Chemistry 11
Santa Monica College
Paper Chromatography: Separation and
Identification of Five Metal Cations
Objectives
Known and unknown solutions of the metal ions Ag+, Fe3+, Co2+, Cu2+ and Hg2+ will be analyzed
using paper chromatography. An unknown solution containing some of these cations will be
identified by comparison to the Rf values and colors of the stained spots of known solutions.
Background
Most chemists and many other scientists must routinely separate mixtures and identify their
components. The ability to qualitatively identify the substances found in a sample can be critical.
For example, an environmental chemist investigating samples of polluted ground water will want to
know which toxic ions might be present in a sample.
Chromatography is one of the first tools used in such situations. In this technique, many types of
mixtures can be separated into the component pure substances; by comparison to a standard
sample, each component substance can also be tentatively identified.
Many varieties of chromatography exist, each one designed to separate specific types of mixtures.
The common feature of each type of chromatography is that a mobile phase (a liquid or gas) is
pushed through a stationary phase (a solid). Table 1 lists several varieties of chromatography and
typical identities of the phases. Paper chromatography will be used in this experiment.
TABLE 1
Type of Chromatography
Gas (GC)
Liquid
(LC ,HPLC, column)
Paper
Thin-Layer (TLC)
Mobile Phase
Stationary Phase
inert gas
(helium)
solvent/solvent Mixture
(organic or aqueous)
solvent/solvent Mixture
(organic or aqueous)
solvent/solvent Mixture
(organic or aqueous)
waxy liquid or silicone
inside narrow tubing
solid packing
(silica, alumina)
paper
silica/alumina coated glass,
plastic or metal
The example of column chromatography (Figure 1) demonstrates the typical features found in this
analytical technique. The diagram shows an experiment where a two-component mixture is
subjected to column chromatography. The column is packed with a solid material called the
stationary phase. A liquid solvent or eluting solution is poured into the column and completely wets
the solid packing material. Then the mixture is loaded onto the top of the wet column and more
eluent is added. Gravity pulls the mobile phase down through the stationary phase and the
components in the mixture start to move through the column at different rates. In the diagram,
component A moves faster than component B; thus component B is retained on the column for a
longer time than component A. Usually this is due to a difference in solubility of the two compounds
in the solvent and/or to a difference in attraction to the solid packing material. As more eluent is
added to the top of the column, the components will eventually exit the column separately. The
time taken to exit the column, called retention time, will be reproducible for each component under
the given set conditions—mobile and stationary phase identities, temperature and column width.
Once the components exit the column, the solvent can be removed by evaporation and the pure
components can be further analyzed or identified.
Paper Chromatography
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Chemistry 11
Santa Monica College
Component B
Component A
Stationary Phase
with Eluent
Figure 1. A typical column
chromatography experiment
demonstrates the separation
of a two-component mixture.
Tentative identification of the components can be achieved by
comparing the unknown mixture a carefully prepared known
mixture: if a known component has the same retention time as
an unknown component under the same conditions, it is
probable—but not conclusive—that the two components are
the same. Further analysis may be needed to confirm this
hypothesis. If the known and the unknown have different
retention times, then it is not likely that the two components are
identical.
Other variations of chromatography use capillary action—the
attraction of a liquid to a solid surface—to pull a solvent
through solid material. An informal version of paper
chromatography can be observed when an ink-written page
comes in contact with water or other liquids. The ink runs and
several colors are separated in the ink streak.
The diagram below (Figure 2) shows the result of a thin-layer
chromatography experiment. Two black ink spots on the solid
surface have had a solvent passed through them. The solvent
is water or another liquid that is pulled through the stationary
phase by capillary action. In this example, a piece of plastic
coated with a powdered solid is used as the stationary phase.
Alternatively a piece of filter paper can be used as the
stationary phase. The experiment shows that the black ink is a
mixture containing several different colored substances. Each
component has a slightly different solubility in the mobile
phase, so when the liquid is pulled through the stationary
phase, each component moves at a different rate, separating
the ink into spots of different colors.
In this experiment, similar principles are used to separate
several metal cations by a paper chromatography procedure.
The metal ions—Ag+, Fe3+, Co2+, Cu2+, and Hg2+—have
differing solubility in the mobile phase—aqueous HCl with
ethyl and butyl alcohol—and will move at different rates up
the paper. The different metal-ion solubilities are probably
due to the formation of various compounds with the chloride
ion and their varying ability to dissolve in the organic solvent.
A diagram showing how to prepare the paper is shown below.
Standard solutions containing each of these ions will be
spotted onto the paper using a capillary tube, along with a
standard solution containing all five ions. An unknown will
also be spotted onto the paper. Once the paper is prepared,
it will be developed by placing the paper into the eluent. After
75-90 minutes, the paper is visualized by wetting it with an
aqueous solution containing potassium iodide, KI, and
potassium ferrocyanide, K4[Fe(CN)6]. The unique color
observed for each ion is produced by a chemical reaction
with the visualization solution. This is one useful way to
identify which ions are present in an unknown mixture.
Paper Chromatography
Figure 2: Thin-layer chromatography of
black ink after development. This picture demonstrates a common problem
where the spots widen as they move up
the plate, eventually merging at the top
of the plate.
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Chemistry 11
Santa Monica College
20 cm
Your Name(s)
Tape
12 cm
(5 single ions, 1X each)
mixt. (3X) (2X and 4x, same unknown)
Known ions
1.5 cm
Unknown Mixture
Figure 3: Diagram showing how to prepare the paper for the chromatography experiment
The distance the ion moves up the paper can also be used to identify the ion. However, since
students will develop their chromatography experiments for different amounts of time and under
slightly different conditions, each student will have somewhat different measured distance for a
given ion. The ratio of the distance moved by an ion (D) to the distance moved by the solvent (F,
solvent front) is characteristic and should be nearly the same for all students. This ratio is called Rf,
or “retention factor.”
D
Rf =
F
Procedure
Safety
Avoid contact with the metal ion solutions, the eluting solvent, and the visualizing solution. Wear
disposable gloves to touch your chromatogram after the elution occurs and for the remainder of the
experiment. Do not breathe the vapors of the eluting solvent or the visualizing solution. Place the
wet chromatogram on a paper towel, not directly on the laboratory bench. Use the visualizing
solution only in the space provided by your instructor. Dispose of the gloves and chromatogram in
the specified waste container after the experiment is finished. Wash your hands thoroughly after
contact with all solutions in this lab.
Paper Chromatography
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Chemistry 11
Santa Monica College
Materials and Equipment
Chemicals: 0.1 M aqueous solutions of AgNO3, Hg(NO3)2, Fe(NO3)3, Co(NO3)2, and
Cu(NO3)2, each with dedicated capillary tubes; eluting solution (aqueous HCl with ethyl and
butyl alcohol); visualizing solution (aqueous solution of KI and K4[Fe(CN)6]).
Equipment: Clean piece of chromatography paper; disposable Latex gloves (nitrile gloves
are vailable in the stockroom for people with allergies to Latex); 600 mL beaker; plastic
wrap; forceps or beaker tongs; ruler *
*Items obtained from stockroom
Preparation of the paper for chromatography
1. Each pair of students should obtain a piece of filter paper with the dimensions shown in Figure 3.
Make sure the paper is clean and without tears or folds. Use a pencil—not a pen—and a ruler to
draw a line across the paper one cm from the long edge of the paper. You will spot the metal ion
solutions on this line. Write your name in pencil in the upper left-hand corner of the paper.
2. Practice spotting water and/or ion solutions onto a strip of filter paper so that you know how to
create spots of the correct size. Use glass capillary tubes to spot the ions onto the paper. Solution
is applied by lightly and quickly touching a capillary tube containing the solution to the line you drew
on the paper. The spots should be between 5–8 mm in diameter. Spots larger than this will
excessively spread out during the experiment and make analysis difficult.
3. Known 0.1 M aqueous solutions of AgNO3, Hg(NO3)2, Fe(NO3)3, Co(NO3)2, and Cu(NO3)2 are
provided in test tubes, each containing two or three capillary tubes. Starting on the left, mark the
identity of the ion underneath each spot with a pencil; then spot each known ion carefully onto the
line. Be careful to avoid contaminating the capillary tube with other ions and replace the capillary
tubes back into the correct test tube. A test tube containing a known mixture of all five ions is also
provided with a set of capillary tubes. Spot this mixture onto the line as well. Because this solution
is more dilute than the single-ion known solutions, apply the known mixture three times, letting the
spot dry between each application. A heat lamp will help to dry the spot more quickly.
4. Several unknowns are also provided in test tubes, along with capillary tubes. Your instructor will
tell you which unknown should be used. The unknowns will contain between one and four cations,
and are more dilute than the single-ion known solutions. The unknown will also need to be applied
two and four times for the two trials, letting the spot dry between each application. In case of error,
you should spot the unknown in two places along the line so that two trials are available for
analysis.
Developing the chromatography paper
1. Place a piece of tape along the upper right edge, as shown in Figure 3. Then form a cylinder by
connecting the two short edges of the paper with the tape. Make sure the edges do not touch. The
paper should look similar to Figure 4.
2. Obtain 15 mL of the eluting solution. Carefully pour
some of this solvent into a 600 mL beaker and carefully
swirl for a second or two. Caution: Do not breathe the
vapors from this solution! Make sure that the level of the
liquid will be below the spot line on the paper once the
paper is placed in the developing chamber.
Figure 4. Folded paper should look like this
prior to developing the experiment.
Paper Chromatography
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Chemistry 11
Santa Monica College
3. Place the paper cylinder into the beaker with the marked edge down. The spots should be above
the level of the solvent. The paper should not be touching the sides of the beaker. Carefully cover
the beaker with plastic wrap and place it in the hood for 75-90 minutes. The solvent should start to
move up the paper. Once the beaker is covered, make sure it is level and do not disturb it during
the development period. Your instructor may have an assignment for you to work on while you
wait.
Visualization and analysis of the paper
1. Once the development period is over, wear disposable gloves and remove the paper from the
beaker. Latex gloves are available in the lab and nitrile gloves are available in the stockroom for
people with Latex allergies. Let any solvent drip back into the beaker, then remove the tape. Lay
the chromatography paper on a paper towel and immediately mark the solvent front with a pencil.
Pour the used eluting solvent into the waste container provided. Dry the paper under a heat lamp in
the hood. Caution: Do not breathe the vapors! Be careful not to burn the paper under the lamp.
2. Once the paper is dry, bring it to the visualization station on the paper towel. Briefly dip the
paper into the visualizing solution located in a shallow dish in the fume hood. Lift the paper out of
the solution immediately and let any excess drip off at the station. Place the wet paper onto a dry
paper towel and dry it under a heat lamp immediately, then carry it to your bench for analysis.
3. Find each known single-ion first and record the colors you observe. Some spots may fade over
time, so record the colors while the paper is still wet. Measure the distance each spot moved, D,
with a ruler. Measure to the center of each spot. Record your data in the data table.
4. Measure the distance to the solvent front, F. The value of F should be approximately the same
across the entire paper. Use these values to calculate the Rf for each ion. Make your
measurements as shown in Figure 5. Each observed spot has its own Rf value. Record your
results in the data table.
5. In the lane containing the mixture, find each ion and
record the distance moved by each ion. Calculate the Rf
for each ion in this lane. The values should closely
Solvent Front
match those observed in the single-ion knowns.
F
D
Spotting Line
Figure 5. Measurement of distances
used in the calculation of Rf for a spot.
6. In the lane containing the unknowns, locate the center
of each spot observed and record its distance and
calculate the Rf values. Use the lane that has the
clearest spots. The color and Rf values for the unknown
spots should closely match some of the known ions.
You should now be able to identify which ion or ions are
found in your unknown. Record your data in the
corresponding table.
7. Make a sketch of your chromatogram in the space provided on your lab report form, being sure
to indicate the position and approximate size and shape of each spot on the paper. Dispose of the
paper in the designated waste container.
Cleanup
Place the chromatography paper and the used gloves in the waste container provided. The used
eluting solution should already have been placed into another waste container. Note that two
different waste containers are provide for this experiment so be sure to read the labels so you will
use the correct one! Be sure to wash your hands thoroughly before leaving the laboratory.
Paper Chromatography
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