Experiment 2 – Chem 276 – Fall Semester 2010 Page 1 of 5

Experiment 2 - Thin Layer Chromatography

Introduction
Chromatography is an experimental technique by which a mixture of compounds can be
separated into its individual components. In thin layer chromatography, there is a
stationary phase as well as a mobile phase. For this experiment, the TLC plate consists of
an aluminum backing on which is placed a thin layer of silica gel. The silica gel is the
stationary phase. The eluding solvent is the mobile phase which carries with it the
components of the mixture. Thin layer chromatography is used mostly for analytical
purposes. Microgram to picogram quantities can be separated by this technique and then
characterized by their Rf value.
The retention factor, or Rf, is defined as the distance traveled by the compound divided
by the distance traveled by the solvent.

distance traveled by compound

Rf =
distance traveled by solvent front

In the example shown below, a compound travels 16.2 cm while the solvent front travels
23.0 cm, the Rf is 0.704:

solvent front

new position
of compound

23.0 cm

16.2 cm

origin

16.2
R = = 0.704
f 23.0

The Rf for a compound is a constant from one experiment to the next only if the
chromatography conditions below are also constant:

 solvent system
 adsorbent
 thickness of the adsorbent
 amount of material spotted
 temperature
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Since these factors are difficult to keep constant from experiment to experiment, relative
Rf values are generally considered. “Relative Rf” means that the values are reported
relative to a standard, or it means that you compare the Rf values of compounds run on
the same plate at the same time.

The larger an Rf of a compound, the larger the distance it travels on the TLC plate. When
comparing two different compounds run under identical chromatography conditions, the
compound with the larger Rf is less polar because it interacts less strongly with the polar
adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in
a mixture, you can predict that a compound of low polarity will have a larger Rf value
than a polar compound run on the same plate.

The Rf can provide corroborative evidence as to the identity of a compound. If the

identity of a compound is suspected but not yet proven, an authentic sample of the
compound, or standard, is spotted and run on a TLC plate side by side (or on top of each
other) with the compound in question. If two substances have the same Rf value, they are
likely (but not necessarily) the same compound. If they have different Rf values, they are
definitely different compounds. Note that this identity check must be performed on a
single plate, because it is difficult to duplicate all the factors which influence Rf exactly
from experiment to experiment.
 Experiment 2 – Chem 276 – Fall Semester 2010 Page 3 of 5

Procedure

1. Cut six 1½ × 4 inch pieces of TLC plates.

2. Next you will need to draw a line 1 cm from the bottom of your TLC plates
with a pencil. See diagram in introduction.
3. To one plate, spot on the line with any two of the five compounds listed in
Table 1. The spots should be about 1 cm apart and on the line. To insure that
your plate is properly spotted use an UV light. Remember the identity of each
spot.
4. To another plate, spot on the line with any two remaining compounds listed in
Table 1. The spots should be about 1 cm apart on the line. To insure that
your plate is properly spotted use an UV light. Remember the identity of each
spot.
5. To final plate, spot on the line with final compound from Table 1 and the
unknown mixture. The spots should be about 1 cm apart on the line. To
insure that your plate is properly spotted use an UV light. Remember the
identity of each spot.
6. Prepare the following eluding solvents in separate 100 mL beakers.
A) Prepare a 1:1 mixture of ethyl acetate and hexanes. Do this by mixing 25
mL of ethyl acetate with 25 mL of hexanes.
B) Prepare a 1:4 mixture of ethyl acetate and hexanes. Do this by mixing 10
mL of ethyl acetate with 40 mL of hexanes.
7. Pour enough of the1:1 mixture of eluding solvent in three separate developing
chambers until the depth is approximately ½ cm. Be sure that the solvent
surface is below the application points. (i.e. The spots should not be in the
solvent.)
8. Place each TLC plate in a developing chamber containing the 1:1 mixture of
ethyl acetate and hexanes and allow the solvent to climb the plate until in
comes with in a ½ cm of the top of the plate. Once this occurs, remove the
plates from the chambers and mark the solvent fronts with a pencil.
9. Allow the TLC plates to dry.
10. Take the TLC plates to a dark room and with the use of a UV light circle the
illuminated spots with a pencil.
11. Stain your TLC plate by dipping it in a stain solution and drying over heat gun
as directed by your TA.
12. Calculate the Rf values for each of the spots.
13. Repeat steps 7 thru 12 with the 1:4 ethyl acetate and hexanes eluding solvent.
14. Dispose of used TLC plates and elution solvent as directed by your TA.

Table 1
Acetophenone
Anisole
Benzoic Acid
Benzyl Alcohol
Benzaldehyde
 Experiment 2 – Chem 276 – Fall Semester 2010 Page 4 of 5

Questions
1. Draw the structures of the following compounds.

Acetophenone

Anisole

Benzoic Acid

Benzyl Alcohol

Benzaldehyde

Compound Rf in 1:1 Rf in 1:4 Appearance in Appearance in

EtOAc/Hex EtOAc/Hex
___________ stain __________ stain
Acetophenone

Anisole

Benzoic Acid

Benzyl Alcohol

Benzaldehyde

2. Calculate the Rf values of each spot.

 Experiment 2 – Chem 276 – Fall Semester 2010 Page 5 of 5

3. What would occur if you had added too much sample to your TLC plate?

4. From your results, which compound is the least polar? Do the structures of these
compounds support your experimental date? Explain.

5. From your results, which eluding mixture is the most desirable? Explain.

6. From your Rf values, are all the compounds pure? If not, identify what the
impurities might be in the contaminated sample.

7. From your Rf values, what are the identities of the compounds in the unknown
sample?