Experiment C

Separating a Mixture by Thin Layer Chromatography

Objectives

- To select an appropriate solvent to separate a mixture of biphenyl, 4-phenylphenol, and

benzophenone by thin layer chromatography.
- To calculate the Rf values for reference materials and the unknown samples.
- To identify the components of unknown samples from Rf values.

Introduction

Thin Layer Chromatography

Thin layer chromatography (TLC) is an analytical technique frequently used in organic

chemistry to separate sample mixtures. It can be used to determine the number of components
in a mixture, purity of sample and identity of sample components by analyzing the
chromatogram obtained. TLC analysis can be carried out with relatively inexpensive and
sample equipment: a TLC plate and a developing chamber with appropriate solvent. Besides,
TLC is used frequently to follow the progress of a reaction, to select a suitable solvent before
attempting a column chromatography, and also to monitor the separation during a column
chromatography.

Chromatography refers to the separation of a mixture of two or more different compounds by

distribution between two immiscible phases: stationary phase and mobile phase.
Depending on the nature of these two phases, we have different chromatographic techniques
including thin layer chromatography, paper chromatography, and gas chromatography. In
each technique a sample mixture is placed into a liquid or gas, called a mobile phase. The
mobile phase carries the sample through a solid support, called stationary phase, which
contains a solid adsorbent or another liquid. The different compounds in the sample mixture
move through the stationary phase at different rates, due to different attractions for the mobile
and stationary phases. Thus, individual compounds in the mixture separate as they move
through the stationary phase. The separate compounds can be collected or detected,
depending on the particular chromatographic technique involved.

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Process of Separation

In TLC, sample separation is performed on a thin layer of adsorbent spread on a solid support
which can be a glass, an aluminium foil or a plastic (the adsorbent acts as the stationary
phase). The sample, which is dissolved in a suitable solvent, is spotted onto the adsorbent
plate near its lower edge. The plate is then placed into a developing chamber containing a
shallow layer of solvent (the mobile phase). The solvent will creep up and carry the
components of the sample to move along the plate by capillary action. Due to differences in
their relative attractions for the mobile and stationary phases, the components will move up
the plate at different rates. This results in separation of compounds. The whole process is
referred to as development and the developed plate is called a chromatogram.

solvent

Fig. 1 A typical developing chamber for TLC

Separation by Differential Retention

Adsorption is a surface process caused by the electrostatic interactions between sample

components and adsorbent molecules. As solvent travels along the plate, different
components of the sample are attracted to the surface of the adsorbent more or less strongly,
depending on their polarity and other structural features. The more strongly a component is
attracted to the adsorbent (or the more strongly the component is retained by the adsorbent),
the more slowly it will move up the plate. The differential retention of sample components
hence leads to sample separation.

solvent front

Fig. 2a TLC plate before development Fig. 2b TLC plate after development
(The mixture components are separated)

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Selection of Adsorbent and Solvent

The selection of the stationary and mobile phases in TLC determines the performance of a
separation. Silica gel (SiO2∙xH2O) and alumina (Al2O3) are two commonly used adsorbents in
TLC and column chromatography. Silica gel is generally used for polar samples, as its polar
Si–O and O–H bonds allow it to interact with polar solutes by dipole-dipole interaction and
H-bonds formation. Alumina is applied when less polar or non-polar sample is
encountered.

Water content affects adsorbent activity by occupying polar sites of the absorbent surface.
The greater the water content, the lower the adsorbent activity. For reproducible results, the
plates are dehydrated by heating in an oven and stored in a desiccator.

In selecting an appropriate solvent for separation, the polarity of solvent used should be
comparable to that of solute. A polar solvent can displace polar solutes from adsorbent
surface and move them over the plate. For non-polar solutes, a non-polar solvent should be
used. As if a polar solvent is used in the case of non-polar solutes, the solvent molecules
instead of the solute molecules will bind tightly to the adsorbent, leaving most of the solutes
suspended in the mobile phase, resulting in no differential retention and hence no separation.
Solvent selection is usually a process of trial and error until a desired separation is achieved.

In this experiment, you will perform thin layer chromatography using silica gel as the
adsorbent for sample mixture separation. You will have to try out the various solvents
provided to select a suitable solvent system for your sample analysis.

Polarity of Selected Solvents

Solvent Dielectric constant Remarks

ethyl acetate 6.02 A

hexane 1.89 B
methanol 32.7 C
dichloromethane 8.93 D
toluene 2.38 E

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Visualization of Chromatogram

After developing a TLC plate, visualization of sample spots is needed if the sample
components being analyzed are colourless. UV fluorescence is a common visualization
method. Commercial TLC plates are coated with specially formulated silica gel or alumina
for UV fluorescence visualization. These plates contain evenly distributed fluorescent
indicators that are chemically inert to most solvents. As UV radiation shines on the developed
plate, the indicators absorb radiation at 254 nm (short wave) or 365 nm (long wave) and emit
green or blue fluorescence. If the sample absorbs UV radiation, the spots will appear dark as
no light reaches the indicators. Use a pencil to mark those spots appear in UV light.

Exposure to iodine vapour is another commonly used visualization method. Iodine reacts
with many organic materials to form complexes that are either brown or yellow. After
development, the plate is placed in a chamber containing iodine crystals and vapour. The
spots of components that can react with iodine to form complexes will appear as brown or
yellow spots. After this iodine staining, the spots should be marked by a pencil immediately
as the complexes may not be stable and the colour of the spots may fade.

Calculation of Retention Factor for Identification

The identity of a sample spot can be determined through a comparison of its retention factor
(Rf value) with that of a reference material. Rf is defined as follows:

distance travelled by sample

R =
distance travelled by solvent front

Under an established set of conditions, a given compound always travels a fixed distance
relative to the distance the solvent front travels (i.e. same Rf value). The distance travelled by
a sample is measured from the origin (the point where the sample is spotted on) to the centre
of the spot (see Fig. 3 next page). Identical Rf values for a sample and a reference suggest
that both compounds may be identical. However, since two different compounds can have the
same Rf value under the same experimental conditions, further analysis needs to be conducted
if there is no prior information about the composition of the sample mixture.

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 solvent front

distance travelled by solvent front

distance travelled by sample

Fig. 3 TLC plate after development

In this experiment, you will be given with an unknown sample. By TLC, you will first
determine the Rf values of three reference materials: biphenyl, 4-phenylphenol and
benzophenone. Then you will analyze your sample and determine the following:

(1) the number of components present in your sample;

(2) if your sample contains one or more of those three reference materials.

In the process, you will test the various solvents provided to select an appropriate solvent
system for each of the TLC analysis.

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Experimental Procedure

Part I) Setting up the Developing Chambers

1. Label five glass bottles (A-E) and place a piece of filter paper along the inner walls of
each of the bottles.
2. Pour 5mL of solvent (A: ethyl acetate, B: hexane, C: methanol, D: dichloromethane,
E: toluene) into each of the labeled bottles.
3. Stopper and set aside the glass bottles.

Part II) Determining the Optimal Solvent System

1. Label five TLC silica gel plates (A-E) at the plate top with pencil.
2. On the TLC plates, draw a line and mark positions for sample spotting on the line (2
points for each plate). The line should be 1cm from the bottom, and the sample spots
should be 0.6cm away from each other.
3. Use a 0.3mm capillary tube to spot the reference mixture (mixture of biphenyl,
4-phenylphenol and benzophenone) onto all of the TLC plates. Each spot should not be
larger than 2mm in diameter.
4. Wait for the solvent to evaporate. Repeat step 3 to the right spot of each plate to increase
the sample concentration.
5. Wait for the solvent to evaporate completely.
6. Ensure the solvent level in chamber is lower than the sample line of TLC plates. Use
forceps to place the spotted TLC plates into the corresponding developing chambers. The
plates should be tilted and supported by the wall of chamber.
7. Stopper and set aside the developing chambers.
8. When the solvent front almost reaches the plate top (less than 1cm from the top), take the
plate out and mark the solvent front immediately.
9. Wait for the solvent to evaporate completely.
10. Put the developed TLC plates under the UV light and mark any visualized spot.
11. Measure the distance travelled by the solvent front and the component spots.
12. Sketch the chromatogram for each of the five solvent systems on the Data Sheet.
13. Determine the solvent system that can give the best separation of the reference mixture.

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Part III) Obtaining the Chromatograms for Reference Materials

1. Prepare and spot TLC plates (M: biphenyl, N: 4-phenylphenol, O: benzophenone) as in

Part II.
2. Apply the most efficient solvent system identified in Part II to develop the TLC plates.
3. Visualize and mark the spots. Measure the distance travelled by the solvent front and the
component spots.

Part IV) Unknown Sample Analysis

Repeat Part III with the unknown sample provided.

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