THIN – LAYER CHROMATOGRAPHY
Purpose:
The purpose of this lab is to gain familiarization with TLC procedures and to identify an
unknown analgesic using a set of standard analgesics.
Discussion:
 What is thin-layer chromatography? How does it work?
 How are white or colorless compounds identified on a TLC plate?
 What is the Rf value? How is it calculated?
 How do impurities affect the Rf value?
 How does the data help interpret the purity of the compound?
Objectives:
In this lab, you will...
1. gain familiarization with TLC procedures.
2. use TLC to identify an unknown analgesic by quantitatively comparing it to
known standard analgesics using Rf values.
3. Use TLC to evaluate the purity of the caffeine extracted in a previous lab.
4. Use TLC to evaluate the purity of the aspirin synthesized in a previous lab.
Safety:
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Wear safety goggles at all times during the lab.
Ethanol and ethyl acetate are flammable. Do not use any open flames.
Do not look directly into the UV lamp. It will damage your vision.
Ethyl Acetate and Acetic Acid are corrosive and irritating. Keep them in the fume
hood and handle with care.
Materials:
4 Chromatography Plates (TLC Plates)
Graphite Pencil
8 Small Test Tubes
250 mL Beaker
Watch Glass
100 mL Graduated Cylinder
8 Toothpicks
UV Lamp
95% Ethanol
Ethyl Acetate
Glacial Acetic Acid
Standard Analgesics
A1 – Aspirin
A2 – Acetaminophen
A3 – Caffeine
A4 – Ibuprofen
A5 – Sodium Naproxen
Unknown Analgesic
Your caffeine sample
Your aspirin sample
Procedure:
1. Choose an unknown. Make sure every group has as different unknown.
2. Place a very small amount (< 0.01 g) of each standard, the unknown and your caffeine
and aspirin into separate small test tubes. Add 5 – 10 drops of 95% ethanol to dissolve
the solids. Much will remain undissolved, but enough of the analgesic will dissolve to
make a spot on the TLC plate.
3. Draw a light pencil line 2.00 cm from the end of a chromatographic plate. Measure this
distance carefully. Due to the thinness of the TLC plates, place only two samples on each
plate.
a. Plate 1 – A1 and your aspirin
b. Plate 2 – A3 and your caffeine
c. Plate 3 – A2 and A4
d. Plate 4 – A5 and your unknown
4. Dip a toothpick into one of the sample test tubes and allow the liquid to soak into the
wood. Remove the toothpick from the test tube and place a small, tight drop of the
sample along the pencil line drawn on the TLC plate. Repeat with the other samples, and
be sure to evenly space out the samples. Place your spotted TLC plate under the UV
lamp to make sure you can see the spots.
5. Repeat step 4 once the spots have dried. Place the second spots directly on top of the first
spots.
6. The two chemicals in this step should be kept in the fume hood. Using a 50 mL
graduated cylinder, measure out and pour 19.0 mL of ethyl acetate into a 250 mL beaker.
Using a 10 mL graduated cylinder, add 1.00 mL of glacial acetic acid to the 250 mL
beaker containing the ethyl acetate. This is your solvent.
7. Carefully place your spotted chromatography plate into the solvent and cover with a
watch glass. Do not disturb the developing process. Allow the plate to “develop.” The
solvent should pass over the plate and over the samples as it moves up the plate by
capillary action.
8. When the solvent front has risen to ~1 cm from the top of the plate, remove the plate
from the developing chamber. Watch carefully as the solvent stops moving up the plate.
Once it stops moving, lightly mark the solvent front position with a pencil before it
evaporates. Allow the plate to dry.
9. Examine the plate under a UV light. Mark the spots that you can see by lightly circling
them with a pencil. Measure and record the distances the spots traveled from the 2.00 cm
line to the center of the spots. Measure and record the distance the solvent front traveled.
Each plate should yield two sample distances and two solvent distances. The solvent
front needs to be measured at a point directly above the center of the sample spots.
Cleanup:
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Data:
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Pour the remaining solvent into the organic waste container in the fume hood.
Place the used chromatographic plates in the garbage can.
Clean all glassware used in the lab.
Tabulate the distances each spot moved and the distance the solvent front moved for
each sample.
Analysis/Calculations:
 Calculate the Rf values of each standard analgesic, your caffeine and aspirin, and the
unknown.
 Identify your unknown and assess its purity.
 Assess the purity of your caffeine and your aspirin.
 Calculate the Ea and E% based on the unknown Rf and the Rf calculated for the true
analgesic.