Experiment 1: Thin Layer Chromatography

Experiment Description

In this experiment, you will experimentally determine which solvent is suitable for the
separation of a mixture containing benzophenone, diphenylmethanol and biphenyl by thin layer
chromatography (TLC) using silica gel adsorbent. Structures of these three compounds are
shown below.

O OH

Benzophenone Diphenylmethanol Biphenyl

Background: Thin Layer Chromatography

Chromatographic separations take advantage of possibility that substances will differently
between two phases, a mobile phase and a stationary phase. You have already had some
experience with gas chromatography where the mobile phase is an inert gas, usually helium,
and the stationary phase is a high boiling liquid coating absorbed on the surface of a granular
solid in a column. In thin layer chromatography, or TLC, the mobile phase is a liquid and the
stationary phase is a solid absorbent.

Theory of Thin Layer Chromatography

In thin layer chromatography, a solid phase, the adsorbent (the stationary phase) is a powder
which is coated onto a solid support, as a thin layer (about 0.25 mm thick). Thin plates of
glass are the most common support, but plastic and aluminum can also be used. Like GC, TLC
is usually used as a diagnostic tool it answers questions like “Is my reaction done yet?” You can
watch the starting material go away and the product come in using TLC. “Is my product
clean?” is another good question for TLC. It is also used to develop conditions for larger scale
separations using a technique called column chromatography.

In TLC, the mixture starts as a small spot near the bottom of the plate and the solvent (mobile
phase) carries the compounds up the plate as it travels up from the bottom by capillary action.
In most cases, the stationary phase (adsorbent) is very polar and the mobile phase (eluant) is
fairly non-polar. Molecules that are more polar stick to the polar stationary phase more than
fairly non-polar molecules which are carried along in the mobile phase. Keep in mind that this
is an equilibrium, all molecules do a little of both. Separation occurs because some things spend
a higher percentage of the time standing still, adsorbed on the stationary phase than others do.

Several factors determine the efficiency of a chromatographic separation. The adsorbent and
solvent system chosen are the easiest factors to change. Silica gel (SiO2) is a very commonly
used, strongly polar adsorbent material that you will be using for these experiments. Other
common polar adsorbents include alumina, charcoal and Florisil. Non-polar adsorbents may
also be used with relatively polar solvent systems, this is known as “reverse phase”
chromatography because the nature of the stationary and mobile phases is inverted. Drug
purification frequently employs this technique.

The most common factor that is adjusted to achieve good separation is the solvents used in the
mobile phase. As you can see in the list provided below, there are many choices for solvents
and solvent mixtures are quiet common. The substances being separated are adsorbed onto the
stationary phase, but polar solvent molecules are also adsorbed by the stationary phase.
Molecules that are already adsorbed are displaced and “pushed along” by polar solvent
molecules. Thus, everything moves up the plate faster in more polar solvent systems. The
“Eluting power” of a solvent is largely a measure of how well it is adsorbed onto the stationary
phase, displacing other molecules.

Eluting solvents for chromatography

Note: This is not a complete list of possible solvents, but it does include the most commonly
used ones.

Least Eluting Power Hexane or Pentane

Cyclohexane
Benzene
Dichloromethane
Chloroform
Ether (anhydrous)
Ethyl acetate (anhydrous)
Acetone (anhydrous)
Ethanol
Methanol
Water
Pyridine
Greatest Eluting Power Acetic Acid

The non-polar solvents at the top of the list are often used as a base and a few percent of a
stronger eluting, more polar solvent is added. As the eluting power of the added solvent
increases, the amount that is generally added decreases. “Medium polar” solvents like diethyl
ether and ethyl acetate may be used 1%-50% combination with hexane making these mixtures
very tunable and common. Stronger eluting solvents like methanol cannot be used as more than
10% of the solution or the silica gel will dissolve in them causing problems with the separation.
More than 1% of pyridine or acetic acid isn’t often necessary, a drop or two is more common,
while these two additives are next to each other on the list, they can have very different effects
on a separation depending on the functional groups in the molecules being separated. Water is
very strongly eluting and its presence as an impurity in your solvent can be problematic.

The functional groups of the molecules in your mixture effect how strongly they are adsorbed
by the stationary phase. Very “greasy” non-polar substructures, usually made entirely of
carbon an hydrogen, are hardly adsorbed by silica gel at all. Polar groups, with oxygen and
especially nitrogen are more strongly adsorbed. The ability to hydrogen bond with the silica
gel creates a strong adsorbing interaction in alcohols, carboxylic acids and amines.

Table 3. Adsorbability of organic compounds by functional group

Least Strongly Adsorbed Saturated hydrocarbons; alkyl halides

Most Strongly Adsorbed
Unsaturated hydrocarbons; aIkenyl halides
Aromatic hydrocarbons; aryl halides
Polyhalogenated hydrocarbons
Ethers
Esters
Aldehydes and ketones
Alcohols
Acids and bases (amines)

TLC is useful because it is reproducible. For a particular adsorbent/solvent/compound

combination, the ratio of the distance the compound travels to the distance the solvent travels
remains constant. This ratio is called the Rf value.

Rf value = distance traveled by substance

distance traveled by solvent front)

While having the same Rf value (under the same conditions) does not prove that two
substances are the same, having different Rf values demonstrates that they are different.

Techniques: Thin Layer Chromatography

Spotting
Thin Layer Chromatography takes only a very small quantity of compound. The
mixture that you are separated is dissolved in a solvent. Using a very thin glass tube, called a
capillary you can put a tiny amount of solution in a specific spot on the plate. First, using a
pencil gently draw a line across the bottom edge of the plate, 0.5-1.0 cm up from the bottom,
as shown in the figure. You may also want to label the positions where your spots will be if
you’re using more than one. A simple number or
letter code is probably best. Be careful not to
scrape off too much adsorbent from the plate.
Dip the tip of the capillary, (sometimes TLC Plate Before it has Been
called a “TLC Spotter”) into the solution of your Developed
mixture and then briefly touch it to the plate.
Keep the spot small, just a few millimeters in
diameter. If you need a heavier spot, wait for the
solvent to evaporate and spot again.

Developing
Make sure that the jar you’re using is dry
inside. If it isn’t, rinse it with the solvent you’re
going to put in it. Do not rinse it with water. Spots of Compound
As shown in the diagram above, place a
clean dry piece of filter paper (cut if necessary) Pencil Line
standing up against the wall of the jar. The paper Std Mix
will soak up some of the solvent and help to
saturate the atmosphere inside the jar with solvent
vapor. This will make your plate run faster by
slowing the evaporation of solvent from the plate.
Pour a small amount of solvent into the
jar. Just a few milliliters, you don’t want it to be

Developing a TLC Plate

Cap the Jar

Filter paper
wet with solvent

TLC Plate
Leans against Wall Solvent level,
below spots
so deep that you’ll dunk your spots! Tip the jar to wet
the paper with the solvent.
Place the TLC plate gently into the jar, you
may want to use your forceps for this. Be careful not TLC Plate After it has Been
to let the solvent level get above the line at the bottom Developed
of the plate. Place a cap on top of the jar. There is no
need to screw it on.
Wait and watch the solvent front travel up the
plate. This could take a while, especially if you have Pencil Line
a less volatile solvent. Remember, if the plate runs to mark
Solvent Front
too far, you have to start over, so be patient and keep
an eye on it. When the solvent front has gotten 0.5- Separated
Compounds
1.0 cm from the top of the plate, pull it out with your (May not be visible)
forceps and immediately mark the solvent front with a
pencil line.

Visualization Std Mix

One of the most common ways to visualize

compounds on a TLC plate is to use a UV light. The
TLC plates that we are using have been treated so that
they fluoresce a green color under UV light. The
aromatic rings in the compounds that we are
separating absorb the UV light before it reaches the
fluorescent compound, causing dark spots to appear
where the compound is. Take your plate and a pencil Visualization of a TLC Plate
to the UV lamp provided in the lab and look at it with a Hand-held UV lamp
under the light. Trace around any spots that you see
so that you’ll know where they are later.
Compounds that are not UV active can be seen
by staining the TLC plate. The plate is sprayed with
or dipped into a solution and then heated. Reactions
which cause a color change occur with the compound
on the plate, and spots appear.
If you can’t see anything, you either
accidentally washed the spots off in the solvent or
didn’t spot enough to start with. If your compound
streaked, or have long tadpole tails, or your spots are
huge, try again spotting less compound this time.

You should draw each plate in your notebook as

part of your record of the experiment.
Calculating Rf Values

The distance that a compound travels in a specific chromatography system, compared to how
far the solvent has traveled, is a constant. The relationship between the distance traveled by the
solvent front and the substance is usually expressed as the Rf value:

Rf value = distance traveled by substance

distance traveled by solvent front)

Rf = B/A

Distance the
Solvent Traveled
=A
Distance the
Compound
Traveled = B
Std Mix

Using a Ruler measure both distances in millimeters and record the Rf values
Experimental:
O OH

Benzophenone Diphenylmethanol Biphenyl

Pre-lab Questions:

1. From the solvents listed below, select a single solvent that you think would give you
the best chance of separating benzophenone, diphenyl methanol and biphenyl
(structures above) using TLC on silica gel. Explain the reasoning behind your
choice. Keep in mind that phenyl groups are fairly “greasy” and non-polar.

2. Assuming you separate the above compounds by TLC using your solvent of choice,
predict the order in which the compounds will move up the plate from highest Rf
(fastest moving) to lowest Rf (slowest moving).

Table: Eluting solvents for chromatography

Least Eluting Power Hexanes (a mixture of isomers)

Toluene
Dichloromethane
Diethyl Ether
Ethyl acetate
Acetone
Ethanol
Methanol
Water
Greatest Eluting Power Acetic acid

Note: Bring a ruler and pencil to lab for this experiment

 Based on your answers to pre-lab question 1, choose two solvents to start with.
Prepare a TLC chamber with each solvent, as described in the Techniques section.

Spot two TLC plates using the dichloromethane stock solution of the mixture
containing benzophenone, diphenylmethanol, and biphenyl. Develop the plates
simultaneously, one in each of the different solvents. After developing the plates, remove them
from the chamber, indicate the position of the solvent front with a pencil mark, and allow the
plates to air dry for a few minutes. Examine them under UV light, gently circle the spots that
you see with your pencil and make a sketch in your lab notebook of the appearance of each
plate showing the positions of all spots. Calculate Rf values for each spot, measuring in
millimeters to the center of the spots.

Depending on the results of this first TLC experiment, design and conduct a second TLC
experiment using two other solvents:

1. If neither of the first two solvents separated all three compounds on the TLC plate, then,
based on the appearance of each plate, decide if the solvent used was too polar or not
polar enough. Then select two new solvents that you believe will change the polarity in
the right direction to achieve separation. Test the new solvents by preparing and
developing two additional plates.

2. If one of the first two solvents separated the three compounds on the TLC plate, but the
other didn’t, then, based on the appearance of each plate and the relative polarity of the
two solvents, consider how the polarity might be changed from the solvent that worked
to improve the separation. Test two new solvents to see if you can improve the
separation. There is more than one solvent which will achieve this separation.

3. If both of the solvents you tested in the first place provided separation of all three
compounds, conduct a second experiment to determine if the separation can be
improved by increasing or decreasing the polarity of the solvent from the two solvents
that worked.

In the second experiment, as with the first, make sketches of each TLC plate showing the
positions of the spots, and calculate Rf values.

After conducting at least two pairs of TLC experiments (developing four plates) as described
above and finding one or more solvents that can be used to separate the three compounds in the
mixture, prepare one final TLC plate in which you spot the mixture of three compounds side-
by-side with each of the three standards. The standards are separate, identified solutions of
each compound. Be careful not to contaminate the standards. The plate you prepare will then
be spotted four times, once with the mixture and once with each of the three standards. Be sure
to label the spots. Then develop this final plate using the best solvent from your earlier
experiments and compare Rf values to confirm the identity of the three spots in the mixture.
If you were not able to achieve a very good separation of the compounds in the first or second
experiment, conduct additional experiments (as time permits) until you achieve a good
separation of all three compounds. Be sure to carefully consider the results of all previous
experiments to make the best choice of solvents, and make sketches of all plates in your lab
notebook.

In your lab report:

Report the Rf values of the spots in each plate that you ran.

Discuss the decisions that you made to choose a solvent system which separated the mixture.
You should have used your knowledge of the principles of chromatography to guide your
choices.

Identify the components of the mixture. Explain why they elute in the order that they do.

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