Paper Chromatography
(using a learning cycle approach)
Welcome: This is the second of a four-lab experiment sequence, covering four important
aspects of chemistry, and utilizing a learning cycle.
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Lab 1 Making Experimental Observations: chemical reactions and properties
Lab 2 Paper Chromatography: chemical interactions and separation techniques
Lab 3 Conservation of Mass
Lab 4 Limiting Reagents
Learning cycle: to help you better understand the chemical concepts in each lab
experiment, we will employ a three-step learning cycle.
Step 1: We will ask you and a partner to go into the lab, follow a procedure, and
make observations.
Step 2: You will then be asked to review material that provides an explanation about
what you observed in lab.
Step 3: Before going back into the lab, you will be asked to apply your understanding
by designing experiments. You and your partner will then go back into the lab to conduct your
experiments and gather experimental results. Designing and carrying out your own experiments
can be a very challenging part of the lab and requires very careful thought.
Paper Chromatography: to help you better understand the chemical concepts in
each lab experiment, we have incorporated descriptive information about paper chromatography
into today’s lab activity.
Learning Goals:
a) To understand the chemical names and symbols for some common metal ions.
b) To use the laboratory technique of paper chromatography to separate and identify metal
ions in an aqueous (water) solution.
c) To learn about paper chromatography
d) To design an experiment to determine the metal ions present in an unknown solution.
Lab 2 LC 1
Paper chromatography. Chemistry has a wide application in many real-world
uses. Have you ever watched a crime-scene investigation TV show that dealt with identification
of a written document, such as a ransom note? Aside from their handwriting, the criminal can
also be identified by the type of pen they use. Scientists can utilize a method known as
chromatography to separate different components of a pen’s ink. Since each model of pen has a
different makeup of ink, this separation technique can be used to narrow down the possibilities
for the type of pen used.
Paper chromatography is a specialized form of chromatography that is relatively fast and
inexpensive yet powerful enough to separate a range of different chemicals. This type of
chromatography uses a piece of paper and a solvent to separate the components of a mixture.
You will use paper chromatography in this experiment to separate and identify metal ions in an
aqueous solution. In this experiment, a strip of chromatography paper will be spotted with
several known samples of metal ions and developed using a mixture of acetone and hydrochloric
acid. After measuring the distance that the solvent moves up the strip of paper (the solvent front
distance), the paper is dried and then treated with several solutions that cause the samples to
change color. The distance that the sample has moved can be measured and the Rf values (ratio
of distance traveled by sample to distance traveled by solvent) for each sample can be
determined. The paper strip with the developed sample spots is called a chromatogram.
Atoms can readily gain or lose electrons. If electrons are removed or added to a neutral atom,
a charged particle called an ion is formed. Because opposites attract, positively and negatively
charged ions are attracted to each other and form compounds called ionic compounds. The
positively charged ion (called a cation) is often a metal and the negatively charged ion (called
an anion) is often a nonmetal. Many of these ionic compounds will dissolve in water to form
the individual ions of the compound. For instance, sodium chloride (NaCl) dissolves in water to
form sodium ions (Na+) and chloride (Cl-) ions. In fact, natural water sources contain metal ions
including sodium (Na+), magnesium (Mg2+), calcium (Ca2+), and iron (Fe3+). They can also
contain harmful metal ions such as lead (Pb2+). In this experiment, you will be determining how
metal ions move and interact with solvent and paper.
Lab 2 LC 2
Pre-lab Assignment
In your lab notebook, after reading the lab procedure, please prepare the following information:
At the beginning of lab, hand in the copy of your Pre-Lab Assignment and be prepared to discuss it.
a) Please write a 2-3 sentence introduction to the lab.
b) A table of safety information including the chemicals used in the lab and any safety
handling precautions. This information can be obtained using the list of chemicals found
below, looking up the appropriate MSDS (material safety data sheets), and understanding
how the chemicals will be used in this experiment. The MSDS can be accessed by the
link attached to each chemical in the Chemicals & Equipment Table below.
c) Please answer the following questions:
i. Describe your familiarity with separating things by chromatography. Perhaps you have a
water softener in your home or you may have done an experiment using a coffee filter to
separate colors in markers. Explain.
ii. Imagine a strong versus a weak chemical interaction. On the atomic level, draw
what you think might be happening with a strong or weak chemical interaction.
Table of Chemicals and Equipment
Chemicals
0.25 M copper (II) nitrate for the
2+
Cu ion solution
0.25 M nickel (II) nitrate for the
2+
Ni ion solution
0.25 M iron (III) nitrate for the
3+
Fe ion solution
0.25 M cobalt (II) nitrate for the
2+
Co ion solution
Unknown consisting of one or more
of the above solutions
Chemicals
Acetone
6 M Hydrochloric acid (HCl)
Equipment and Supplies
Chromatography filter paper,
paper towel, pencil, ruler,
capillary tube, beaker, 3 pint
bottles with corks
8M Ammonia Hydroxide
(NH4OH)
1 % dimethylglyoxime (DMG
in ethanol) solution
Reagent Ethanol
Instructions before going into lab:
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You should work in pairs for this experiment. If there are an odd number of students, then
one group can have three people. Write the name of your partners in your lab notebooks.
Each pair of students should discuss and answer questions as they encounter them. These
questions are set off in the experiment (i.e. “Q:”). The questions offer guidance during
the lab. Answers to these questions do not need to be reproduced in your lab report.
Each student is to record notes and observations and all other data in their own lab notebook.
Each student is responsible for collecting the information needed to complete the Lab Report
and Post Lab Portions of the Experiment.
Goggles are required at all times in the lab. There are no exceptions. Gloves and foot
covers are available.
Lab 2 LC 3
Step 1 of our Learning Cycle: the following activities should
be completed in the lab:
a) Acquire three glass pint bottles with cork stoppers, three 8-inch (20.5 cm) strips of
chromatography paper and a solvent mixture of 90 mL of acetone and 10 mL of 6 M HCl.
b) Hold the paper vertically. Towards the bottom (about 1.5 cm or 5/8” inch away from the edge),
draw a straight line across the paper using a PENCIL. Draw two small X’s spaced evenly on
the line to mark the initial spots for your samples. Label these marks "Cu" and "Ni".
c) Place the end of a clean capillary tube in the Cu2+ solution, hold the tube perpendicular to the
strip, and gently touch the tip of the tube to the previously marked X on the paper. Allow the
solution to form a small spot that is about 3 mm in diameter (if the spot is any larger, you will
get poor resolution). Allow the spot to dry. Dip the capillary tube in the Cu2+ solution again
and dab it on the spot that was just created. Let the spot dry and repeat a third time. The goal is
to make a small but concentrated spot. Then clean the capillary tube by rinsing it thoroughly
with distilled water in your wash bottle and then repeat the spotting procedure on the other X
with the Ni2+ solution.
d) When handling these strips, avoid contamination with the solutions and chemicals present in the
laboratory. Place them on a clean paper towel and always hold the strips at the sides with your
fingertips.
e) Repeat the spotting procedure on the second piece of paper, using the
Fe3+and Co2+ solutions.
f) Repeat the spotting procedure a third time, selecting two known metal
ions. This will allow you to gather some duplicate data. You may choose
from among any of the known metal ions.
g) While the strips are drying, prepare the solvent mixture. Mix 90 mL acetone and 10 mL 6M
hydrochloric acid in a beaker. Pour about one third of the mixture into each of the three glass
pint bottles.
h) Insert one of the prepared chromatography strips into
each of the bottles so that the bottom of the strip just
touches the liquid. Make sure that the solvent does
not touch the ion spot. (You do not want the spots to
be submerged, as the ions will dissolve in the solvent
and the experiment will be ruined.) The strip should not
touch the sides of the bottle and the top part of the
paper should extend out of the top of the bottle. Fold
the top part of the paper over the edge of the bottle and secure the strip by inserting the cork.
Observe your sample as it develops. Repeat this procedure using the other two strips, so they
Lab 2 LC 4
are all developing at the same time.
Q: What is happening to the solvent level on the rectangular piece of paper? Is it moving and if
so, in what direction?
Q: What is happening to the marked spot you placed on the paper as the solvent moves up the
paper? Do you think this is related to anything else going on in the experiment (direction of
solvent, etc.)?
i) Allow the solvent to move approximately 10 cm up the strip (just to the shoulder of the
bottle). Do not allow the solvent front to come near the cork in the top of the bottle. If any
colors appear on the strip while it is in the solvent be sure to record the colors you observe.
Remove the cork and carefully pull out the paper. Immediately draw a straight line at the
highest point of the solvent level (the solvent front) with a PENCIL. Let your sample dry for
a few minutes in open air. Mark the leading edge of any spots (the end of the spot closest to
the solvent front) that you observe once you take the paper out of the bottle. Some of the
spots may not be visible yet.
Health Warning: the procedure below is to be done under the hood. Exposure to ammonia
at low concentrations may produce rapid skin or eye irritation. Higher concentrations of
ammonia may cause severe injury and burns.
j) Perform the following procedure under the hood. Move each chromatogram back and forth
above an open bottle of Concentrated Ammonia (NH4OH) solution. You will see the vapors of
the ammonia gas reacting with the HCl on the paper to form a white gas of ammonium
chloride (NH4Cl). If a spot develops, mark its leading edge and record the color. Finally, spray
each of your chromatograms with a mist of 1% dimethylglyoxime (DMG in ethanol) solution.
Again, mark the leading edge of any new spots observed, and record their colors, if any.
Lab 2 LC 5
Step 2 of our Learning Cycle: The following activities should
be completed out of the lab (e.g. in the break-out room).
Chromatography Exercise. Before analyzing the results of the paper
chromatography experiments you did in Step 1, we’d like you to do the following exercise to
help you think about analyzing chromatography strips. (Please work with your lab partner.)
Consider the following three chromatography strips, representing the same sample at three
different times in the developing process: t = 10 min, t = 12 min, and t = 14 min (from left to
right in the figure below). Note that in the strips, some of the measurements are indicated to
give you an example of the type of data you need to record.
Using a ruler, measure the distance, in centimeters, from the starting line to the solvent front
on each of the strips and record the data in the table below. [Note: since the figure may print
at a different scale, use the distances consistently from your ruler.] Next measure the distance
from the starting line to the leading edge of the spot and record the data in the table below.
Finally, calculate the ratio of the distance the spot moves to the solvent front; this ratio is
called the Rf value.
Time
Distance
Spot Moves
Distance Solvent
Moves (solvent front)
Ratio: distance spot moves to
distance solvent moves [Rf value]
Q: What characteristic of the spot is independent of the time at
which the spot is observed? (Hint: think about the values in your
table.)
Q: Now consider the two samples 1 and 2 shown at the left. Can you
make an educated guess which sample would be the same metal ion
used in the exercise above? Explain your reasoning
Lab 2 LC 6
Paper Chromatography Reading. Please continue working with your lab
partner for the next activity, which involves reading the passages below and answering the
questions as a way to understand chromatography. Each student in your group will be asked to
fill out a separate worksheet in completing the following activity but you are encouraged to discuss it
together. The worksheet should be included in your lab report. Please note that you will be
asked to draw a picture, which may be drawn on a separate sheet of paper. This drawing
should also be included in your lab report.
[The following information is based on Chemistry in the Laboratory, 5th Ed, by Postma et al, 2000
and Introductory Chemistry, 2nd Ed Lab Manual by Gloffke & Kimbrough, 2002]: “Chroma-“ means
color. Chromatography was first described by a Russian botanist named M.S. Tswett, who used the
technique to separate plant pigments. He packed a glass column with an absorbent powder and then
poured a solution containing the pigments down the column. The solution separated into different
colored bands. Many of today’s techniques don’t produce visible colors, but are still called
“chromatography”.
What all chromatographic techniques have in common is the separation of components in a mixture
by their varying attractions to two materials: the moving phase and the stationary phase. In today’s
paper chromatography lab the components of the mixture are the metal ions (Ni+2, Cu+2, Fe+3, and
Co+2), the stationary phase is the paper, and the moving phase is the solution of acetone and HCl. As
the moving phase travels, the metal ion components spend some of their time on (adsorbed on) the
paper, and some of their time in the solution. Metal ions that have a greater attraction for the solution
will be carried along quickly. Metal ions that have a greater attraction for the paper will be slowed
down. Chromatography takes advantage of differences in properties between the moving phase and
the stationary phase. For example, biochemists may need to separate proteins with very different
sizes. They often use a column packed with a very porous stationary phase. Small proteins can fit
into the pores easily: they travel through the column very slowly as they wind in and out of the
pores. Very big proteins can’t fit into the pores at all and move down the column quickly.
The properties of the moving and stationary phases that cause the mixture components to separate
are consistent, so long as conditions remain the same. So if metal “X” travels up your paper
chromatogram faster than metal “Y” today, it will be always be faster. The Rf values will be
consistent as well, so long as all the conditions are the same. This is what makes paper
chromatography a good identifying technique.
Other types of chromatography include thin layer chromatography (TLC), column chromatography,
and gas chromatography (GC). In TLC, the stationary phase is a thin plate of plastic or glass covered
in fine particles. The mobile phase travels up the plate by capillary action, just as in paper
chromatography. TCL is usually much faster than paper chromatography and the resolution of the
spots can be easier. However, it can be difficult to get the fine particles on the plates uniform,
whereas the paper used in paper chromatography is very uniform. This can lead to more uniform or
consistent Rf values with paper chromatography. Column chromatography is what Tswett used. The
Lab 2 LC 7
stationary phase is a fine solid packed into a vertical column. The mobile phase is a liquid that pours
down the column. Gas chromatography is very widely used. In this technique the stationary phase is
a liquid packed into a horizontal tube. The mobile phase is a gas that is pushed through the column.
Please answer the following questions:
Q How did chromatography originate?
Q What do all chromatographic techniques have in common? How do they differ?
Q Suggest an explanation, at the molecular level, on why the metal ions traveled different distances
up the paper.
Q: In your lab experiment, what is the evidence that different metal ions interact differently with
the chromatography paper?
Analyzing Chemical Observations from Step 1: Now we’d like you to
analyze the results from Step 1. Please fill out the table below and answer the questions (you
may do this individually and then compare charts or together). Known Metal Ion
Distance
Spot Moves (specify
units)
Distance Solvent
Moves (solvent
front)
Ratio: distance spot
Observations?
moves to distance
solvent moves [Rf value]
Q: What claims can we make from our laboratory observations (Step 1)? Explain your
reasoning.
Lab 2 LC 8
Paper Chromatography Worksheet Name: ______________
When metal ions are spotted on the paper, placed in a bottle with solvent, what is observed? Please
provide an explanation for how this occurs.
Draw a picture of your explanation of what happens during paper chromatography.
Comparing two different metal ions, how would you explain differences in the ratio of the distances the
metal ion moves to the distance the solvent moves?
Lab 2 LC 9
Step 3 of our Learning Cycle. You may start this step in the
breakout room before proceeding to lab.
Based on what you have learned in the first two steps of the learning cycle, we
would like you to design an experiment to identify metals in an unknown
solution containing 1-3 unknown metal ions.
Write out a procedure to determine your unknown and explain it to your TA.
Obtain an unknown, then go back into lab to test your procedure. Good luck!
Answer the following questions once your investigation is completed:
Q: Based on your procedure, were you able to successfully identify your unknown solution? This
should be part of your claim in your lab report. If you are able to make a claim of identifying the
unknown solution, explain your evidence and reasoning.
Q: Based on the calculated Rf values, rank the interaction of the metal ions in your unknown
solution with the stationary phase (the paper) from least (starting with 1) to greatest. If you have
a larger Rf value, does it mean that you have more or less interaction with the stationary phase?
Staple your chromatograms into your lab notebook and make a drawing for
your lab report.
PLACE ALL EXCESS and USED CHEMICALS IN THE PROPERLY MARKED WASTE
CONTAINERS.
Lab 2 LC 10
Designing Experiments Worksheet – Hand in at end of lab
You have been asked to design an experiment to identify metals in an unknown solution
containing 1-3 unknown metal ions. Please fill out this form and turn it in at the end of lab.
Lab Partners: ________________ _______________ Lab performed on date: _________
Signatures:
________________ ________________
Before going
into lab:
Designing Experiments: Procedures, Observations, and Results
Describe experiments & predictions: (attach extra pages if needed)
Please describe your
proposed
experiments before
performing any
experiments
Any predictions?
We recommend
explaining your
experiments to your
lab instructor before
performing them.
While in lab:
Observations and results: (attach extra pages if needed)
Describe data in lab
and changes in
procedures. Can you
support any claims?
While in lab:
Any special issues to
report?
Lab 2 LC 11
AFTER LAB: Post-Lab Discussion and Assignments:
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Before starting the post lab discussion, groups should have already discussed their
evidence to identify their assigned unknown, answering the Q: What claims can we
make about our experiments? Also, the Q: Were their any issues with our
experiments?
Next, groups should discuss with the lab instructor and other groups how they
designed their investigation and their claims. Particular care should be made to
communicate how the experiments were conducted and the reasoning behind the
analysis to make their claim.
Groups may want to note variations in how experiments were conducted and if
some experiments worked out better because of their design.
Using your understanding of paper chromatography, describe the chemical
interactions that lead to one chemical moving at different rates along the paper.
Laboratory Report
Instructions for completing your lab report are posted at:
http://umaine.edu/generalchemistry/files/2011/08/Lab_Grading_Guide_Spring_2012_V1.0.pdf
Remember to include your paper chromatography worksheet as well as your drawings
in your lab report. Please attach a copy of your worksheet to your lab report.
Here are some points to include in your lab report:
a) An introduction, which includes a description of your experiment to identify your
unknown.
b) Your claim, underlined, following the introduction.
c) The reasoning your group used to make your claim.
d) In practical terms, describe how paper chromatography works
e) Answering the question: How challenging was it to develop a procedure to identify an
unknown?
Remember that scientific explanations have three components: claim, evidence and
reasoning. More information about claims, evidence, reasoning, and instructions for lab
reports can be found at:
http://umaine.edu/general-chemistry/files/2011/08/Lab_Grading_Guide_Spring_2012_V1.0.pdf
Lab 2 LC 12