(CHE 276) Organic Chemistry Lab Experiment 1

Thin Layer Chromatography

"Adapted from: N. I. Totah, CHE 276 Organic Chemistry Laboratory Course Reader, Syracuse
University, Fall 2011"

Principle of Thin Layer Chromatography :

As the solvent containing a mixture of organic molecules (polar and less polar)
moves up the silica gel due to capillary action, the polar organic molecules will
interact stronger with the silica gel than the less polar organic molecules, and
thus the polar organic molecules will move slower than the less polar organic
molecule on the silica gel.
This almost magical difference in the interactions between different
molecules with the silica gel gives separation of a mixture of organic molecules,
ranks the relative polarities of the organic molecules, and to some extent, helps
in the identification of organic molecules.
Background:
Chromatography is one of the most ubiquitous methods of analyzing and purifying
organic compounds. While originally used to separate plant pigments, today this process includes
a variety of sophisticated techniques which allow for the separation, isolation, and identification
of the components of a mixture. While there are many types of chromatography, the fundamental
basis for this technique concerns the distribution of the individual components of a mixture
between two phases: the stationary phase and the mobile phase. For any given compound (A), an
equilibrium is established such that the compound is either adsorbed on the stationary phase or
dissolved in the mobile phase. In thin layer and column chromatography this equilibrium is
determined by the polarity of the stationary and mobile phases, and by the polarity of the
compound itself. In gas chromatography, however, the equilibrium is determined primarily by
temperature.

As the name implies, the stationary phase is a non-moving substance (often SiO2 or
Al2O3) to which the components of a mixture may adsorb. It can exist in a variety of forms, but is
commonly contained in a column or spread in a thin layer over a glass or plastic backing. The
mobile phase percolates over the stationary phase and may be either a gas, as in gas
chromatography, or a liquid as in column or thin layer chromatography. A compound dissolved in
the mobile phase is carried along in the direction of the flow. A compound adsorbed on the
stationary phase does not move.
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(CHE 276) Organic Chemistry Lab Experiment 1

The individual components

mixture also exist in equilibrium
between stationary and mobile
phases. In many cases, the mixture
loaded on the stationary phase, then
mobile phase is added. As the
phase flows over the stationary
it carries with it all components of
mixture in the direction of flow.
each component has a different
affinity for the stationary phase,
adsorbed to a greater or lesser extent
relative to the other components of
mixture. Compounds which favor
stationary phase are held longer, and
result move more slowly than do
compounds which favor the mobile
It is these differences in equilibrium
allow for the separation of
compounds in multi- component
mixtures.

of a
is first
the
mobile
phase
the
Since
each is
that
the
as a
phase.
which

Figure 1: Chromatographic separation of a

In the example shown here
two component mixture. As the mobile phase
(Figure 1), compound A interacts
more
moves,
it
carries
blue
molecule
faster
than
strongly with the stationary phase
than
red molecules because silica gel interacts
does compound B. As a result, more
of
stronger with red molecules than with
compound A is adsorbed on the
stationary phase at any given time. It blue molecules.
follows that compound B thus spends more time in the mobile phase than does compound A, and
is carried more quickly in the direction of the flow. As such, these two compounds will
eventually separate, and the separation will increase the longer the mobile phase travels.

Science of Thin layer chromotography.

(This section perhaps requires the most thought-intense reading for an understanding,
and is central to many questions in the quiz and final examination.)
The plate for a TLC is made of silica gel, which is a very polar material. For organic
solvents used for running a TLC, they are not considered as polar as silica gel. (1) Otherwise,
if the solvent is too polar, such as water, the silica gel will be disolved. In addition, the silica
gel binds to polar molecular stronger than non-polar molecules through noncovalent
interactions. These two facts form the basis for many logics explaining how TLC (and
column chromotography, LAB 5) works.

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(CHE 276) Organic Chemistry Lab Experiment 1

Consider two cases. First, for a given eluent solvent, how would a polar and
nonpolar molecule (to be analyzed) behave on a TLC plate. Second, for a given molecule (to
be analyzed), how would a polar and a nonpolar eluent solvent impact and compare the
movement of the molecule on a TLC plate.
For a given eluent solvent and comparing the behavior of a polar and nonpolar
molecule, the interaction between the silica and the molecule dominate over the interaction
between the solvent and the molecule. Why? see (1) above. For this reason, it is more
important to consider that the silica gel interacts with the the polar molecule stronger than
with the nonpolar molecule. Thus, for any eluent solvent (polar or nonpolar), the polar
molecule will always move slower than the nonpolar molecules.
For a given molecule and comparing the effect of a polar and a nonpolar solvent, the
interaction between the silica gel and the molecule is the same for both solvents. Thus, one
only needs to consider that the molecule will interact stronger with the polar solvent than
with nonpolar solvent. As a result, the molecule will always move faster when polar solvent
is used than when nonpolar solvent is used. This fact stays true for both polar and nonpolar molecules, because nonpolar molecules still interact stronger with a polar solvent
than with a nonpolar solvent.
To make it more concise, the following Tables are used to compare different
scenarios described above.
Sample for analysis
Polar molecule
Nonpolar molecule

Polar solvent
Slow(a)
Fast

Sample for analysis

Nonpolar solvent
Polar molecule
Slow(b)
Nonpolar molecule
Fast
Case (a) will be faster than case (b), see the two Tables below.
Sample for analysis
Polar molecule

Polar solvent
Fast(c)

Non polar solvent

Slow

Sample for analysis

Polar solvent
Non polar solvent
(d)
Nonpolar molecule
Fast
Slow
Case (c) will be slower than case (d), see the previous two Tables.
Note that, of course, the relative polarity (i.e. which is more polar) of two molecules
does NOT change in different solvents. The relative polarity of the common organic
molecules and solvents are shown in Table 1 and Table 2.
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(CHE 276) Organic Chemistry Lab Experiment 1

How to compare and determine relative polarity between two different organic molecules?
The polarity of a small organic molecule is determined by the functional groups the
molecule possesses. The symmetry of the molecule also plays a small role in influencing the
polarity of the molecule. Below is a list of general guidelines for ranking the polarity from
high to low.
1. Charged groups such as R4N+ or COO- are the most polar functional groups.
2. Functional groups with hydrogen bonding capability are the second category of
polar functional groups.
3. Atoms in functional groups with high electronegativity introduce more polarized
bonds (and thus more polar molecules) than those with low electronegativity.
4. The more polar functional groups a molecule possess, the more polar the molecule
will be.
5. In general, a polar functional group has a more dominating effect over less polar
functional groups on a molecule. For example, a carboxylate (RCOO-) is likely more
polar than a sugar-based surfactant that has five hydroxyl (OH) groups.
6. All else being the same, an asymmetric molecule is more polar than a symmetric
molecule.
The eluting power of various organic solvents parallels the order shown in Table 2. Thus,
the greater the polarity of the solvent, the greater its ability to dislodge and displace a compound
from the stationary surface, and the faster the compound will move along. This property of
solvents is quantified somewhat by listing them in order of their ability to displace solutes from
adsorbents. This listing is known as an "eluotropic series" and will vary somewhat from
adsorbent to adsorbent. An eluotropic series shown in Table 2 is for silica gel.
Operation of Thin Layer Chromatography:
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(CHE 276) Organic Chemistry Lab Experiment 1

Thin layer chromatography

(TLC) is used primarily as an analytical
technique to determine the purity of a
compound, the status of an ongoing
reaction, or as a preliminary means of
identification. The sample is spotted near
the bottom of a glass or plastic plate
which is coated with a thin layer (hence
the name) of dry adsorbent (Figure 2).
The plate is then placed in a covered
beaker or jar that contains a small
amount of the appropriate solvent. The
level of the solvent in the jar must be
Figure 2: TLC developing chamber. Note that the
below the level of the sample spots, and
spots are all above the level of solvent. This figure
the atmosphere in the jar should be
shows the TLC plate immediately after it is put in to
saturated with solvent vapors. A filter
the chamber.
paper is used to help with the saturation.
Capillary action draws the solvent up the
plate (If the jar is not saturated with solvent vapors, the solvent will not run all the way up the
plate). When the solvent front is near the top of the plate, it is removed from the beaker and the
location of the solvent front is marked with a pencil. If the compounds are colored, and the plate
can be read easily, no other method of visualization is needed. If the compounds are not colored
then they can be visualized using an ultraviolet lamp or a chemical stain, such as iodine.
For each spot on the TLC plate a characteristic value called the ratio to front, or Rf value
can be calculated (Figure 3). Rf is defined as the ratio of the distance traveled by a spot
(measured from the center of the spot) to the distance traveled by the solvent:
Rf =

Distance traveled by the compound

Distancetraveled by solvent

Although the Rf is characteristic for a

given compound, it depends greatly on the
solvent and the type of adsorbent used.
Consequently there are no tables of Rf values in
the chemical literature. However, under standard
conditions Rf can be used to identify the
components of a mixture, to determine the purity
of a compound, and as an indicator of whether or
not a reaction has gone to completion.
The difference in Rf values between two
spots on a plate, Rf, will also vary with the
solvent, and is used as a measure of the
performance of the separation. The choice of

Figure 3: TLC plate after developing and

staining. Note that the distances are
measured from the initial spot location, not
from the bottom of the plate.

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(CHE 276) Organic Chemistry Lab Experiment 1

developing solvent is crucial. With too polar a solvent, all of the 'spots' will run to the top of the
plate, and Rf will be zero. With a very non polar solvent, the spots will not move from the
baseline, and again Rf = 0. Often times, mixtures are used to adjust the polarity of the
developing solvent, in order to achieve good separation. The information gained by TLC in terms
of the relationship between solvent polarity and separation is also quite useful when choosing a
solvent for column chromatography.
Procedures:
A. Understanding Rf Values
In this portion of the experiment you will explore how the Rf value of an organic
compound varies with increasing length of TLC plate. An important component of this
experiment is to develop proper technique when spotting the TLC plate.
Obtain two TLC plates of different lengths. Record the length of each plate. Draw a light
pencil line about 1cm from the bottom of each TLC plate. Do not use ink! (why?) Using a
micropipet, spot each plate with a solution of benzyl alcohol. Remember that the best results are
obtained from small, compact spots. You may want to practice making small spots on a paper
towel before spotting your TLC plates.
While the spots are drying, prepare a TLC developing chamber as follows: Fold and tear
a filter paper in half and place it in a 100mL beaker. Add enough eluent A to the developing
chamber so that the solvent covers the bottom of the beaker to about 0.5cm. Do this in the hood!
Get the filter paper soaked with solvent. This filter paper acts as a wick to keep the developing
chamber saturated with solvent vapors.
Once the spots have dried, carefully place the TLC plate in the beaker. IMPORTANT! To
insure even movement of the solvent, the TLC plate should not touch the filter paper. Watch the
solvent move up the plate. This may take some time. When the solvent front is about 0.5cm from
the top of the plate, remove it from the developing chamber and immediately mark the location
of the solvent front with a pencil. Visualize your plates using an iodine chamber. Circle the
spot(s) with pencil. Measure and record the distances traveled by the solvent front as well as the
distances from the origin to the center of each resolved spot. Be sure to also record a copy of
your TLC plate. You can do so by tracing the shape of the TLC plate and mark where is the
baseline, solvent front, and the positions of the spots of the compounds. Calculate the Rf value
for each spot.
B. Rf Values & Solvent Polarity
In this portion of the experiment you will evaluate how the Rf value of an organic
compound is affected by solvent polarity.
Obtain three TLC plates of the same length. Draw a light pencil line about 1cm from the
bottom of each TLC plate. Using a micropipet, spot each plate with a solution of phenol. Once
the spots have dried, develop one plate in eluent A, one plate in eluent B, and 1 plate in eluent C.
After each elution is complete (e.g. when the solvent front is about 0.5cm from the top of the
plate), remove the plate from the developing chamber and immediately mark the location of the
solvent front with a pencil. Visualize your plate with UV light, and circle any spots with a pencil.
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(CHE 276) Organic Chemistry Lab Experiment 1

Record a copy of your TLC plates. Calculate the Rf value of phenol for each of the three runs.
Based on your findings, arrange the eluents A, B, and C in order of increasing polarity.
C. Rf Values & Compound Functionality
In this portion of the experiment you will evaluate how the structure of a compound (e.g.
what functional group it contains) can impact it's Rf value. You will examine a series of organic
compounds by TLC, each of which contains a different functional group.
Draw a light pencil line about 1cm from the bottom of a thin layer chromatography
(TLC) plate. Mark six equally spaced points along this line and label them A, B, C, D, E, F; one
for each solution of the following compounds:
A: phenol
D: acetophenone
B: anisole
E: benzoic acid
C: phenethyl alcohol F: phenyl acetate
Using a micropipet, put a small spot of solution at the appropriate mark. Use a clean
micropipet for each sample. Take care not to cross contaminate the samples! Once the spots have
dried, develop them in a TLC chamber containing eluent B. After elution is complete remove the
plate from the developing chamber and immediately mark the location of the solvent front with a
pencil. Visualize your plate with UV light, and circle the spots with a pencil. Record your
observations. Calculate the Rf value for each spot.
D. Identification of Commercial Food Dye Components by Thin Layer Chromatography:
In this portion of the experiment you will be investigating the makeup of some common
dyes used in commercial food colorings. You will first examine the commercial solutions and
identify the number of components in each. Next you will identify these components. Some of
the most widely used food dyes are shown below:

Draw a light pencil line about 1cm from the bottom of a thin layer chromatography
(TLC) plate. Mark four equally spaced points along this line and label them R (red), G (green), B
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(CHE 276) Organic Chemistry Lab Experiment 1

(blue), Y (yellow), one for each solution of commercial food color. Using a micropipet, put a
small spot of solution at the appropriate mark. Take care not to cross contaminate the samples!
Remember that the best results are obtained from small, compact spots.
While the spots are drying, prepare the developing chamber. Use a 3:1 mixture of 2propanol and ammonium hydroxide as the developing solvent. Do this in the hood! Be sure the
plate is dry, then develop as usual, again making sure that the TLC plate does not touch the filter
paper. Watch the solvent move up the plate. This may take some time. When the solvent front is
about 0.5cm from the top of the plate, remove it from the developing chamber and immediately
mark the location of the solvent front with a pencil. Measure and record the distances traveled by
the solvent front as well as the distances from the origin to the center of each resolved spot. Be
sure to also record a copy of your TLC plate. Calculate the Rf value for each spot.
Run a second TLC as above, this time using the (five) known F,D &C dye solutions.
Compare the Rf values of the known dyes with those of the commercial dye solutions. What
components can you identify? What other information can you use besides Rf?
References:
Pavia, D. L.; Lampman, G. M.; Kriz, G. S. "Introduction to Organic Laboratory Techniques", 3rd
ed. McGraw Hill Book Co: New York, NY, 1989, pp 268-273.
Mohrig, Hammond and Schatz "Techniques in Organic Chemistry", 3rd ed., Ch. 17: Thin Layer
Chromatography, pp 219-235.
Dickson, H.; Kittredge, K. W.; Sarquis, A. M. "Thin Layer Chromatography: The 'Eyes' of the
Organic Chemist" J. Chem. Ed. 2004, 81, 1023-1025.

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