Student Instructions

1
Chromatographic Purification and Analysis of a Mixture of Compounds



The objective of this exercise is to separate a mixture of two compounds into two pure
samples. This will be accomplished using flash chromatography in the next lab period, but
before you can run the flash column, you need to discover the optimal solvent system by using
thin layer chromatography (TLC). This is done by trial-and-error, using a mixture of two
solvents having different polarities. The most common polar solvent for chromatography is
ethyl acetate (EtOAc) and the most common non-polar solvent is hexanes. You will use 0%,
10%, 20%, 30%, 40% and 50% mixtures of EtOAc in hexanes to find the optimal conditions.
Once you have discovered the optimal solvent combination for ferrocene and acetylferrocene,
you will use it to purify a mixture of the two compounds using flash chromatography in the next
laboratory period. The flash chromatography will be performed using prepacked silica
cartridges.

Hazards

Ferrocene is an irritant and is hazardous to the eyes and skin. It is hazardous if ingested.
Acetylferrocene may be fatal if swallowed, is toxic if absorbed through the skin, and may cause
eye, skin and respiratory tract irritation. Ethyl acetate and hexanes are flammable, and hexanes
are a neurotoxin. Appropriate personal protective equipment should be used at all times, and
the reagents should only be handled in a well-ventilated fume hood. MSDS sheets are freely
available from the vendor at http://www.sigmaaldrich.com.


Day One: Thin Layer Chromatography (TLC)

Preparing the TLC Chamber

For the TLC portion of this experiment, you may work with a partner, but you will need to work
individually for the second portion of the experiment. Each partner should run three TLCs using
different percentages of the solvents to total the six possibilities listed above for each pair of
students. For example, one student would use 0%, 10%, and 20% EtOAc in hexanes and the
other would use 30%, 40%, and 50% EtOAc in hexanes. Make 10 mL of each of your three
mixtures and prepare a TLC chamber for each of them. This is described nicely in this video:

http://www.youtube.com/watch?v=EUn2skAAjHk.

Student Instructions
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For this laboratory, TLC chambers will be prepared by adding the 10 mL of solvent to a 250 mL
beaker and using a watch glass as a cover.

Preparing the Spotting Solutions and the GC Sample

Obtain approximately 10 mg each of ferrocene and acetylferrocene. There is no need to weigh
the compounds at this point—simply use the tip of a spatula. Mix the two powders in one test
tube and add approximately 2 mL of EtOAc. Remove approximately 1 mL of the homogeneous
solution and add it to a gas chromatography (GC) vial. The vial should be at least half full. Be
sure to clearly label the GC vial with both partners’ names or initials.

Gas chromatography (GC) is an excellent method for analyzing the purity of a compound. As
opposed to TLC, it is quantitative, but it is also expensive and more time-consuming than a
routine TLC. We will collect GC samples of your mixture of ferrocene and acetylferrocene and
will then analyze the purity of the ferrocene and acetylferrocene that you will separate using
flash chromatography in the next lab period.

Your TA will show you how to operate the instrument. To use your time effectively, analyze the
samples whenever time allows instead of waiting until the end of class. The remainder of the
solution in the test tube will be used for your TLC experiments.

Spotting the TLC Plates

In another test tube dissolve approximately 10 mg of pure ferrocene in 1-2 mL of EtOAc and do
the same for acetylferrocene in another test tube. You should now have three test tubes—one
containing a solution of pure ferrocene, one containing a solution of pure acetylferrocene and
one containing a mixture of the two compounds.

Before spotting the TLC plates, draw a pencil line at the bottom using a ruler approximately 1
cm from the bottom of the plate. (You must use a pencil, as the ink from a pen will elute up the
TLC plate when it contacts the organic solvents.) This line will be above the solvent level in your
TLC chamber; otherwise, your compounds will dissolve into your solution, and the TLC would
need to be repeated. Draw pencil marks on this line and label them for pure ferrocene, the
mixture, and pure acetylferrocene. To spot the TLC plates, use a micropipette which will draw
the spotting solution into itself by capillary action. Make small spots on the pencil line of the
TLC plate by using the solutions prepared earlier. Spot the plate several times for each
compound and blow onto the plate to dry it between applying the spots. The spots should be
nor more than 3 mm wide. Place the plate in the TLC chamber containing one of your TLC
solvents. The eluent will move up the TLC plate and move the spots. Once the solvent has
moved near the top of the TLC plate, remove it from the chamber and quickly draw a line with a
pencil to indicate the distance the solvent moved. Repeat this procedure with the other solvent
systems.

Student Instructions
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F = Ferrocene, M = Mixture, and A = Acetylferrocene


Visualizing the Spots

Once the solvents have eluted up the plates, visualize the spots using a UV lamp, and circle the
spots using a pencil. While the spots may be visible without it, the UV lamp will make the spots
easier to see.

Calculating R
f


Calculate the retention factor (Rf) for each of the spots using the following equation:

R
f
= (distance spot moved)/(distance solvent moved).

R
f
values do not have units, but you should still be aware of significant figures. Compare your R
f

values with those of your partner. An optimal solvent system will provide a good separation of
the compounds and an ideal Rf of approximately 0.3 for the lower compound. Draw
representations of your three TLC plates as well as those of your lab partner in your laboratory
notebook.




Day Two: Flash Chromatography

Flash chromatography is the most common method for purifying organic compounds in
research laboratories. The procedure is performed after nearly every reaction to remove
reagents, catalysts and unwanted byproducts. Consequently, all modern organic chemists are
experts at separating compounds by flash chromatography.
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The traditional apparatus for performing flash chromatography involves a glass column that
contains a stationary phase (usually silica gel or alumina). Mixtures of solvents are then forced
through the stationary phase, making a homogeneous slurry. The compounds to be separated
are then loaded onto the top of the slurry, more solvent is added, and finally pressure is applied
to force the compounds to move through the stationary phase. Polar compounds adhere to the
polar stationary phase, so they move slowly; non-polar compounds are not attracted to the
stationary phase, so they move more quickly down the column and are collected in test tubes
from the bottom of the column.

For more information on the theory and practice of flash chromatography, see the following
videos:
http://www.youtube.com/watch?v=gzp2S0e9o8s (TLC and column chromatography)
http://www.youtube.com/watch?v=EytuRMS1154 (practice of traditional flash
chromatography)

Our apparatus is significantly different from that shown in the YouTube videos. It was designed
as a substitute for traditional flash chromatography. Our goal was to construct an apparatus
that would eliminate the students’ exposure to silica gel, which can cause serious respiratory
problems if inhaled. For a demonstration of how to prepare and operate the flash
chromatography apparatus, please see the accompanying video on the course webpage.

The Flash Chromatography Apparatus


3-way stopcock connects to
disposable silica column
Connect to air or N
2
line.
Control flow with regulator.
Eluent (usually
EtOAc/hexanes) goes here.
Collect purified fractions
here.
Student Instructions
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Filling the Apparatus and Preparing the Silica

When using the apparatus, be sure to check all connections between the pieces to be sure they
are secure. Loose connections will cause the apparatus to either leak or spray solvent. If this
does occur during use, loosening the cover of the bottle will release the pressure and should
stop the leaking. The silica gel column must be filled with eluent before the sample is loaded.
To do this, first turn on the air flow lightly and make sure that the 3-way stopcock is turned so
that the opening to the side is closed but the other two are open (see figure below). Add
approximately 100 mL of your optimal eluent (determined in the previous lab period) to the
bottle, screw on the cap and connect it to the air line. Gradually begin to increase the air flow.
Use the regulator to control the flow of the solvent. You should be able to see solvent coming
through the line and entering the silica cartridge. After a moment, there should not be any air
bubbles. At this point, solvent should be flowing through the line and the 3-way stopcock,
through the silica cartridge, and into a waste beaker. Continue allowing solvent to flow into the
waste beaker until the silica cartridge is entirely wet and no air bubbles are present in the line
from the bottle to the silica column. The original white color of the silica cartridge will change
almost entirely to a gray color as the solvent flows through it.

Positions of a 3-Way Stopcock




Loading the Silica Column Cartridge

Weigh and record about 25 mg each of ferrocene and acetylferrocene. Mix them together and
dissolve them in a minimal amount about of the solvent system that you used to pack the
column (~ 1 mL of the EtOAc/hexanes mixture). Adjust the 3-way stopcock so that the openings
are all closed and the flow of solvent is stopped. Carefully remove the silica column cartridge
from the 3-way stopcock and inject the sample to be separated into the top of the cartridge
using a 1 mL syringe with no needle. Be sure that the syringe is placed firmly into the top of the
cartridge, or the sample will spill over when injected. Once the sample is in the silica column
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cartridge, firmly reattach it to the 3-way stopcock on the apparatus and adjust the 3-way
stopcock to continue the flow of solvent through the cartridge.

Chromatographic Separation

Continue to run solvent through the column until both compounds have eluted from the
column (refilling the reservoir if necessary). Collect the solvent that comes out of the bottom of
the silica cartridge in culture tubes. Your two compounds are in these culture tubes. Do not let
the silica column cartridge run dry. You should be able to see the two compounds run through
the cartridge as colored bands. You will be finished after approximately 12 culture tubes have
been collected and when there is no more color visible in the silica cartridge.

Isolation of Materials

Take TLCs of each test tube (you can put a bunch of them on the same plate). If the last test
tube contains one of the two spots, mix more EtOAc/hexanes eluent and continue to run the
column until no more compound remains in the column. Your TLC plates should look similar to
the drawing below, where some of the early tubes contain pure ferrocene and later tubes
contain acetylferrocene.

TLC of Purified Fractions




Use a rotary evaporator to isolate solid ferrocene and solid acetylferrocene. Combine the tubes
containing the pure ferrocene into one round bottom flask (test tubes 2 and 3 from the above
TLC plate) and then combine the tubes containing pure acetylferrocene (test tubes 5-9 from the
above TLC plate) into a different round bottom flask. Be sure to record the weights of the
round bottom flasks in advance when clean, dry, and empty for comparison. Evaporate the
samples using the rotavap until entirely dry. Record the weights of the round bottom flasks
again. Calculate % recovery:

% recovery = (g of isolated product)/(g of starting mixture) x 100%

See this video for an example of rotovap operation:
http://www.youtube.com/watch?v=7c0XL-ZQn5I.
Student Instructions
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Analysis of Purity by GC

Add a small amount of EtOAc to each of your flasks and then transfer the two solutions to two
GC vials. Analyze the purity of each of your compounds by GC.