CHEM 211-2007
Experiment 6
How Can an
Impure Solid
Substance Be Purified
and How Can Purity Be
Measured?
TLC CHROMATOGRAPHY
A. Pre-lab Preparation Assignment:
1. Read: This handout
2. Read: Thin-layer chromatography (Lehman Op 19: pp. 104-109)
Column chromatography (Lehman Op 18: pp. 93-102 we will use the Microscale
approach.)
3. Work through the material and links on the Experiment 6 page of the course website.
4. Complete the Prelab Questions linked to the Experiment 6 page on the course website by
5:00 PM, Wednesday, October 10.
5. Consider possible responses for the QOD (See below)
6. In your lab notebook: (See Lab Manual pp. 19-21 for format.)
a. Enter this experiment into the Table of Contents with the initial page number.
b. Enter the title of this experiment, your group mates’ names (See the group
assignments for Experiment 6 on the Lab Section Lists page on the course website)
and the QOD on the appropriate first left-hand page.
c. Write the procedure for the experiment.
7. Bring your notebook and your ideas to your lab section meeting October 11 – 16 and be
prepared to respond to the Question of The Day.
B. Objectives:
1. To master column and thin layer chromatography techniques for separating and analyzing
solid organic samples.
2. To discover a logical approach for selecting a solvent system for purification of a solid
compound from a crude sample.
C. Question of the Day:
How is an appropriate solvent chosen for column
chromatographic purification of a crude sample of a solid
compound?
D. Key Terms/concepts/techniques:
Column chromatography:
 Chromatography
 Distribution and partition
 Adsorption (and adsorptivity) vs. desorption
 Solubility
 Polarity: polar vs. non-polar
 Intermolecular forces: London Dispersion forces vs. dipole-dipole interactions
 Eluates vs. eluents
CHEM 211 Experiment 6
2
Chromatography
Thin Layer chromatography
 Ascending vs. descending chromatography
 Capillary action
 Application vs. development vs. visualization vs. identification
 Rf values
E. Introduction:
In the experiments over the last few weeks, we have developed an understanding of the
relationships between molecular structure and intermolecular forces. In Experiment 6 we
apply this understanding to develop chromatographic methods for purifying and analyzing
organic compounds.
1. Chromatography:
Chromatography is defined as the separation of a mixture of two or more different
compounds or ions by distribution between two phases, one of which is stationary and the
other moving. Various types of chromatography are possible, depending on the nature of
the two phases involved. Thus there is
 solid-liquid (column, thin layer or tlc, paper)
 liquid-liquid (liquid chromatography or LC), and
 gas-liquid (gas phase or GC) chromatographic methods (as we saw in Experiment 3).
All chromatographic methods work on the same principle, the differential adsorptivities
of the substances to be separated relative to the two phases. This experiment deals with
column chromatography, which is based on adsorptivity and solubility.
In solid chromatography, the stationary phase is a solid that may be any substance that is
not soluble in the associated liquid phase. Solids commonly used in chromatography are
alumina (Al2O3), and silica (SiO2); both of these compounds are polar. In this experiment
the solid used will be finely divided silica, often called silica gel.
If finely divided silica (or alumina) is added to a solution containing an organic
compound, some of the organic compound will adsorb onto the surface of the fine
particles of silica. This adsorption occurs because of the intermolecular forces created
between the silica and the organic molecules. If the organic molecules are non-polar, the
forces will be weak (London Dispersion forces). Since London forces arise from
electrons in non-polar covalent bonds, non-polar molecules will not adsorb well on the
surfaces of polar silica gel particles unless the organic molecule has many bonds (and
thus must be of high molecular weight). If the molecule is polar, the attractive forces
between the molecule and silica gel particles can be strong (dipole-dipole interactions).
The more polar the compound, the more strongly will it adsorbed onto the silica (or
alumina) surface.
The principle governing solubility is related to that which determines adsorptivities. A
solid substance has a high degree of order because of the presence of many and/or strong
intermolecular forces. When a solid melts the order is destroyed or reduced because
sufficient energy, in the form of heat, has been added to overcome the intermolecular
forces. Similarly, when a solid dissolves, the solvent molecules surround the solute
molecules in the lattice and form solute-solvent interactions that are more stable and/or
more numerous than the forces that are holding the solute in the crystalline lattice. Polar
solvent molecules form good interactions with polar solute molecules; non-polar solvent
molecules form good interactions with non-polar solute molecules. (Refer to the
discussions arising from Experiment 4 on solubility).
In chromatography, the principles of adsorptivity and solubility are important. Solutes
adsorbed onto a solid support (silica or alumina) can be removed by a solvent if the
solvent has a higher affinity for the solute than does the solid support. Initially, the
substance(s) to be separated....referred to as the eluates or elutants ...are adsorbed onto
the surface of the silica or alumina. When solvent...referred to as eluent ...comes in
contact with the adsorbed material, it may form better interactions with the adsorbed
material (eluate), which is removed from the solid support (is desorbed). The process of
adsorption and desorption is a dynamic equilibrium.
Melting Points of Mixtures
3
CHEM 211 Experiment 5
Column chromatography is usually used as a method to achieve quantitative separation
and isolation of the components of a mixture. Thin layer chromatography is most often
used to provide qualitative separation and identification.
2. Column chromatography:
In column chromatography, gravity causes the eluent to pass through a vertical column
packed with silica or alumina onto which has been adsorbed the substance(s) to be
separated. Through a series of desorptions and adsorptions there is preferential separation
based on relative affinities of the eluates for the silica and solvent. THE MORE POLAR
ELUATE HAS A HIGHER AFFINITY FOR THE SILICA AND MOVES DOWN THE
COLUMN MORE SLOWLY THAN DOES A LESS POLAR ELUATE. By gradually
increasing the polarity of the solvent (eluent) all the eluates can be sequentially removed
from the polar stationary phase.
The challenge in column chromatography is to find the solvent or solvent system of
the appropriate polarity to achieve maximum separation.
3. Thin Layer chromatography
Thin layer chromatography (TLC) is an example of ascending chromatography whereas
column chromatography is an example of descending chromatography. The principles
behind both separation methods, however, are identical.
In TLC, a liquid phase travels, by capillary action, up a thin film of solid adsorbent.
Solutes dissolved in the liquid phase will become preferentially adsorbed onto the solid
phase (usually silica gel or alumina). The degree of adsorption will be determined by the
polarity of the solute and the polarity of the solvent. As indicated in the discussion on
column chromatography, polar compounds tend to be more firmly adsorbed onto silica
gel and alumina and thus travel more slowly. Conversely, non-polar solutes have less
affinity for the adsorbent and travel more quickly.
A plate containing a thin and uniform film of silica gel or alumina can be spotted with a
concentrated solution of the substance to be analyzed. The location at which the spot is
applied is noted by drawing a horizontal line across the plate several centimeters from the
bottom of the plate. The application of the solute is critical. The material must be applied
in the minimum size spot. The plate is placed in a covered vessel containing the eluting
solvent. The depth of the solvent must be such that it does not reach the pencil line on
which the material to be analyzed has been spotted. By capillary action, solvent will
travel up the plate. This development is allowed to continue until the solvent is about a
centimeter from the top of the plate. The location of this point is marked by means of a
horizontal pencil line . The critical distance, that over which separation occurs, is that
between the two pencil marks.
The location of the solute(s) on the plate can be determined by several visualization
techniques. The plates used in this experiment have been coated with a fluorescent
material, which imparts a green fluorescence to the plates when they are placed under a
254 nm ultraviolet (UV) lamp. When a substance is adsorbed onto the silica coated with
the fluorescent material, the fluorescence is quenched and a black or blue spot is seen. An
alternative method is to place the plates into a glass chamber containing elemental iodine.
Iodine vapors react with the solutes adsorbed onto the plate to produce complexes which
are brown or yellow in color. In this experiment you will perform both visualization
techniques. Identification of the solutes is usually achieved by comparing their TLC
behavior (location and visualization characteristics) with that of a known substance. The
location of the solute is represented by the Rf value, which is the ratio of the distance
traveled by the solute on the plate to the distance traveled by the solvent. This Rf value is
a constant for a solute in a given solvent system and on a specified adsorbent system.
CHEM 211 Experiment 6
4
Chromatography
F. Chemical Waste Disposal:
1. Silica gel, sand and cotton from the used chromatography column can be thrown in the
trash.
2. Used Pasteur pipets should be placed in glass trash box.
3. Methylene chloride: waste disposal class 3.
4. Hexane, cyclohexane and toluene: waste disposal class 4.
5. Methanol and Acetone: waste disposal class 6.
6. Fluorene: waste disposal class 1.
G. General Overview of the Experiment:
You will work in groups of three or four with a manager, a recorder, a presenter and when
possible a technician.
 Each group will be assigned a solvent system consisting of a polar and a non-polar
solvent. (See the Lab Section Lists page on the course website for group
compositions and individual role assignments.)
 Groups choose a name.
 Group members collaborate to choose various mixtures of 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, 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
In this first part of the experiment, thin layer chromatography (TLC.) will be used to
determine the degree of impurity of the fluorene and the solvent or solvent system to be used
in the column chromatography separation.
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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 mmber.
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 solvents or solvent
systems.
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.
Melting Points of Mixtures
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 and therefore
one component.
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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
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By means of tweezers, place the spotted TLC plate into the chamber, cap the chamber,
and allow the slide to develop to 0.5 cm from the top. (top line on the plate)
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.
CHEM 211 Experiment 6
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6
Chromatography
Examine the dried slides under a short-wave (254 nm) UV lamp; lightly circle the
observed spots with a pencil.
Group members collaborate to determine the group’s the best solvent or solvent system
for separation of the crude fluorene (Figure 6).
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
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Clamp the "column" (Pasteur pipet) to a ring stand in a vertical position.
With the aid of a piece of rigid copper wire, place a very small wad of cotton through
the pipet into the tip. DO NOT JAM IT TOO TIGHTLY!
 To the plugged pipet add 100 mg of sea sand.
 Add 700 mg silica gel(60-100 mesh) to the column so that is sits above the sea sand.
 Add a second sample of 100 mg sea sand.
 Place a 25 mL Erlenmeyer flask that has been labeled (your name, lab section, and
date) and then pre-weighed beneath the column as a receiver for the eluent. Make sure
that the tip of the pipet (the column) is inserted into the neck of the labeled, preweighed flask.
b. Purification procedure:
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Place 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.
 When all the solvent appears to have been removed (by gravity) from the column,
attach a small pipet bulb to the top of the chromatography column and gently depress
the bulb to force the residual chromatography solvent from the column.
 Evaporate the eluted solvent by clamping the 25 mL receiving flask in a hot water
bath in a hood.
 When all solvent has been evaporated, remove the flask form the hot water bath,
allow the flask to cool to room temperature and obtain the mass of the flask and dry
recovered fluorene. Calculate the percent recovery of fluorene.
c. TLC Analysis of the Purified fluorene:
 Analyze your purified fluorene by TLC using your group’s most effective separating
solvent system.
 Collaborate with your group mates to determine the effectiveness and reproducibility
of the purification of fluorene with your group’s solvent system. You should consider
both percent recovery and purity of your recovered fluorene.