Santa Monica College
Chemistry 11
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 employed in such situations. Using this technique many types of mixtures can be
separated into the component pure substances, and 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).
Component B
Component A
Stationary Phase
with Eluent
Figure 1. A typical column
chromatography experiment
demonstrates the separation
of a two-component mixture.
Paper Chromatography
To illustrate the technique, consider the separation of a twocomponent mixture using Column Chromatography, shown
in Figure 1. The column is packed with a solid material
which is the stationary phase. A liquid solvent or eluting
solution – the mobile phase – 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 of the
eluting solution (or 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 Figure 1, 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 the
eluting solution continues to be added to the column,
eventually the two components will exit the column
separately. The time taken for each component to exit the
column is called its retention time. Retention time will be
reproducible for each component under the given set
conditions – mobile and stationary phase identities,
temperature and column width.
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Santa Monica College
Chemistry 11
Once the components exit the column, the solvent can be removed by evaporation and the pure
components can be further analyzed. Tentative identification of the components can be
achieved by comparing the unknown mixture with 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 samples
have different retention times, then it is very unlikely that the two components are identical.
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.
Other types of chromatography might use capillary
action – the attraction of a liquid to a solid surface – to
pull a solvent through solid material. Figure 2 shows
the results of a Thin-Layer Chromatography
experiment. The stationary phase is piece of plastic
which is coated with a powdered solid. A liquid solvent
has been passed through two black ink spots on the
solid surface, pulled through the stationary phase by
capillary action. The results show that the black ink is
actually a mixture containing several different colored
substances. Each component of the ink has a slightly
different solubility in the mobile phase, so that when
the liquid is pulled through the stationary phase each
component moves at a different rate, separating the
ink into spots of different colors. Paper
Chromatography works in a similar manner, using
paper as the stationary phase. An everyday
occurrence of paper chromatography can be observed
when an ink-written page comes in contact with water.
The ink runs and several colors are separated in the
ink streak.
The table below lists several varieties of chromatography and typical identities of the phases.
Type of Chromatography
Mobile Phase
Stationary Phase
Gas
(GC)
inert gas
(helium)
waxy liquid or silicone
inside narrow tubing
Liquid
(LC, HPLC, column)
solvent/solvent mixture
(organic or aqueous)
solid packing
(silica, alumina)
Paper
solvent/solvent mixture
(organic or aqueous)
paper
Thin-Layer
(TLC)
solvent/solvent mixture
(organic or aqueous)
silica/alumina coated glass,
plastic or metal
Paper Chromatography
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Santa Monica College
Chemistry 11
In this experiment, a mixture of 5 metal cations will be separated using Paper Chromatography.
The metal ions – Ag+1, Fe+3, Co+2, Cu+2, and Hg+2 – have differing solubility in the mobile phase
(an aqueous solution of 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 (a chromatograph) is shown in Figure 3. Standard
solutions containing each individual ion will be spotted onto the paper using a capillary tube,
along with a standard solution containing a mixture of 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 results can be visualized by wetting the paper with an
aqueous solution containing potassium iodide (KI) and potassium ferrocyanide (K 4[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.
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: Preparation of 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 chromatographs for different amounts of time and under slightly
different conditions, each student will obtain slightly different measured distances for a given
ion. The ratio of the distance moved by an ion (D) to the distance moved by the solvent front (F)
is characteristic and should be nearly the same for all students. This ratio is called the retention
factor, Rf, where:
Rf =
Paper Chromatography
D
F
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Santa Monica College
Chemistry 11
Procedure
Safety
Avoid contact with the metal ion solutions, the eluting solvent, and the visualizing solution. Use
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 area of the lab designated by your instructor. Dispose of your
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.
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
available in the stockroom for people with allergies to Latex), 600-mL beaker, plastic wrap,
forceps or beaker tongs, ruler
Preparation of the Paper for Chromatography
1. 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 1-cm from the long edge of the paper. 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.
A 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 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.
Paper Chromatography
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Santa Monica College
Chemistry 11
Developing the Chromatography Paper
5. 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 below.
Figure 4. Folded paper should look like this
prior to developing the experiment.
6. 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.
7. 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 lecture or have an
assignment for you to work on while you wait.
Visualization and Analysis of the Paper
8. 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! Also be careful not to burn the paper under the lamp.
9. 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.
10. 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. Make your measurements to the center of each spot, as shown in
Figure 5 on the next page. Record these measurements in the data table on your Lab
Report.
Paper Chromatography
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Santa Monica College
Chemistry 11
11. Measure the distance to the solvent front (F), as shown below in Figure 5. The value of F
should be approximately the same across the entire paper. Record your measurements in
the data table on your Lab Report. Then use your values of D and F to calculate the
retention factor, Rf, for each ion. Each observed spot will have its own Rf -value.
Solvent Front
F
D
Spotting Line
Figure 5. Measurement of distances
used in the calculation of Rf for a spot.
12. 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 match those observed
in the single-ion knowns.
13. In the lane containing the unknowns, locate the center of each spot observed, 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 on your Lab Report.
14. Make a sketch of your chromatogram in the space provided on your Lab Report, being sure
to indicate the position and approximate size and shape of each spot on the paper.
Clean-Up
Place your chromatograph 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 provided for this experiment, so be sure to read the labels and
use the correct one! Finally, be sure to wash your hands thoroughly before leaving the
laboratory.
Paper Chromatography
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