CHEM 211-2008
4
Experiment 5
How Can an
Impure Solid
Substance Be Purified
and How Can Purity Be
Measured?
TLC CHROMATOGRAPHY
G. General Overview of the Experiment:
 Group members collaborate to choose various mixtures of hexane and dichloromethane
developing solvents to test for separating fluorene from the impurities in the crude
preparation that is available.
 Each group member does a TLC analysis of the crude fluorene preparation in a different
solvent system.
 Each group chooses its best solvent mixture for separating fluorene from its impurities.
 Each group member separately purifies a sample of the crude fluorene preparation using
the group’s chosen solvent mixture, recovers the purified fluorene, analyzes the purity of
the recovered fluorene by TLC and calculates the percent recovery fluorene from the
chromatography.
 Group members compare results and present the effectiveness and reproducibility of
purifying fluorene from the crude preparation with their solvent system.
H. Procedure:
Thin Layer chromatography
As we discussed in the pre-lab session, you will first use thin layer chromatography (TLC.) to
estimate the degree of impurity of the fluorene and select the best solvent or solvent system to
be used in a column chromatography separation to obtain purified fluorene.






Group members collaborate to decide on appropriate solvent mixtures to use for assessing
the effectiveness of their solvent system in separating fluorene from its impurities and
then consult with the instructor on their choices.
Manager: Assign a solvent or solvent mixture to each group member.
Each group member: Using acetone as solvent (why???), prepare ~ 0.2-0.3 ml of a dilute
solution of crude fluorene and pure fluorene in separate small test tubes.
Your instructor will have demonstrated the proper technique for spotting plates. Practice
spotting the solutions on scrap TLC plates.
Each group member: Prepare a developing chamber using assigned solvent or solvent
system.
Obtain a good TLC plate from your instructor. Note that the plates should be handled
only along the edges and that the surface should not be touched.
Chromatography
Figure 1
5
Figure 2
CHEM 211 Experiment 5
Figure 3
Figure 4
Figure 1 depicts a TLC plate, which has been marked at the bottom with a pencil to
show the location at which the sample will be applied (point of origin) and where
development begins and at the top where solvent movement has ended.
Figure 2 depicts that same slide under a UV lamp at 254 nanometers.
Figure 3 depicts a slide that has been spotted and viewed under a UV lamp at 254
nanometers.
Figure 4 depicts the same slide that has been developed in the appropriate solvent and
is now viewed under the UV lamp. It shows one spot suggesting that only one
component is present in the sample analyzed.




By means of a ruler and pencil, LIGHTLY draw a horizontal line across the plate at about
1 cm from the bottom. (See Figure 2 above)
Along this line and about 0.3 cm from the edge, mark off two "columns" at 1-cm
intervals. These will be the locations at which you will spot your samples.
On each of the three plates, spot the crude fluorene in the first column and the pure
fluorene in the second column. (See Figure 5). Your instructor will have demonstrated the
procedure.
View the slide under a UV lamp before development to check spots.
Figure 5

By means of tweezers, place the spotted TLC plate into the chamber, cap the chamber,
leave it in a fixed position and allow the slide to develop to 0.5 cm from the top. (top line
on the plate)
CHEM 211 Experiment 5



6
Chromatography
By means of tweezers, remove the developed slide from the chamber and mark the actual
position of the solvent front by means of a pencil.
Examine the dried slides under a short-wave (254 nm) UV lamp; lightly circle the
observed spots with a pencil.
Collaborate with your group to determine the group’s the best solvent or solvent system
for separation of the crude fluorene (Figure 6). If there is a question run additional plate
to determine your optimal solvent system.
Figure 6
2. Column chromatography:
The chromatography column will be constructed from a 5.75 inch Pasteur pipet. Your
instructor will demonstrate the preparation of the column as described below.
a. Chromatography column preparation
 Clamp the "column" (Pasteur pipet) to a ring stand in a vertical position.
 With the aid of a 9 inch Pasteur pipet, place a very small wad of cotton through the
pipet into the tip. DO NOT JAM IT TOO TIGHTLY!
 To the plugged pipet add approximately 100 mg of sea sand.
 Tap the pipet (the column) lightly to level the sand surface.
 Add approximately 700 mg silica gel (60-100 mesh) to the column so that is sits
above the sea sand.
 Add a second sample of approximately 100 mg sea sand.
 Place a 25 mL Erlenmeyer flask that has been labeled (your name, lab section, and
date) and then pre-weighed (tared) beneath the column as a receiver for the eluent.
Make sure that the tip of the column is inserted into the neck of the labeled, preweighed flask.
b. Purification procedure:











Place approximately 100 mg of technical grade fluorene into a small test tube.
Add 2 mL of chromatography solvent to the test tube and gently swirl the mixture to
obtain solution.
By means of a Pasteur pipet transfer the amber solution to the prepared
chromatography column being careful not to overflow the column.
Collect the eluent in the labeled, preweighed 25 mL Erlenmeyer flask.
Rinse the test tube with three 1-mL portions (3 x 1 mL) of chromatography solvent
and add each rinse to the column.
Observe the appearance of the column as the chromatography progresses.
Recall your TLC analyses and continue adding solvent to the column.
Continue collecting eluent until just before the impurity band reaches the bottom of
the column.
Switch collecting flasks and collect the impurity band as a second fraction.
Evaporate the eluted solvent by clamping the 25 mL receiving flasks in a hot water
bath in a hood.
When all solvent has been evaporated, remove the flask from the hot water bath,
allow the flask to cool to room temperature and obtain the masses of the flasks and
calculate the masses of dry recovered fluorene and the impurity. Calculate the percent
recovery of fluorene, total recovery of fluorene and impurity and total percent
recovery of the original sample.
Chromatography
7
CHEM 211 Experiment 5
c. TLC Analysis of the Purified fluorene:
 Analyze your purified fluorene and impurity by TLC using your group’s most
effective separating solvent system as the TLC solvent on the same plate with the
standard “pure fluorene”.
 Collaborate with your group mates to determine the effectiveness and reproducibility
of the purification of fluorene and total recovery with your group’s solvent system.
You should consider both percent recovery and purity of your recovered fluorene.