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3: Paper Chromatography- Separation and Identification of Five Metal Cations (Experiment)

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  • Objectives

    • Known and unknown solutions of the metal ions \(\ce{Ag^{+}}\), \(\ce{Fe^{3+}}\), \(\ce{Co^{2+}}\), \(\ce{Cu^{2+}}\) and \(\ce{Hg^{2+}}\) 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.

    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.

    Type of Chromatography

    Mobile Phase

    Stationary Phase

    Gas (GC)

    inert gas (helium)

    waxy liquid or silicone inside narrow tubing

    (LC ,HPLC, column)

    solvent/solvent Mixture (organic or aqueous)

    solid packing (silica, alumina)


    solvent/solvent Mixture (organic or aqueous)


    Thin-Layer (TLC)

    solvent/solvent Mixture (organic or aqueous)

    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.


    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.


    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.

    In this experiment, similar principles are used to separate several metal cations by a paper chromatography procedure. The metal ions—\(\ce{Ag^{+}}\), \(\ce{Fe^{3+}}\),\(\ce{Co^{2+}}\), \(\ce{Cu^{2+}}\), and \(\ce{Hg^{2+}}\)—have differing solubility in the mobile phase—aqueous \(\ce{HCl}\) 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, \(\ce{KI}\), and potassium ferrocyanide, \(\ce{K4[Fe(CN)6]}\). 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.


    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.”

    \[ R_f = \frac{D}{F} \label{1}\]


    Materials and Equipment

    Chemicals: 0.1 M aqueous solutions of \(\ce{AgNO3}\), \(\ce{Hg(NO3)2}\), \(\ce{Fe(NO3)3}\), \(\ce{Co(NO3)2}\), and \(\ce{Cu(NO3)2}\), each with dedicated capillary tubes; eluting solution (aqueous \(\ce{HCl}\) with ethyl and butyl alcohol); visualizing solution (aqueous solution of \(\ce{KI}\) and \(\ce{K4[Fe(CN)6]}\)).

    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


    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.

    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 \(\ce{AgNO3}\), \(\ce{Hg(NO3)2}\), \(\ce{Fe(NO3)3}\), \(\ce{Co(NO3)2}\), and \(\ce{Cu(NO3)2}\) 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.


    Figure 4: Folded paper should look like this prior to developing the experiment.

    1. 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.
    2. 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.


    Figure 5: Measurement of distances used in the calculation of Rf for a spot.

    1. 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.
    2. 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.
    3. 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.


    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.

    Pre-laboratory Assignment: Paper Chromatography

    1. A two-component mixture is analyzed by paper chromatography. Component A is more soluble in the mobile phase than component B. The following result is obtained. Calculate the Rf for each component and label the identity of each spot.


    1. The mixture from question 1 (above) is analyzed by column chromatography using the same mobile phase and a silica gel stationary phase. Which component has the shorter retention time? Explain your answer. (Assume the same type of binding forces between the components and the stationary phase are present in both paper chromatography and column chromatography.)
    1. An unknown liquid sample is analyzed using paper chromatography using solvent X as the mobile phase. One spot is observed after the plate is developed and visualized. The same unknown substance is re-analyzed using solvent Y as the mobile phase. This time, three spots are observed after the plate is developed and visualized.
    • Is the unknown sample a pure substance or a mixture? Explain your answer, including a possible reason for the different observations in the two experiments.

    Lab Report for Paper Chromatography

    Data, Observations, Calculations and Analysis Known Ions

    Known Ions


    Spot Color (Stained)

    D (Single- Ion)

    F (Single- Ion)


    D (Ion Mixt.)

    F (Ion Mixt.)

    R f






    Unknown ID Code _________________


    Spot Number (from lowest Rr)

    Spot Color (Stained)

    D (Unknown)

    F (Unknown)


    Identity of Spot






    • Sketch of Chromatogram:


    Unknown ID Code Ions Identified

    Questions and Conclusions

    1. What criteria were used to identify the ion(s) found in your unknown? Explain your answer in at least three sentences. Include any difficulties in identifying any ions.
    1. If you let the experiment run for only 30 minutes, what would be the likely result? Would any problems arise in identification of the unknown?
    1. If \(\ce{Co^{2+}}\) and \(\ce{Cu^{2+}}\) spots were the same color, would the identification of an unknown be any more difficult? Explain your answer.