3. Separation of a Mixture into Pure Substances
Paper Chromatography of Metal Cations
What you will accomplish in this experiment
This third experiment provides opportunities for you to learn and practice:

Using the Classification System of Matter to designate materials as belonging in one of two categories:
pure substances (elements or compounds) or mixtures of pure substances.

Separating pure substances that have been physically combined into a homogeneous mixture (a solution)
using one of chemistry’s most sophisticated experimental methods: Chromatography. This separation will
take advantage of differences in the physical properties of these substances.

Using properties of known substances to deduce the identity of unknown substances.
Concepts you need to know to be prepared
The Classification System of Matter
Any sample of matter is either a pure substance or a mixture of pure substances.
Elements and compounds are pure substances. A compound is a chemical combination of two or more elements.
When elements react to form a particular compound, they always combine in a fixed proportion—the composition
of the compound is always constant. The proportion of the elements in a compound is expressed as the chemical
formula of the compound. For example, the chemical formula for water is H2O.
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A mixture is a physical combination of pure substances: elements and/or compounds. The components of a
mixture can be combined in any proportion—mixtures have variable composition.
Heterogeneous mixtures have multiple phases, while homogeneous mixtures (more commonly called solutions)
have just a single phase (they are consistent throughout).
Since mixtures can be made by physically mixing together two or more elements or compounds in any amounts,
the components of a mixture can be separated by physical means. The separation methods are based on
differences in the physical properties of the components.
Chromatography ~ A Method for Separating the Pure Substances in a Mixture
Chromatography was developed in the early 1900s by the Russian scientist Mikhail Tswett, who made an
extensive study of the colored pigments in the green leaves of plants. Tswett was able to demonstrate conclusively
that plants contain more than one type of chlorophyll; he did this by separating the different chlorophyll
compounds from one another.
Tswett managed his separation by grinding the green leaves of plants in a solvent called petroleum ether. He then
let this liquid trickle through a glass tube that was filled with powdered chalk. As the mixture seeped downward,
each pigment (each type of chlorophyll) had a different attraction to the chalk, so the pigments were separated and
became visible as different colored layers in the glass tube.
Twett called this new separation technique “chromatography” because the result of the separation was “written in
color” along the length of the glass tube (chroma is Greek for ―color‖).
Today the word “chromatography” refers to any technique that relies on separating two or more compounds
based on the way they distribute between two phases:

A solid phase that remains stationary

A liquid (or gas) phase that is moving (often called the solvent).
In Twett’s experiment, the chalk was the ―stationary phase,‖ and the petroleum ether solvent was the ―mobile
phase.‖
Chromatographic separation occurs because the various components in the mixture have different affinities for the
two phases, so they move at different rates. A component with a high affinity for the stationary phase moves more
slowly, while one with a high affinity for the mobile phase moves more rapidly.
To see this more clearly, imagine the plant pigments trickling down Tswett’s glass column. As each pigment
moves through the column, it’s asking itself the following questions:

Am I more attracted to the mobile phase or to the stationary phase?

Do I want to keep moving with the solvent?

Or do I want to stay put and stick to the chalk?
Because each type of pigment answers this question in a slightly different way, separation of the components in the
pigment mixture is achieved.
Paper Chromatography
While paper chromatography is one of the simplest types of chromatographic systems, it is highly effective for
many different applications. A strip of porous paper is used as the stationary phase. The mixture to be separated is
applied as a small spot near the bottom of the paper (this is called ―spotting‖ the sample). A fine glass capillary
tube is used for this sample application; when the tube touches the paper, capillary action delivers a portion of the
contents onto the paper, and a small spot is formed.
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The spotted paper is then placed in a chromatography
―chamber‖ (a beaker topped with a watch glass) which
contains the mobile phase. When the spotted paper is
dipped into this solvent, the liquid travels up the paper,
very much the way a paper towel soaks up a liquid spill.
As the liquid climbs the paper, the substances in the
mixture are separated based on differences in their
attractions to the paper (the stationary phase) and the
liquid solvent (the mobile phase).
When the solvent has reached nearly the top of the
paper, the paper is removed from the chromatography
chamber and allowed to dry.
If the mixture has been effectively separated, there will
be a series of spots on the paper, with each spot
corresponding to a particular substance from the original
mixture.
Under a particular set of chromatography conditions (a particular
solid and mobile phase), each substance that’s separated always
travels a characteristic distance relative to the distance that the
solvent travels.
The ratio of the distance the substance travels to the distance the
solvent travels is called the Rf value. The symbol Rf stands for
―Retention Factor,‖ and it is expressed as the fraction:
10.00 cm
12.00 cm
6.80 cm
In the sample paper chromatogram shown to the right, the Retention
Factor values for the two components would be calculated as follows:
Component 1:
Component 2:
Chromatography of Metal Cations
In this experiment, you’ll use paper chromatography to both separate and identify the metal ions in an ―unknown‖
aqueous mixture.
In your lecture course, you’ll have learned that ions are formed when an atom

Gains one or more electrons (forming a negative ion, “anion”), or

Loses one or more electrons (forming a positive ion, “cation”).
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The atoms of metal elements become ―cations‖ when they lose electrons; these positively-charged ions then
combine with negatively-charged ―anions‖ to form ionic compounds, or simply ―salts.‖
The ―unknown‖ aqueous mixture that you’re to separate by paper chromatography will contain some combination
of salts of the following four metals: Ag+ (silver), Co2+ (cobalt), Cu2+ (copper), and Fe3+ (iron).
How will you recognize which of these metal cations are in your unknown mixture?
In addition to ―spotting‖ a sample of your unknown mixture on the paper, you’ll also spot samples of solutions
which are known to contain just one of these metals. You’ll then be able to compare the movement of these known
metal cations to the separation results of your unknown mixture, and identify which of the ions were in your
unknown mixture.
All of the samples you’ll spot near the edge of a piece of filter paper will contain just a few micrograms of metal
cations. When you immerse the edge of that paper in solvent, the solvent will rise up the paper and carry the ions
along with it, to a degree that depends on the relative tendency of each ion to dissolve in the solvent and adhere to
the paper. Because the ions differ in their properties, they’ll move at different rates and thus become separated on
the paper.
The position of each ion on the paper can be recognized during the experiment if the ion is colored, as some of
these ions are. But at the end of the experiment, the position of each ion can be clearly established by treating the
paper with a staining reagent which reacts with each ion to form a colored product.
By observing both the position and color of the spot produced by each ion, and comparing them to the positions
and colors of spots produced by the separation of an unknown mixture containing only some of those same ions,
you’ll be able to identify the ions present in your mixture.
Procedure that you will follow
Paper Chromatography of Metal Cations
NOTE: Nitrile gloves are required for this experiment, as AgNO3 solutions will stain skin (and clothing). DO
NOT touch your gloved hands to your face while you are working. Be sure to dispose of your gloves and wash
your hands before leaving the lab.
1. Prepare a chromatography chamber by adding approximately 5-10 mL of the solvent mixture to a 600-mL
beaker. This solvent is a solution of hydrochloric acid (HCl) and the organic solvents ethanol and n-butanol.
The depth of the solvent in the beaker should be about 5 mm. The solvent must NOT touch the spot when the
paper is inserted into the chamber. Cover the beaker with a large watchglass so that the solvent vapor can fill
the chamber.
2. Place the following volumes of each solution in the corresponding wells of your plastic spot plate:
Well A-1: 8 drops of 0.1 M AgNO3 solution. This is your Ag+ ion.
Well A-2: 8 drops of 0.1 M Co(NO3)2 solution. This is your Co2+ ion.
Well A-3: 8 drops of 0.1 M Cu(NO3)2 solution. This is your Cu2+ ion.
Well A-4: 8 drops of 0.1 M Fe(NO3)3 solution. This is your Fe3+ ion.
Well B-1: 2 drops of each one of the four different metal ion solutions, for a total of 8 drops. Swirl the spot
plate to mix the solutions. This will be your known mixture, since you know it contains all four
metal ions.
Well B-2: 8 drops of one of the unknown test samples. This is your unknown mixture, since you do not know
which of the four metal ions it contains.
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3. Obtain a 10.0 cm by 18.0 cm sheet of chromatography paper. Using a pencil (NOT a pen), carefully draw a
line across the length of the paper, approximately 2 cm from a long edge. Then starting 2 cm from a short
edge, mark six small x’s along this line at intervals of approximately 2.5 cm. Label these as shown below, to
correspond to the six solutions in the wells of the spot plate.
x
Ag+
x
Co2+
x
Cu2+
x
Fe3+
x
All
x
Unk
4. Using a separate open glass capillary tube for each of the six solutions, ―spot‖ each solution at its respective
position on the line. Allow each of the six spots to dry thoroughly, then ―spot‖ each solution again in the same
place (keeping the spots as small as possible; NO LARGER than 2 – 3 mm in diameter). At least five rounds of
spotting are recommended for the pure samples, and twice that many for the mixtures (as these solutions are
more dilute). Be patient with this: the longer you continue spotting and drying (and spotting and drying) to
concentrate each spot, the better your results will be. When you’re satisfied with your spots, allow the paper to
finish air drying thoroughly.
5. When all spots are dry, carefully roll the chromatography paper into a large cylinder, and staple the ends
together. The stapled ends MUST NOT OVERLAP! Then place the rolled paper into the prepared
chromatography chamber, with the spotted end DOWN. Make sure the spots do NOT touch the solvent!
Replace the watchglass on the top of the beaker, then place the beaker on a hot plate turned to a very, very
LOW setting (ex: 2 out of 10 on the dial). The desired temperature is approximately 35oC (only about 10-15o
above room temperature).
6. Allow the solvent front to rise until it is approximately 1 cm from the top of the paper. Do NOT allow it to
reach the top of the paper!
7. Remove the paper, unroll it, and IMMEDIATELY mark the final solvent level with a pencil. Note that the
solvent level is NOT likely to be a straight line, so you must carefully trace any curves of the solvent front with
your pencil. You may dry the paper with the aid of a laboratory heat gun.
8. Then in the hood, and still wearing your nitrile gloves, LIGHTLY spray the dried paper with the staining
solution (a mixture of potassium iodide and potassium ferrocyanide). Be careful not to spray the staining
solution on your bare skin, or touch the sprayed area with your bare fingers.
9. Allow the paper to dry completely, using the laboratory heat gun, if necessary. Then circle each spot with a
pencil and mark its center.
10. Measure the distance from each initial sample spot (the point of origin) to the solvent front (directly above
each spot), and also from the point of origin to the center of each spot. Then calculate the Rf value for each
spot. Be sure to record all of your measurements and observations on the lab notebook portion of your Report
Sheet.
You MUST dispose of all chemical waste as directed by your lab instructor. Do NOT put any chemical waste in
the laboratory sinks or garbage cans – use the liquid waste containers in the hood. You MUST thoroughly clean
your laboratory glassware before replacing it in your equipment locker. Always wash your hands before leaving
the lab.
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Report Sheet 3: Separation of a Mixture into Pure Substances
Student ______________________________ Lab Partner__________________________ Date Lab Performed__________
Section #_________ Lab Instructor__________________________________________ Date Report Received ___________
Lab Notebook: Data and Observations
Results of Paper Chromatography ~ Individual Metal Ions in Wells A-1 through A-4
Ag+
Co2+
Cu2+
Fe3+
Cu2+
Fe3+
Color before staining
Color after staining
Distance traveled by
cation, cm
Distance traveled by
solvent, cm
Rf value (show work)
Results of Paper Chromatography ~ Known Mixture in Well B-1
Ag+
Co2+
Distance traveled by
cation, cm
Distance traveled by
solvent, cm
Rf value (show work)
Results of Paper Chromatography ~ Unknown Mixture in Well B-2
Test Sample # _______
Cations Identified
Color before staining
Color after staining
Distance traveled by
cation, cm
Distance traveled by
solvent, cm
Rf value (show work)
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Formal Report: Results and Conclusions
Discussion Questions
1. Why is it important to allow the solvent front to rise nearly to the top of the chromatography paper, rather than
just half-way? Explain.
2. Why is it necessary to mark the position of the solvent front immediately after removing the paper from the
chromatography chamber? Explain.
3. Which of the four metal ions had the greatest affinity for the mobile phase? __________________
Which of the four metal ions had the greatest affinity for the stationary phase? __________________
Explain your answers in terms of each ion’s Rf value and position on the chromatography paper.
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4. Explain the criteria you used to identify each ion in your unknown mixture. Also, describe any difficulty you
had in identifying the ions.
5. If the stained spots for all of the ions were the same color, would it still have been possible to identify the ions
in your unknown mixture? Explain.
6. Two extreme values for the Retention Factor are 0 and 1. Explain what each value means in terms of the
substance’s affinity for the mobile and stationary phase.
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