Thin Layer Chromatography
Background Information
Mixtures are found everywhere in organic chemistry. Organic molecules present in natural materials are
found in the presence of many other organic molecules. Even when organic chemists carefully design a
reaction to convert one product into another product, side products may form or the conversion of the
one compound into the other may be incomplete so one may have a mixture of the starting material
and the product. Chromatography encompasses many separation techniques that can be used to
separate or analyze mixtures of compounds. One of the most accessible is called thin layer
chromatography (TLC). In learning about TLC you will also explore the polarity and solubility of some
different organic functional groups.
Several common elements are present in all chromatographic techniques: these include the analyte(s)
or the substance(s) to be analyzed, a stationary phase and a mobile phase. The analytes are applied to
the stationary phase and then interact with a mobile phase. The analyte(s) may move to some extent
with the mobile phase or remain attracted to the stationary phase depending upon the characteristics of
the analytes, the stationary phase and the mobile phase. The properties of analytes and their
intermolecular interactions with either the mobile phase or the stationary phase allows different organic
substances to be separated from each other. Refer to your pre-lab lecture, or other course materials for
further chromatography background and applications to organic compound separations.
In this TLC investigation the stationary phase consists of a thin silica gel layer (a purified sand type solid
composed of silicon-oxygen single bond networks) on a plastic film. The adsorbent, sandy, silica gel
layer is the working side of the TLC sheet and the plastic backing simply supports the silica gel on the
plastic sheet. The organic chemicals to be analyzed are applied to the silica gel in a “spot” using a fine
capillary as a “spotter.” Solid samples are dissolved in a small amount of an organic solvent prior to
spotting. The TLC strip will be placed vertically in a small (thin) puddle of mobile phase solvent or
solvent mixture. If the mobile phase travels by capillary action up the strip, and the “spot” should move
with the moving phase but must not be washed into the puddle of moving phase, where should the spot
be placed on the strip?
If you guessed the spot should be clearly above the initial mobile phase level , you are correct. This is
shown in the diagram below. You will want to know at what height you have placed your spot on the
TLC strip so you will mark this height on the side of the TLC strip directly on the TLC strip with a PENCIL.
As soon as the solvent from the analyte evaporates you will not be able to see the spot you just placed
on the TLC strip unless the analyte is colored. It is OK to place more than one sample on a TLC strip
depending upon the width of a given strip, and the size of the spots, with small spots preferred. If two
or more spots are applied to the same strip, record what materials are applied and where on a sketch of
the strip in your notebook. It is possible to re-apply a spot of analyte directly on top of the first
application if the concentration of the analyte is low. (Beginning students usually spot too heavy rather
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than too light. Heavy spots spread excessively, limit separation, and make the Rf difficult to determine.
Spotting too lightly might make some components not visible.)
After applying the analyte(s) to the TLC strip, the strip is placed in a development chamber, a beaker
with a small, low level of mobile phase. To hasten capillary absorption and prevent evaporation, a
watch glass, aluminum foil or other cover may be placed on the top of the developing chamber or
beaker. Folded filter paper may also line the beaker to saturate the chamber with the solvent vapor to
improving capillary adsorption on the TLC strip. Be careful that the paper does not wick mobile phase
onto the TLC strip from the side. The mobile phase is to move on the TLC strip evenly from the bottom
to the top of the strip. Watch carefully as the solvent approaches the top ½ inch of the strip. A diagram
of the TLC strip in a development chamber (beaker) is shown below:
When the mobile phase climbs to about ½ inch of the top of the strip, the strip can be removed from the
chamber. As soon as you remove the strip from the chamber mark the height the mobile phase traveled
on the strip with a PENCIL. Once the solvent evaporates you will not be able to see how far the solvent
climbed on the plate.
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The mobile phase solvent is then allowed to evaporate from the strip and you are ready to visualize the
results of your TLC. If an analyte is colored you may have observed colored spots as the mobile phase
moved on the TLC strip. If the analyte, is not colored, different methods to visualize the results may be
used. These include fluorescence, iodine, and phsophomolybic acid, as well as other stains and
reactants. Some commercial TLC strips contain an Ultraviolet (UV) fluorescent indicator added to the
stationary phase. This means that an organic compound that absorbs some UV may show up as a
darkened spot when the TLC strip is viewed under an UV light. (CAUTION: DO NOT LOOK DIRECTLY INTO
THE UV LIGHT IT CAN DAMAGE YOUR EYES!) Look at the TLC strip when it is illuminated by the UV light
and mark or circle any observed spots with a pencil. Other visualization techniques include exposure of
the strip to iodine in an iodine chamber where many organic molecules will form a brown spot, or
possibly treatment with phosphomolybdic acid. Once the spots have been identified the distance the
spot moves divided by the distance the moving phase solvent moved is determined. This is called the Rf.
The Rf allows one TLC strip to be compared to another as long as the TLC conditions are the same. The
calculation of an Rf is shown below.
Rf = distance the analyte moved = 2 cm = 0.5
distance the solvent moved
4 cm
Thin Layer Chromatography Exploration
In this guided collaborative experiment you will investigate the TLC behavior of two different organic
functional group molecules on commercial silica gel plates in two different solvent systems. Be sure to
answer the investigation questions throughout the course of the activity. Finally, you will apply your
knowledge of TLC to the chromatography of a consumer product. Individual student assignments are
below.
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Student 1 will investigate acetanilide and m-toluic acid. Each of these analytes is available in a
small bottle dissolved in ethylacetate.
Student 2 will investigate benzophenone and p-toluic acid. Each of these analytes is available in
a small bottle dissolved in ethylacetate.
Student 3 will investigate phenyl benzoate and o-toluic acid. Each of these analytes is available
in a small bottle dissolved in ethylacetate.
Student 4 will investigate diphenylmethane and terephthalic acid. Each of these analytes is
available in a small bottle dissolved in ethylacetate.
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Students 1-4: All student run an initial TLC of 2 organic compounds in the same solvent system.
Part A: TLC #1
Begin by preparing a TLC beaker developing chamber by placing 5-7 mL of 80/20 Hexane/Ethyl acetate
solution in a 100 mL beaker lined with a piece of folded filter paper.
Student 1: Spot one TLC strip ¾ inch from the bottom of the strip with acetanilide on one side of
the strip and m-toluic acid on the other side of the strip. After applying the analytes to the strip
place the strip in the developing chamber. Cover the top with a watch glass or other cover.
● Student 2: Spot one TLC strip ¾ inch from the bottom of the strip with benzophenone on one side
of the strip and p-toluic acid on the other side of the strip. After applying the analytes to the strip
place the strip in the developing chamber. Cover the top with a watch glass or other cover.
● Student 3: Spot one TLC strip ¾ inch from the bottom of the strip with phenyl benzoate on one
side of the strip and o-toluic acid on the other side of the strip. After applying the analytes to the
strip place the strip in the developing chamber. Cover the top with a watch glass or other cover.
● Student 4: Spot one TLC strip ¾ inch from the bottom of the strip with diphenylmethane on one
side of the strip and terphthalic acid on the other side of the strip. After applying the analytes to
the strip place the strip in the developing chamber. Cover the top with a watch glass or other
cover.
While the first strip is developing, conduct the following solubility studies with your laboratory partner.
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NOTE: While doing the solubility studies, be sure to check on the progress of the mobile phase in your
TLC developing chamber. If it runs too long, the components of the analyte may run all the way to the
top edge of the strip and merge together. When the mobile phase is near the top, remove the TLC strip,
mark the solvent front line, allow the mobile phase to evaporate from the surface of the strip. Finally
visualize and mark the spots. Before determining the Rf value, set up the next developing TLC
experiment.
Part B: Solubility and Polarity
1. Mix 1 ml hexane with 1 mL colored water in a small test tube. Describe the miscibility of these
two solvents?
2. Mix 1 mL ethyl acetate with 1 mL colored water in a small test tube. Describe the miscibility of
these two solvents?
3. Mix 1 mL propanol with 1 mL colored water in a small test tube. Describe the miscibility of
these two solvents?
Students 1-4 (All students answer the following questions.)
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1. Order the following solvents from most poplar to least polar: Ethyl acetate, Hexane, Propanol,
Water
2. Would you expect a mixture of ethyl acetate and ethanol to be miscible? Why or why not?
Part C: TLC #2
All students will run another TLC of the same 2 organic compounds in a different solvent system.
Prepare a second TLC beaker developing chamber by placing 10-15 mL of 50/50 Hexane/Ethyl Acetate
solution in a 250 mL beaker lined with a piece of folded filter paper.
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Student 1: Spot another TLC strip ¾ inch from the bottom of the strip with acetanilide on one
side of the strip and m-toluic acid on the other side of the strip. After applying the analytes to
the strip place the strip in the 50/50 solvent developing chamber. Cover the top with a watch
glass or other cover.
Student 2: Spot another TLC strip ¾ inch from the bottom of the strip with benzophenone on
one side of the strip and p-toluic acid on the other side of the strip. After applying the analytes
to the strip place the strip in the 50/50 solvent developing chamber. Cover the top with a watch
glass or other cover.
Student 3: Spot another TLC strip ¾ inch from the bottom of the strip with phenyl benzoate on
one side of the strip and o-toluic acid on the other side of the strip. After applying the analytes
to the strip place the strip in the 50/50 solvent developing chamber. Cover the top with a watch
glass or other cover.
Student 4: Spot another TLC strip ¾ inch from the bottom of the strip with diphenylmethane on
one side of the strip and terephthalic acid on the other side of the strip. After applying the
analytes to the strip place the strip in the 50/50 solvent developing chamber. Cover the top
with a watch glass or other cover.
Students 1-4:
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While waiting for this TLC to develop, measure the analyte and solvent distances and determine
the Rf for the previous TLC in 80/20 hexane/ethyl acetate solution. Record the observed Rf in
your laboratory notebook, along with the first mobile phase composition.
Be sure to check on the progress of the mobile phase in your TLC developing chamber. When
the mobile phase is near the top, remove the TLC strip, mark the solvent front line, allow the
mobile phase to evaporate from the surface of the strip. Finally visualize and mark the spots.
Measure the analyte and solvent distances and determine the Rf for the this TLC of your two
organic compounds. Record this observed Rf in your laboratory notebook along with the
second mobile phase composition.
Combine and look at the relative polarity of some of the compounds used in this laboratory
investigation by filling in the following table. In this table you will also need the Rf values of the
TLC and the polarity descriptions of the compounds of all the students in your group. It is also
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helpful if you draw out the structural formulas of the compounds, and identify the functional
group. Which compounds are least polar? Which compounds area most polar?
Compound
Name
Compound
Structure
Compound
Functional
Group
Classification:
Very Polar
Polar
Slightly Polar
Nonpolar
Very
Nonpolar
Rf
80/20 Hexane
/ Ethyl
acetate
Rf
50/50
Ethyl
acetate/
Ethanol
Hexane
Ethyl Acetate
Propanol
Acetanilide
m-Toluic Acid
Benzophneone
p-Toluic Acid
Phenyl Benzoate
o-Toluic Acid
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Diphenylmethane
Terephthalic Acid
m-N,NDiethyltoluamide
DEET
1. Why was 100 % hexane not suggested as a mobile phase in this investigation?
2. A compound related to m-Toluic acid is m-N,N-Diethyltoluamide, which is otherwise known as
DEET. From the functional group classification of DEET do you expect DEET to be polar or
nonpolar? Explain your choice and reasoning.
TLC Challenge
1. You have been asked to determine whether a mixture contains, m-toluic acid, DEET (m-N,N,diethyl toluamide) or a mixture of m-toluic acid and DEET by TLC, which solvent system would
you use?
2. Before you try your choice, consider how DEET appears in insect repellent lotions or sprays.
Many skin products contain water as well as organic compounds, including DEET containing
insect repellants. How would applying a sample with water possibly change the TLC behavior of
organic substances?
3. To get around this, apply a sample of a DEET containing insect repellant to a half of piece of
small filter paper. After a few minutes, dip the filter paper containing the insect repellant into a
small amount 5 mL of ethyl acetate or dichloromethane. Spot this solution of the commercial
insect repellant next to the pure m-N,N-Diethyltoluamide and run the TLC of these samples in
the solvent system you have chosen.
4. Report the Rf of DEET, m-toluic acid and the commercial insect repellent containing DEET.
Explain how TLC can be used to identify the presence of the specific organic chemical, DEET in a
consumer product such as an insect repellant spray or lotion.
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5. Does the insect repellant you analyzed contain DEET? Does it contain m-toluic acid? How
confident are you of your results?
6. Currently another insect repellant on the market is Icaridin (World Health Organization, WHO
approved name where the initially suggested name was picaridin.) The structure of Icaridin, [1piperdinecarboxylic acid2-(2-hydroxyethyl)-1-methylpropylester] is below:
7. Describe how you might distinguish between two insect repellents containing DEET or Icaridin?
What polarity differences do you predict between DEET and Icaridin?
8. Rank the following compounds from least polar to most polar: Ethyl acetate, Hexane, DEET, mToluic Acid.
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