CHEM 211-2008
Experiment 5
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: Chromatography (Padías pp. 150-151)
Thin-layer chromatography (Padías pp. 153-160)
Column chromatography ((Padías pp. 160-166)
3. Work through the material and links on the Experiment 5 page of the course website.
4. Complete the Prelab Questions linked to the Experiment 5 page on the course website by
7:00 PM, Tuesday, October 7.
5. Consider possible responses for the QOW (See below)
6. In your lab notebook: (See Lab Manual pp. 16-19 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 and the QOW on the
appropriate first left-hand page.
7. Bring your notebook and your ideas to lab discussion on Wednesday, Oct. 8 and be
prepared to respond to the Question of The Week.
8. After the lab discussion, write the procedure for the experiment.
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 Week:
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 5
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 5 we
apply this understanding to develop chromatographic methods for purifying and analyzing
organic compounds.
1. Chromatography:
As we saw in our discussion of gas chromatography, in general, 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 2).
All chromatographic methods work on the same principle, the differential affinities of the
substances to be separated between the two phases. This experiment deals with column
and thin-layer chromatography, which are based on adsorptivity of a solid surface and
solubility in a liquid solvent.
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 very 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. As we have seen in our studies of boiling
points and solubility, 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 a large surface area (usually high molecular
weight compounds). 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. These
interactions are very similar to those we saw affecting solubility in Experiment 3.
In chromatography, the principles of adsorptivity and solubility are important because
they are competing for the components of the sample being separated. 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, 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 (eluates), which is removed
from the solid support (is desorbed). The process of adsorption and desorption is a
dynamic equilibrium.
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
Melting Points of Mixtures
3
CHEM 211 Experiment 5
separated (eluates). Through a series of desorptions and adsorptions there is preferential
separation of the eluates based on their relative affinities for the silica and solvent. THE
MORE POLAR ELUATE HAS A HIGHER AFFINITY FOR THE SILICA (stationary
phase) SO IT MOVES DOWN THE COLUMN MORE SLOWLY THAN DOES A
LESS POLAR ELUATE. By gradually increasing the polarity of the solvent (eluent) all
of 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 repeatedly adsorbed onto the solid phase
(usually silica gel or alumina) and desorb into the solvent. 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 stationary phase 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.
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. Dichloromethane: 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.