g aspirin) dissolved in 20 mL of the ethyl acetate-ethanol-acetic acid solvent mixture.

Developing the TLC Plates:
After spotting your plates, obtain a 16-oz wide-mouthed screw-cap jar for use as a development
chamber; make sure it is DRY. Using a graduated cylinder, measure 10-15 mL of Development
Solvent (0.5% glacial acetic acid in ethyl acetate) and pour it into the jar. Recall that the solvent
level must not be above the spots on the plate or the samples will dissolve off the plate into the
reservoir instead of developing. Place the spotted plates in the chamber, back-to-back with the
spots facing out, secure the lid, and allow the plates to develop without disturbing the jar.

When the solvent front has risen to a level about 0.5 cm from the top of the plates, remove the
plates from the jar (in your hood) and, using a pencil, mark the position of each solvent front. Set
the plates on a piece of paper towel in your bench-top hood to dry. It may be helpful to place a
small object under one end to allow optimum air flow around the drying plates. Dump the
Developing Solvent into the waste container and return the jar to the counter to air dry. Do not
rinse the developing jar with water as the presence of water hurts the developing process.
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TLC Analysis of Analgesic Drugs
Observing the TLC Sample & Reference Plates:
You will need to observe both of your sample plates and the reference plate provided for you
under a short-wavelength UV lamp. The reference plate can be viewed while you are waiting for
your sample plates to develop and/or dry, and the first sample plate can be observed while the
other is developing. Be sure not to use the UV lamp outside of the box and wear the safety
glasses at all times while viewing.

Observe the reference plate under the UV lamp. This developed plate has been spotted with
solutions of acetaminophen, aspirin, caffeine, and salicylamide, and finally the reference solution
in the last position. (The spotting has been done in alphabetical order from left to right.)

Carefully notice any differences in behavior between salicylamide and the other substances.
Before proceeding, make a sketch of the plate in the space provided on below and note the
differences in appearance that you observed.

Observe your sample plates under UV illumination and lightly outline the visible spots with a
pencil. In the space provided below, sketch each plate.

Using your ruler, measure, in millimeters, the distance traveled by each spot on both sample
plates as well as the distance traveled by the solvent front. Use these measurements to calculate
R
f
values for each spot. With the R
f
values and the information taken from the reference plates,
you will be able to identify the contents of each tablet. Complete the data tables below.



RESULTS

1. Provide sketches of the TLC plates. Label each spot by identifying the substance.
Reference Plate Sample Plate Sample Plate














AC ASP CF SAL REF 1 2 REF 3 4 REF


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TLC Analysis of Analgesic Drugs
2. Based on your experimental results, list the substances found in each of the analgesic drugs
you analyzed. Record the R
f
values calculated for each substance found in each tablet.

Sample
Number
Drug Name Analgesics Found R
f
Values
1
2
3
4


3. Summarize your findings for each of the standard compounds in the table below. Calculate
the R
f
values from the reference mixture spots on both sample plates and report the average
R
f
value in the table.
ANALGESIC DRUG R
f
VALUE
BEHAVIOR WITH
UV LIGHT
Acetaminophen
Aspirin
Caffeine
Salicylamide


4. Calculate the R
f
value of a spot that travels 5.7 cm, while the solvent front travels 13 cm.
Show your calculations.

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TLC Analysis of Analgesic Drugs
Example TLC Reference Plate

The standard solutions are spotted at the marks along the dotted line at the bottom of the plate.
As the plate is developing, the Development Solvent carries the standard compounds (Aspirin,
Caffeine, etc.) up the plate. The properties of the compounds and the experimental conditions
determine the rate at which each compound is carried up the plate, which means the compounds
have different rates and are therefore separated as they travel up the plate. The figure below
shows what the developed reference plate will look like under a UV lamp.

The retention or retardation factor (R
f
) is the distance traveled by an analyte relative to the
distance traveled by the solvent front. Below is a sketch of a reference plate that indicates how to
measure the specified distances. Note: It is essential for you to use a pencil to mark your TLC
plate as ink will smear on the plate.


R
f
=
distance traveledbythe substance
distance traveled by the solvent front





Solvent Front
S
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F
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D
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A
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A
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C
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S
a
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A
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Standard
Distances
Fluorescent Spot

























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