23

Thin Layer Chromatography and Column Chromatography

Introduction
Contrary to popular belief TLC does not stand for Tender Loving Care. At least in the
chemistry world, TLC stands for Thin Layer Chromatography. Chromatography is the set of
laboratory techniques for the separation of mixtures
TLC is used for several reasons:
Identifying compounds by comparison
Determining purity or lack thereof
Monitor reaction progress
Monitor Column Chromatography

It is an important and fast qualitative analysis of a mixture.
If a compound is pure you should see one spot on the developed TLC plate. If it is impure you
should see multiple spots.
TLC is a very simple process that takes into account many scientific theories. All
chromatography techniques are based on the same simple concept of a stationary phase and a
mobile phase.
Stationary Phase The stationary phase (like the name implies) does not move. It is the
white powder, in the case of TLC; the stationary phase is typically silica (SiO2). Occasionally a
more basic stationary phase is needed and alumina may be used. The stationary phase is what
creates separation between spots.
Mobile Phase this is the liquid inside the container. It is the job of the mobile phase to
push the sample along the plate.
TLC works based on the polarity difference between the
sample and of the solvent system. Polarity, as you recall, refers
to the separation of electric charge leading to a molecule or its
chemical groups having a dipole moment, such as water. All
molecules have some degree of polarity (Figure 3) and choosing
the right solvent system depends on polarity of the compound
you are analyzing.
Almost always, the stationary phase is more polar than
the mobile phase. This is because the stationary phase is usually
the very polar adsorbent Silica (SiO2). Because of the Like
Dissolves Like the more polar compounds will stick to the plate
and not travel as quickly with the solvent front as a non-polar
compound will. Therefore, more polar compounds will stick to
the bottom, while non-polar compounds will travel further up the
plate with the solvent front.

Figure 3: Polarity of organic compounds
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A key to a successful TLC is choosing the correct solvent or
solvent system. Typically a solvent system involving a mixture of a
polar and non-polar solvent is implemented. Figure 4 lists solvents
in order of decreasing polarity. A typical solvent system would be
one that combines two solvents far apart from each other on the
chart, such as Ethyl Acetate and Hexanes or Dichloromethane and
Methanol. The ideal solvent system will place the compound being
analyzed in the middle of the plate.
Thin Layer Chromatography and Column Chromatography
separate compounds using the same principles. The main difference
between the two is that the solvent moves UP the plate via capillary
action in Thin Layer Chromatography and DOWN the column due
to gravity in Column Chromatography. In both cases compounds
are separated based on their polarity and therefore how much they
prefer the solvent to the stationary phase.
Experimental Procedure Techniques
Thin Layer Chromatography Technique
1. Pick a solvent system
2. Dissolve/dilute your sample in an appropriate solvent; usually something of equal or
greater polarity.
3. Prepare your TLC plate.
a. Draw a baseline across the plate 2mm from the bottom.
b. Then, draw a hash mark where you plan to place your sample(s).
c. This should only been done in PENCIL. Never use ink on a TLC plate.
(Youll see why soon!) Make sure not to disturb the white powder
(silica) on the TLC plate.
4. Use a capillary to spot your sample onto the pre-drawn hash mark.
a. Make sure the spot is not too large, if necessary spot several times to
ensure a sufficient amount of compound is on the plate.
b. Visualize the plate under a UV lamp to verify the presence of the
spotted compound. Compounds with conjugated double bonds are
considered UV active and will show up purple under the UV light
on the TLC plate.
5. Place the plate in the TLC developing chamber (or a beaker with a watch glass on top).
The chamber must contain the pre-determined solvent system (Step 1) and a piece of
filter paper. The purpose of the filter paper is to soak up the solvent and super
saturate the chamber with solvent vapors.
6. Make sure that there is enough solvent to completely cover the bottom of the
chamber but not go above the pre-drawn baseline. If the solvent level is above the
baseline of the TLC plate your sample could wash away in the solvent.
7. Also make sure you replace the watch glass. If you leave the lid off the chamber
the solvent will evaporate and drag your sample with it and give a higher false
reading.
8. Do not adjust or move the TLC plate once it is placed in the solvent.
9. Allow the solvent to elute up the TLC plate. You will be able to see the silica dampening.
Figure 4: Solvent Polarity
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10. Remove the plate from the developing chamber and mark the solvent front. (Where the
solvent stopped on the plate) This should be 2-3mm from the top of the plate.
11. Visualize the TLC plate. This means to look at the TLC plate. Most of the time you will
not be able to see anything on the plate because the compound is not visible to the naked
eye (visible spectrum). To see the location of the sample on the plate you can do one of
two things:
a. Utilize a UV light. This is the easier of the two solutions. Look at the
plate under UV light. Most scientific UV lights come equipped with
both short and long wavelength filters. Make sure you look at the
plate under both wavelengths.
b. Use a stain. There are several stains that can be used depending on
your preference and sample type. Typically iodine or
phosphomolybdic acid are used. If it is necessary your TA will
instruct you on how to use these.
12. After you have looked at the plate make sure to circle the spot(s) you see so that you can
look at them later. The visualization ability does not last forever. So it is important to
circle (with pencil) where your spot(s) are located.
13. Calculate the Rf for each spot using the equation below.
R =
istoncc spot tro:clcJ
istoncc sol:cnt tro:clcJ

*Each distance is measured from the baseline.

So you ran your TLC but something went wrong; what do you do?
So youve followed all the above steps and go to look at your TLC and you see:
1. One giant smear along the plate
2. Nothing
3. A single spot at the top
4. A single spot on the base line

What happened and how do you fix it?
1. Youve spotted too heavy. This means you have put too much compound on the TLC
plate. You can fix this either by spotting the same sample less times (on a new plate), or
you can dilute the sample. This means adding more solvent to the dissolved test sample.
2. One of two things could have happened:
a. You sample is not visible by UV. You can fix this by using a stain like Iodine to
visualize the sample.
b. You havent spotted heavy enough and you need more compound. You can fix
this by spotting the sample more heavily and running the plate again.
3. Your solvent system is too polar, so everything ran to the solvent front. You can fix this
by increasing the non-polar ratio of the solvent.
4. Your solvent system is too non-polar, so everything stayed on the polar baseline. You
can fix this by increasing the polar ratio of the solvent.
*In every case a new TLC must be run. You can never put the same TLC back in the TLC
chamber.
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Column Chromatography Technique:
1. Set up your column like Figure 5 shows.
a. You will be using the long chromatography column, fitting one end with stopcock
(be sure it has a frit in it) and the other end with the funnel.
b. Into a beaker weigh out enough silica to fill the column half full; take extreme
care not to breathe in the silica. Silica is ground glass and you do not want it in
your lungs.

2. Pour the silica into the column and then wet it with your chosen solvent system.
a. Use enough to wet all of the silica and run it through the column so that the wet
silica packs into place with no air pockets. Air pockets will disrupt the flow of
your compounds and could cause mixing of your spots.
b. Once the column is wetted, take care that it does not run dry until you are done
with your separation.
3. Now that your column is set up and your stationary phase is loaded and prepped you are
ready to load your sample.
a. Dissolve your sample in a minimal amount of your running solvent and pipet it
onto the top of the column.
b. Drain some solvent so that top of the sample is now even with the top of the silica
and place a protecting layer on top (either sand or cotton).
c. If using sand pour a small layer on top of the silica. If using cotton insert a small
plug of cotton and slide it down to the top of the silica. The sand or cotton will
protect the silica when you add additional solvent.
4. Now you are ready to run your column. There are two ways to run a column. Gravity
and Flash. The gravity method uses the force of gravity to determine the flow rate of the
mobile phase. The flow rate can be increased to some degree by extending the mobile
phase above the top of the stationary phase (additional mass for gravity to work on). The
flash method involves using some method such as a pump (a pipet bulb for example) or
gas flow to push the solvent through the column. Even though the solvent is moving
more quickly through the stationary phase the same equilibrium principles still apply and
so this is a way of accomplishing simple separations fairly quickly.
Figure 5
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*You will be using the Flash method and your TA will demonstrate how you will use the
Nitrogen lines to force the solvent through the column. Take care not to turn up the pressure
to high as there is a risk you can cause the glass column to explode.
5. Collect 1-2ml fractions in reaction tubes. TLC the fractions and elute the TLC plates in
the same running solvent as the column. You want to keep your fractions small to avoid
collecting two compounds in the same fraction. At the beginning, if there is nothing in
the fraction when you TLC it, you can dispose of it. This is because the first few
fractions are just running solvent from loading the column.
Practical Tips for both TLC and Column Chromatography
Remember that solvents evaporate at different rates; this means that a solution that started
out 80% Hexanes and 20% Ethyl Acetate will not have that same ratio after hour of sitting
in a beaker or graduated cylinder.
o If you are using the solvent system to run TLCs to monitor the progress of a
reaction then the changing ratio will give the same compound a new Rf value
each time a new TLC is run. This will prove to be a problem if you are
tracking the compound based on its initial Rf value.
o There are two ways to deal with this issue: Remake the solvent system every
time a TLC is run (this can be very time consuming and wasteful) or run a
comparative TLC each time.
A comparative TLC is one where compound or reaction mixture is
spotted next to the pure starting material(s) for reference. The plate is
then run as usual and this way any Rf changes due to a change in
solvent system is negated by the ability to identify all spots on the
plate.
Use TLC to determine the ideal solvent system before setting up the column so that it
wont dry out.
Always label the TLC plate.
o At the baseline under each hatch mark indicate what compound will be
spotted there (i.e. P=product, C=Crude, P=Pure, Rxn=Reaction, B=Biphenyl,
etc). Be consistent from plate to plate.
o In the space above the solvent from indicate the solvent system and if it is a
TLC monitoring a reaction, the time point. For example 20% EtOAc/Hexanes
5min.
When visualizing the plate circle everything that you see, not just the spots you expect.
This means if you see a spot at the baseline and two spots in the running lane you should
circle all three spots and calculate three Rf values.




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Experimental Procedure: Thin Layer Chromatography
Part 1: Identifying compounds by comparison:
The FDA separates color additives for foods into two categories: Certifiable or Exempt
from certification. Certifiable color additives are man-made and must be tested for consumption
safety and approved by the FDA. There are 9 certified color additives on the approved list.
Certified food color additives (or dyes) are polar and water soluble with a variety of uses.
The food coloring 4-packs that are purchasable in the grocery store contains vials of various
combinations of Yellow #5, Red #40, Blue #1, and Red #3. The dye combinations in each color
are shown in the chart below. They can be combined to make any color imaginable. Dye
standards purchased from a chemical supplier have only one dye present in each color and can be
used as references to identify the dyes present in commercial sources.






Procedure: Using a 1% NaCl solution run a comparative TLC of all the Standard colors.
Calculate the RF values for each of the spots present on the plate. Choose two of the store food
colorings. Run a comparative TLC of these two items in a 1% NaCl solution. Calculate the RF
values of each of the spots present on the plate. Compare the RF values of the Standards and the
commercial sources to identify the dyes present in each of the commercial sources. Your TLC
plates should look like the examples below. Record the Rf values in the chart on the next page.
Observations:





Color Dye name
Red Red #40
Blue Blue #1
Yellow Yellow #5
Dye Standards
Color Dye name
Red Red #40, Red #3
Blue Blue #1, Red #40
Yellow Yellow #5
Green Yellow #5, Blue #1
Black Red #40, Blue #1, Yellow #5
Store Food coloring
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Please fill in the store dye color you chose in the appropriate blank. In your report please make a
chart identifying the dyes present in each of the commercial sources you tested. It should look
similar to the Store Food Coloring chart in the introduction.
Part 2: Determining Purity of a compound:
In most synthesis experiments a TLC will be performed to determine if the compound is pure or
not. One mistake new chemists will make is to always use the same solvent system. If they only
see one spot they assume their product is pure (even if they dont see much movement from the
baseline) and carry on with the experiment. When running a TLC it is always a good idea to
adjust your solvent system until your spot has an RF of ~0.5. Doing this helps you to see if the
compound is pure in a variety of ways.
1. If your solvent system is too non-polar (or too polar) you may several spots overlapping
at the baseline (or the solvent front). Adjusting the system to move the spot to the middle
of the plate will cause these spots to spread out as it is unlikely they will have the same
RF values.
2. You can see if what you thought was a streak is actually two spots.
You have been given a mixture of compounds. It is up to you to find a solvent system that will
separate the spots from one another on the plate. Ideally one of the spots will be in the middle
(RF =0.5) but the key here is that you see three distinct spots. You will have to run several
TLCs to get this right; but practice makes perfect and this is a very useful skill. We have given
you a starting point; it is up to you what adjustments you make from there.
Procedure: Draw and label the TLC from each trial. The three compounds in the mixture are
Benzoic Acid, Benzophenone, and 3-nitrobenzoic acid. Indicate which spot you believe each
compound to be based on polarity.


Name Rf Value(s) Name Rf Value(s)
Red Store Dye 1________
Blue Store Dye 2________
Yellow
Standards Commercial
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Observations:
Your ideal plate Trial #1: 50% Ethyl Acetate Trial #2: Ethyl Acetate
50% Hexanes Hexanes
Rf of spots Rf of spots

Trial #3: Ethyl Acetate Trial #4: Ethyl Acetate
Hexanes Hexanes
Rf of spots Rf of spots

Trial #5: Ethyl Acetate Trial #6: Ethyl Acetate
Hexanes Hexanes
Rf of spots Rf of spots

Trial #7: Ethyl Acetate Trial #8: Ethyl Acetate
Hexanes Hexanes
Rf of spots Rf of spots
.
Part 3: Using Column Chromatography to Purify a Mixture of Compounds:
Procedure: Once you have identified the solvent system that gives you the ideal TLC in Part
2 set up a Column and isolate each of the compounds in the mixture. Typically once you have
isolated each compound you would boil off the solvent, verify the purity of the solid sample by
melting point and obtain a mass and percent yield for each individual compound. In this
situation since you are just learning the technique once you have isolated each compound and
verified this by TLC you can dispose of the fractions in the organic waste.
Record the TLCs from the Column purification on the next page. Record a comparative
TLC of the Isolated compounds to verify their purity. Examples of each type of TLC are shown
below.
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Observations/ Data: For all TLCs done, be sure to include a drawing of the plate
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Observations/ Data: For all TLCs done, be sure to include a drawing of the plate
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Observations/ Data: For all TLCs done, be sure to include a drawing of the plate
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Post Lab Questions
1. Rank the following compounds from most polar to most non-polar: amide, ether,
carboxylic acid, hydrocarbon, and ketone.



2. Which of these compounds is NOT UV active? Circle all that qualify.





3. Match the spots to the compounds







4. How would you fix this TLC plate?





5. How would you fix this TLC plate?