Demonstration 4: Separation of the Components of a Mixture by Column Chromatography

             Introduction

                The purpose of this activity is to separate the components of a mixture by column chromatography, a very
                common method for separating a mixture.  It is based on the fact that different materials are attracted to
                adsorbents with varying strengths. These attractions are based on the types of forces of attraction; very polar
                solutes are strongly attracted to some column media while nonpolar solutes are not. The solvent also plays an
                important role in the proper operation of the column. If the solvent attracts the solutes too strongly, all of the
                solutes travel close to the solvent front (high but similar R _f values) and good separation is not obtained.

             Safety

        Proper laboratory safety procedures must be followed. Any substances spilled should be
        immediately washed off with large amounts of water.

    Materials

        Support stand and clamps
        Chromatography column*
        Cellulose packing*
        Glass wool*
        Dyes to be separated*
        Distilled water should also be available.
        *These materials are contained in a kit available from Sargent-Welch; stock
        number S-18818-10. The refill materials are also available; stock number
        S-18818-20.

    Procedure

        It is important that you do the demonstration prior to doing it before a group.  It will require several hours to set up and perform.

1. Support the column with clamps on a support stand. Be sure that the column is vertical and that there is sufficient room to place a collecting flask under it.

2. Place a small quantity of glass wool in the bottom of the column.

3. Close the stopcock and fill the column with distilled water (solvent).

4. While tapping the column (use your thumb or the eraser head on a pencil) sprinkle the
cellulose into the column slowly. The purpose of the tapping is to help the packing material settle uniformly in the solvent. It will take about 15-20 min to fill the column.

5. Once the column is filled, place a plug of glass wool on the top. The purpose of this plug is to protect the surface of the packing while you add sample and solvent. Open the stopcock allowing solvent to run out of the column until the top level of the solvent is even with the top of the upper glass wool plug, and then close the stopcock.

6. Dissolve a small amount of the solute in 10-15 mL water; use a dropper to place it carefully onto the column.

7. Allow the solvent to run out until the level of the pigment is even with the top of the upper glass wool plug.

8. Carefully fill the column with solvent, then allow the column to run.  CAUTION: You must maintain the solvent above the top of the glass wool plug atop the column. Do not allow the column to run dry.

9. Once separation is evident you can show students and explain the system.

                    1. What evidence do you have that separation occurred?
                    2. Which colored substances moved the fastest? The slowest?

            Extensions
                    1. If you continue running the column, and collect the different fractions in separate beakers you
                        can demonstrate how complete separation is attained.
                    2. Try other samples and solvent systems.

Suggestions for Other Demonstrations

        CAUTION: Use appropriate safety guidelines in performing these activities.

                Dilution: CEPUP (Chemical Education for Public Understanding Program, Lawrence Hall of Science,
                    University of California, Berkeley) has a semiquantitative dilution experiment in which dilution is continued on a
                    multi-well strip or plate until the solution appears colorless.

            Supersaturation: Doing Chemistry DMEX C22 (Videodisc side C, Frame20792, and Teacher’s Manual,
                p. C22-48) provides details for the classical sodium acetate hydrate supersaturation demonstration. (See also The Chemistry of Rocks, Minerals,
                and Gems module.)

            Distillation: ChemCom (Chemistry in the Community) has a distillation activity using ethylene glycol and water that can be done either as a laboratory
                exercise or as a demonstration.

            Conductivity of Solutions: Doing Chemistry DMEX C26 (Videodisc Side C, Frame 25883, and Teacher’s Manual, p. C26-62) provides details for a
                small-scale electrical conductivity demonstration. However, a simpler small-scale electrical conductivity meter can be constructed from a battery and a
                low-voltage light bulb. Either type is safer than using 120-volt electricity as the electrical circuit, although the use of direct current has the disadvantage
                that electrochemical deposition can ruin the electrodes if used for long time periods (see Periodicity module).

            Ice Cream: A fun activity is to study colligative properties through the making of ice cream. The students should undoubtedly perform the activity in
                the kitchens of the school’s Home Economics facilities or at home rather than in the chemistry laboratory. Use a thermometer to show the depressed
                freezing point of the ice salt bath etc.

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