AP Chemistry Lab: Separation of Molecules by Paper Chromatography
One of the practical applications of intermolecular forces is the separation of a mixture of
chemical compounds by chromatography. This technique uses differences in intermolecular
forces of attraction between the mixture molecules and either a stationary phase or a mobile
phase to separate the components of the mixture.
Pre-lab: The following items must be completed and turned the day before the scheduled
laboratory session:
1. Write up the normal pre-lab components: heading, objective, safety, summary of
procedure, and a suitable table to record your data.
2. Using any computer with internet access, you will follow the steps below to perform a
series of simulations on molecular attraction and answer questions on these simulations.
a. Go to http://mw.concord.org/modeler/
b. Select “More” at the bottom of “selected Curriculum Modules”
c. Select “Intermolecular attractions” from the Chemistry Column
d. Run each of the ten simulations. At the end of each simulation, take a snapshot of
any diagrams and answer all questions.
e. On the final page, click on the bottom “Create a report of my work”
f. Print out a copy of your report and attach it to your pre-lab.
3. Read the attached background section.
4. Answer the following questions. Please note that running the simulations and answering
all of the required questions is more than one evenings work. It is strongly suggested that
you begin work on the pre-lab several days before the due date. If you do not turn in a
complete pre-lab assignment, you will not be able to do the lab and will receive a zero
grade.
5. Questions:
a. What does the Rf value describe on the molecular level?
b. Describe how you measure and calculate an Rf value.
c. If a molecule has very high affinity for the stationary phase, would the Rf value be
high or low? Explain.
d. List the following mobile phases from least polar to most polar: acetone, ethanol,
hexane, 2-propanol, water.
e. If Compound A is very polar, in which mobile phase would it have the highest Rf
value? The lowest?
NFA: AP Chemistry: W. Istone: 01/2014
Background:
There are two phases in paper chromatography, a stationary phase (the paper) and
a mobile phase (the solvent). A molecule can have a greater affinity for either the
paper or for the solvent. The filter paper is made of cellulose, a polymer. Cellulose
will attract water molecules to the exposed hydroxyl (OH-) groups along the
polymer. This interaction makes a thin layer of water on the paper that competes
for the attraction of the molecules being separated. Alternately, the molecule can
be attracted to the solvent and travel with the solvent up the paper. When doing
chromatography, a small amount of solvent is placed in a sealed container. The
mixture being separated is put on a piece of paper, the starting point is marked,
and the paper is put into the solvent. The container must be sealed so the solvent
saturates the paper and does not evaporate first. The level of separation is measured
by a ratio that compares the distance that the molecule travels to the distance the
solvent travels. This ratio is called the Rf value. To get the R, value, the experimenter
must identify the distance that the solvent traveled on the paper and measure the
distance. Second, the experimenter must identify the distance that each molecule
traveled and measure that distance. It is best to run the test more than once to
reach the best separation values possible. The Rf value is a ratio of the distance of
the molecule divided by the distance of the solvent. The greater the distance the
molecule travels, the greater its affinity for the solvent and the greater the Rf value.
In this experiment, students will have a choice of different solvents to use. When
they propose the best solvent for separation of the mixture, they will also need to
evaluate it in terms of "greenness:' In modern chemistry, chemists use principles
of green chemistry to evaluate the solvents that are used in a chemical process for
their level of toxicity to humans and the environment. Solvents are also evaluated in
terms of their life cycle or how long the molecule remains in the environment and if
the molecule breaks down to become more benign or more toxic. The overall focus
of green chemistry is to be more efficient in chemical production, producing less
waste, using fewer toxic molecules, and producing waste that biodegrades and does
not pose a risk to the environment. See Tables 1 and 2 in the GSK solvent selection
guide, available at http://pubs.rsc.org/en/contentiartic/e/anding/2011/gc/cOgc00918k
under "supplementary information;' for an evaluation of solvents based on these
guidelines. To understand the differences between solvents, look up the green
rating for hexane (found in chromatography solvent) and compare it to 2-propanol.
The food dyes that are in the mixture have their own green chemistry issues. For
example, the molecules used may have a life cycle that is longer than previously
anticipated and possibly increased toxicity. When scientists evaluate the toxicity
of molecules based on the experimental data, efforts to understand the origins of
toxicity often look at the molecular structure of the substance. A key strategy for
NFA: AP Chemistry: W. Istone: 01/2014
looking at molecular structure is to identify functional groups that are present.
There are specific functional groups that are known to create toxic by-products
when they are metabolized in the human body, such as acetaminophen that can be
converted to N-acetyl-p-benzoquinone imine. All three food dyes used in this lab
are azo dyes, which means that they contain a double-bonded nitrogen connecting
multiple aromatic carbons. While the molecules resemble one another, only red #40
has been linked to allergic reactions in some people, but the FDA has not found
conclusive evidence that such a dye is unsafe. In Europe these food dyes are not
used and natural pigments are used instead.
Procedure: Each team of two will analyze a mixture of the three dyes shown above
(Blue 1, Red 40, and Yellow 5) and two paper chromatography strip. Each team will run
a chromatogram in two different solvents. The “A” and “C” Teams will use water and
chromatography solvent (a mixture of hexane and acetone). The “B” and “D” teams will
use ethanol and 2-propanol. The two teams at each lab bench will share data so that
every student has chromatography data for all four solvents.
1. Safety: ethanol, 2-propanol, acetone, and hexane are all flammable, so no open
flames should be used. These four solvents all have high vapor pressures, so they
NFA: AP Chemistry: W. Istone: 01/2014
should be kept in closed or covered containers as much as possible and the
ventilation fan should be used.
2. Only write on the chromatography strips with pencil. Draw a line across the
strips 1.0 cm from the bottom and a second line 1.0 cm from the top. Label the
strips with the solvent name (or abbreviation) at the top.
3. Place a very small drop (use a toothpick) of the dye mixture in the center of the
line near the bottom of the strip.
4. Your teacher will demonstrate how best to hang the strip in an appropriate
container. Attach the hangar above the line near the top.
5. Pour about 5 mL of the solvent in the bottom of the container.
6. Hang the strip so that the bottom of the strip dips into the solvent, but so that the
solvent level is below the 1.0 cm line.
7. Allow the chromatogram to run until the solvent reaches the upper line.
Immediately remove the strip from the solvent and hang it to dry.
8. When dry, carefully measure the distance that the solvent has traveled from the
lower 1.0 cm line and the distance that each dye has traveled from this line.
9. Calculate the Rf value for each dye.
10. Prepare a table which displays the Rf values for each dye in each of the four
solvents.
11. The used chromatography solvent must be poured into a waste solvent container
in the fume hood. All other solvents may be rinsed down the drain.
Post-lab Questions:
1. Based upon your pre-lab information and your lab date, which dye is the most
polar? Which is the least polar? Explain.
2. Which solvent(s) provided the most useful analysis of the dye mixture? Which
provided the least useful? Explain.
3. Which parts of the three dye molecules are polar? Which parts are non-polar?
Reference: “AP Chemistry Guided-Inquiry Experiments: Applying the Science
Practices”, The College Board, 2013.
NFA: AP Chemistry: W. Istone: 01/2014