Experiment #5: TLC of Analgesics
Chemistry 102
Background:
In this experiment, thin-layer chromatography (TLC) will be used to determine
the composition of various over-the-counter analgesics. For this task you will be given
two TLC plates (the same type of plates as in the spinach lab). On one TLC plate, you
will spot four standard compounds contained in analgesics. In addition, a standard
‘reference’ mixture containing all four of these same compounds will be spotted. The
purpose of this TLC plate is to determine the order of elution (Rf values) of the known
substances and to index the standard reference mixture. The reference compounds are:
Acetaminophen, Aspirin, Caffeine and Ibuprofen. The structures are at the bottom of
the page.
On the second TLC plate you will spot solutions of 2 different ‘unknown’
commercial analgesic tablets along with the reference mixture. After this TLC plate is
developed you will compare the Rf values of the unknown analgesics to those of the
reference mixture and from this information you will be able to determine the unknown
analgesics’ identity. Keep in mind that some analgesics contain more than one of the
substances.
In the spinach lab all the spots were visible to the naked eye. The substances
that we are detecting are colorless compounds and cannot been seen with the naked
eye. In order to visualize the spots we will view them under ultra violet (UV) light.
Under UV light, some of the spots will appear as dark areas on the plate, while others
will fluoresce brightly. This difference in appearance under UV illumination, along
with the distances the spots travel (Rf value), will help to distinguish the substances
from one another.
O
H
N
OH
O
O
HO
O
Aspirin (acetylsalicylic acid)
Acetaminophen
O
OH
O
Ibuprofen
N
N
O
N
N
Caffeine
Materials:
Acetaminophen, aspirin, caffeine, ibuprofen, standard reference mixture, 2
unknown over-the-counter analgesic tablets, 0.5% glacial acetic acid in ethyl acetate,
absolute ethanol, methylene chloride, TLC chamber, 2 TLC plates (3.5 cm), micro
capillaries, UV-lamp, ruler, 2 test tubes, spatula, hot plate, small beaker, pencil
Safety Precautions:
Dichloromethane is not flammable, nor is it considered carcinogenic, but it can
cause liver damage if ingested, or with repeated inhalation or skin exposures. It should
be used in the fume hoods (inhalation of vapors can cause nausea or drowsiness).
Avoid spills; it can also pass through both latex and nitrile gloves. Do not look directly
at the UV lamp, the UV light can damage your eyes.
Procedures:
TLC of reference compounds:
1. Obtain two TLC plates and micro capillaries. Using a lead pencil (not a pen) lightly
draw a line across the plates (short dimension) about 1 cm from the bottom. On the first
plate, using a centimeter ruler, start about 0.5 cm in from the edge of the plate and
lightly mark off five 0.5-cm intervals on the line. These are the points at which the
samples will be spotted.
2. Starting from left to right spot first acetaminophen, then aspirin, caffeine and
ibuprofen. This order is alphabetic so you can’t forget where you spotted what. The
standard reference mixture is spotted in the last position. Only a small amount of
material is needed. It is important that the spots be made as small as possible and that
the plates not be overloaded. (Note: in the spinach lab we spotted 2-3 times—in this lab
the solutions are highly concentrated so only a small amount need be added). If these
cautions are disregarded, the spots will tail and will overlap one another during
development. The applied spot should be about 1-2 mm in diameter.
3. Place the spotted plate in a development chamber. The development chamber
should be filled with the development solvent of 0.5% glacial acetic acid in ethyl acetate.
Fill the chamber with the development solvent to a depth of about 1/4 inch. Place the
spotted plate in the chamber, put the top on, and allow the plate to develop.
4. When the solvent has risen to a level about 1 cm from the top of the plate, remove
the plate from the chamber and, using a lead pencil, mark the position of the solvent
front. Let the plate dry. When the plate is dry, observe it under a short-wavelength UV
lamp. Lightly outline all the observed spots with a pencil. Before proceeding, make a
sketch of the plate in your notebook and note the differences in appearance that you
observed. Measure the distance that each spot has traveled relative to the solvent front.
Calculate the Rf values for each spot and record next to your sketch. By matching the
Rf values from your reference compounds to those of the standard reference mixture,
determine the identity of each spot in the standard reference mixture.
Analysis of two unknown analgesics:
1. Next, obtain half a tablet of each of the two analgesics to be analyzed. Place the
tablet on a piece weigh paper (fold the paper diagonally first, then unfold it, so that you
can easily pour the powder into the test tube after crushing), and crush it well with a
spatula. Transfer each crushed half-tablet to a small, labeled test tube.
2. Using a 10 mL graduated cylinder, measure out 5 mL of absolute ethanol and 5 mL
of methylene chloride and mix the solution well in a small beaker. Add 5 mL of this
solvent to each of the crushed half-tablets and then heat each of them gently for a few
minutes in a warm water bath. Not all the tablet will dissolve, since the analgesics
usually contain an insoluble binder. In addition, many of them contain inorganic
buffering agents or coatings that are insoluble in this solvent mixture.
3. After heating the samples, allow them to settle and then spot the clear liquid extracts
on the second plate. Using a centimeter ruler, start about 0.5 cm in from the edge of the
plate and lightly mark off 3 1-cm intervals on the line. Spot the unknown analgesics at
positions 1 and 2. At the third position spot the standard reference solution. Develop
the plate in 0.5% glacial acetic acid-ethyl acetate as before.
4. Observe the plate under UV illumination and mark the visible spots as you did for
the first plate. Sketch the plate in your notebook. Note the differences in appearance
and measure the distance traveled by each spot relative to the solvent line. Calculate
the Rf values for each spot and record next to your sketch. Match the R f values of the
unknowns to those of the standard mixture and record your conclusions about the
contents and identity of each tablet.
Waste Disposal:
Place all waste into the organic waste container. Put the used capillary tubes into
the glass waste container (do not put them into the regular trash!).
Pre-Lab:
1. Prepare your notebook as described in the lab notebook handout. Include: name,
partner’s name, title of experiment, date, purpose, structures of all reference
compounds, procedures and tables for your data and observations for each of the two
TLC experiments.
2. Answer the following pre-lab questions:
a) Rank the four reference compounds by polarity (from least to most polar):
b) Predict the relative Rf values for the reference compounds (from lowest to highest):
Report:
1. In your notebook, write a brief discussion of the experiment. In this section you
should summarize your results (identity of unknowns and results that back up those
choices), note any interesting observations and make any possible conclusions about the
experiment (successful vs. unsuccessful and reasons why). Be sure to discuss the Rf
values of the reference compounds vs. their relative polarities (did your results match
your pre-lab predictions?). Also be sure to include a comparison of the R f values of the
standards to those of the unknowns as part of your discussion.
2. Answer the post-lab questions.
3. Make a copy of your lab notebook pages (do not tear out the originals) and attach the
question sheet with your answers. Staple all pages and turn it in.
Post-Lab Questions:
1. Why did we use 0.5% glacial acetic acid in ethyl acetate instead of 70:30
hexane/acetone for the TLC development solvent?
2. If a less polar TLC development solvent was used, how would that affect the R f
values of the reference compounds?