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Myeeka Hammond
May 31,2017
Chromatography

Purpose: The purpose of chromatography is to separate mixtures, more or less homogenous, into their
individual substances.

Introduction: Chromatography is a technique used for the analysis and separation of chemical mixtures.
There are several ways to accomplish this but one thing that all chromatography has in common is the
use of a mobile phase and a stationary phase. The stationary phase is motionless and is the actual
medium that performs the separation. The mobile phase is what moves (usually a liquid solvent or a gas)
and carries the mixture through the stationary phase. The different individual compounds in the mixture
all interact with the stationary phase differently and move through it at different speeds, thus giving rise
to the separation. Thin layer chromatography (also known as TLC) is a physical separation of a mixture
into its individual components by distributing the components between a stationary phase which is the
porous TLC plate and a mobile phase in which the solvent moves through the stationary phase and
carries the material that needs to be separated. The driving force to separate these components is
called capillary action. This method can be used to determine how many different components (usually
non-volatile) are in a sample. The following are some common uses of Thin layer chromatography: to
determine the number of components in a mixture, to determine the identity of two substances and to
monitor the progress of a reaction. The difference each molecule travels along the absorbent in relation
to how far the mobile phase has traveled is called the Retention factor (Rf) and can be used to identify
molecules, as the value is molecule specific. The Rf value for any given molecule will vary depending on
the mobile and stationary phase used. Another type pf chromatography is Column chromatography.
Column chromatography is a method to physically separate all of the components (also usually non-
volatile) of a mixture. The driving force to separate these components is gravity. Both methods work
based on polarity differences between components in a sample. The columns stationary phase and it is
contained in a column while the mobile phase: liquid; passes though column gravity or pressure. In a
typical column, the stationary phase, a solid absorbent normally like silica gel (SiO2) or alumina (Al2O2) is
placed in a vertical glass column. The mobile phase, a liquid, is added to the top of the column and flows
down through the column by either gravity or an external pressure (flash chromatography). Separation
of compounds is achieved through the varying absorption on and interaction between the stationary
and mobile phase.

Part 1: Thin Layer Chromatography

Analysis of a mixture of colored compounds
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Myeeka Hammond
May 31,2017
Chromatography

Materials:

1. TLC plate
2. Pencil
3. Capillary spotters
4. 50 mL beakers
5. 250 mL beaker
6. Filter paper
7. 2:1 hexane (C6H14) ether (C2H5)20
8. 2-nitrophenol (C6H5NO3)
9. 2-nitroaniline (C6H6N2O2)
10. 4-nitroaniline (C6H6N2O2)
11. Ferrocene (C10H10Fe)
12. UV light
Procedure: After we obtained the TLC plate, we drew a line across the bottom about 1 cm from the
bottom and on that line, we drew 5 dots. The second thing that we did was obtain the compounds that
we were going to use for the chromatography and dissolved them in a 2:1 mixture of hexane and ether.
Once the solutions were dissolved, we used a capillary spotter, in each compound, and dapped it the
TLC plate. We then poured a few mL of the 2:1 hexane and ether mixture into a big enough beaker to
cover the entire plate, and then placed the plate in the beaker so it can absorb the solution. After the
solution made its way to the top of the plate, we took the plat out and placed it under a UV light so we
could see how far the compounds moved up the plate. To see the actual distance that the compounds
traveled, we used a ruler and measured from the line we drew on the plate to spot that it stopped
traveling.

Analysis of Analgesic Drugs

Materials:

1. Aspirin (C9H8O4)
2. Acetaminophen (C8H9NO2)
3. Caffeine (C8H10N4O2)
4. UV light
5. Hot water bath
6. TLC Plate
7. Pencil
8. Filter paper
9. CH2Cl2: methanol mixture
10. Ch2Cl2: methanol: acetic acid mixture
11. Big beaker
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Myeeka Hammond
May 31,2017
Chromatography

procedure: The first thing we did was obtain a tablet for our unknown and a few samples of the other
three compounds to use for testing. We then crushed the tablet on a filter paper and placed the powder
into a test tube. Then, we added 1 mL of a 1:1 mixture of CH2Cl2 and methanol. We then set up a water
bathe in a fume hood and placed the test tube in the bath, heating gently and stirring the mixture for a
few minutes. Once it was done boiling, we used a capillary spotter to spot our unknown and the other
compounds on to a TLC plate. After spotting the compounds, we placed the TLC plate in a beaker which
contained a solution of CH2Cl2: Methanol: acetic acid mixture. When the solution in the beaker reached
the top of the TLC plate, we took it out and then placed the TLC plate under a UV light to see how far the
compounds traveled. To get the actual distance that the compounds traveled, we used a ruler and
measured from the line at the bottom of the plate to spot that the compounds stopped at.

Data and observations: For the first part of the experiment the results were a little weird. Some of the
compounds, 2-na and 4-na, resulted in an Rf value of 0 because the compounds did not move on the TLC
plate. There are a few possibilities on why this could have happened, but I believed is that the solution
that we used in the beaker was not strong enough to move the substances up the TLC plate. Another
possibility is that not enough compound was placed on the TLC plate s resulting in it not reacting with
the solution from the beaker. Another observation that can be made is that out 5 compounds tested
Ferrocene was the most polar compound because it reached the highest on the TLC plate. The other
compound that reached high on the TLC plate, basically the same height as Ferrocene, was the
unknown. Based on that observation I would say that the unknown compound that was used in the
experiment is like Ferrocene or it is the same compound. For the TLC of the analgesic drug the most
polar substance ended up being the antacid because it traveled the most distance on the TLC plate. On
the other hand, the Tylenol ended up being the least polar because it traveled the least distance. For the
unknown that we choose, it had the same polarity as Excedrin and it traveled the same distance as
Excedrin, so it is safe to say that they are the same compound or are very similar.

Part 2: Column Chromatography (Separation of a Fluorene/Fluorenone mixture)

Materials:
1. Glass wool
2. Sand
3. Alumina (Al2O3)
4. Chromatography column
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Myeeka Hammond
May 31,2017
Chromatography

5. Ring stand
6. Hexane (C6H14)
7. 0.2 grams of Fluorene(F2)/fluorenone(C13H8O)
8. 0.5 mL of toluene (C6H5-CH3)
9. Glass rod
10. Pipet
11. Beaker of any size
12. 9:1 Hexane (C6H14)/acetone (C3H6O) mixture
13. TLC plate
14. UV light
Procedure: The most important step in the column chromatography is preparing the column. Once we
obtained the column, we added glass wool, pushed all the way to the bottom with a wire, then added
some sand followed by alumina and some more sand on the top. After preparing the column, we
obtained 0.2 grams of the fluorene/fluorenone mixture and dissolved it in about 0.5 mL of toluene. The
first time we poured only hexane into the column and immediately after we added the mixture of
toluene and fluorene/fluorenone on top of the hexane and allow it to run through the column and into a
test tube. After this we added hexane, in 1mL increments, to the column for about 5 or 6 more times to
drain out all the fluorene. Once all the fluorene was out, we eluted with a 9:1 hexane/acetone mixture
and allowed the column to drain in more test tubes. After all the yellow disappeared from the column
we obtained a few TLC plates, drew the line and the dots, we placed the solutions from all the test tubes
onto the dots and placed the plates in a beaker containing the 9:1 hexane/acetone mixture. Once we
ran the TLC chromatography, we placed the plates under the UV light to see if the column
chromatography was a success or not.

Data and observations: After we added the fluorene/fluorenone mixture to the column, the alumina
turned a yellow color but It did not change the color of the solution going in to the test tubes until the
4th or 5th time. To get that yellow color out we had to change the eluant to a different mixture. When the
solution coming out was clear the compound fluorene was being separated and when the solution
started coming out yellow the compound fluorenone was coming out of the mixture. If the hexane
eluant is not ran through the column a decent amount of times it will result in wrong results, I learned
that the hard way and had to do it twice. Also, the first time I did the experiment I did not weigh out the
correct amount of the fluorene/fluorenone mixture and I think it caused the results to be wrong as well.
But the second time around I got it right. When I tested the solutions from the test tubes on the TLC
plate, the first spot had nothing, showing it was only the hexane, the next couple of spots had two
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Myeeka Hammond
May 31,2017
Chromatography

different compounds until it went down to only one compound, the fluorenone, which is the more polar
than fluorene. The most difficult part of this experiment is knowing when to switch the eluant to get the
other substance out of the column.

Overall Conclusion: Overall the experiment was a good one, a little confusing as part A of the TLC
chromatography did not have any procedures and we had to choose which steps to do and had to ask
the professor for help. Another thing that was a little confusing was the column chromatography. I think
it can be a little easier to understand with clearer instructions. For the column chromatography, the first
time that I did it the TLC plate showed two compounds and then on the very last one it only showed one
compound but it was the least polar compound that it was showing. Based on that I knew I did
something wrong because the very last spot should be the one that is most polar. The second time
around, I ended with more test tubes and more of a range of colors from clear to yellow. After doing the
TLC plate for the second time around, the last spot from the very last test tube resulted in a polar
substance and it only showed one substance on the plate. My prediction for this error the first time
around, is that I did not do the eluting of the column correctly or I did not elute enough times with the
hexane only.

Post lab questions:

1. Identify the components of your mixture of colored compounds, and, in two sentences,
explain the basis for your conclusion.
2. Identify your unknown analgesic drug, and, in two sentences, explain the basis for your
conclusion.
The unknown analgesic drug is Excedrin. This is because the polarity of the unknown matches the
polarity of the Excedrin tablet.
3. On the basis of the results of your TLC analysis, list the analgesic standard compounds in order
of decreasing polarity. In one sentence, explain your reasoning.
From lowest to highest in polarity: Acetaminophen, caffeine, and aspirin. The reason behind this is
that the higher the spot traveled the more polar it is.
4. Why is it important to apply the sample to the column in the smallest amount of solvent
possible? What do you think would have happened if you had used too much solvent in this
step?
Excessive solvent may cause your sample to begin moving through the column before youve started
to run your gradient. If this were to happen it throw off your chromatography later on; indicating
your compound came off the column before it actually did and would change how you would
categorize the compound.
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Myeeka Hammond
May 31,2017
Chromatography

5. What is the purpose of the sand on the top of the column? On the bottom of the column?
The purpose of the sand at the bottom of the column is to create an even bedding for the stationary
phase foundation that then narrows towards the end of the stopcock. The sand at the top prevents
disruption of the well packed stationary phase when additional solvent (eluent) is added to the
column.
6. Now that you have experienced both techniques, compare and contrast melting point
determination and TLC as methods for detecting impurities I a solid compound? Explain your
answer
A melting point can tell you if an impurity is present through depressed melting points that are
broadened. Since the depressed melting point is a result of disrupted intermolecular forces, the
impurities could be nearly anything. In TLC, you can determine not just the impurities present, but
how many depending on the number of spots observed. Additionally, you may identify the given
impurities by Rf where this is less likely with melting point impurities. One benefit to melting points
however is that if two compounds have identical melting points and are mixed, the depression will
still show an impurity. If two impurities have the same Rf value, there will be no way to observe this
as there is with melting points.
7. An orange compound, dissolved in chloromethane, is added to a chromatography column. The
elution is begun using hexane as the solvent. After 6 L of the solvent was passed through the
column, the orange band had still not traveled down the column appreciably. What should be
done to make this experiment better?
Hexane is an extremely nonpolar solvent with little to no ability to move polar compounds along the
stationary phase. Increasing the solvent polarity is ideal in this case. It is important to do this in
increments and not all at once. Switching to something as strong as methanol could flush the whole
column out with no separation giving the opposing effect.
8. A sample was placed on a chromatography column. Dichloromethane was used as the eluting
solvent. No separation of the components in the sample was observed. What might have gone
wrong? How would you change the experiment in order to overcome this problem?
As stated in the previous problem, a solvent that is too polar is also problematic. In this case the
samples likely all got flushed off the column and did not separate. Reducing the polarity may help.
Try 50/50 mix oh hexane to CH2.