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2.4: Chromatography

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    Chromatography tank

    Credit: Theresa Knott [CC BY-SA 3.0 or GFDL]

    Chromatography is a collective term for a set of analytical techniques used to separate mixtures. Chroma means color and graph means to write or draw. Paper chromatography is an analytical technique used to separate mixtures of chemicals (sometimes colored pigments) using a partitioning method. The paper in this method is called the stationary phase because it does not move and serves as a substrate or surface for the separation. Analytes (substances being analyzed) are separated from each other based on a differential affinity to a solvent. The solvent dissolves and carries the analytes along the matrix of the stationary phase. Since the solvent moves through a wicking action, it is called the mobile phase.

    The distance that the analyte migrates along the paper related to the total distance that the solvent or mobile phase moves is called the Retention Factor or RF.

    R_{F} = \frac{D_{A}}{D_{S}} = \frac{migration distance of analyte}{migration distance of solvent}

    Are The Food Colorings Used in Colored Candy the Same as the FD&C Approved Chemicals?

    How many colored spots do you expect to see for each reference standard?

    1. Obtain a 25 cm square piece of chromatography paper that will fit into the beaker that will serve as the chromatography chamber.
    2. Draw a pencil line across the lower end of the chromatography paper about 2 cm from the bottom.
    3. Draw additional vertical tick marks along this line every 2 cm.
    4. Place colored candy in a flask with 2 ml ethanol until the color dissolves into the solution.
    5. Using an applicator, create a very small spot on a tick mark and allow to dry.
    6. Repeat application on the spot to make a very small and dark spot.
    7. Continue to spot reference standards along other tick marks. These reference standards are food coloring.


    8. Place approximately 1 cm of mobile phase solution (a very polar saltwater solution) into the beaker.

    9. Roll the filter paper into a cylinder and fix with staples.


    10. Place cylinder into the beaker and cover for 20 minutes or until the mobile phase reaches 2 cm from the top of the paper.


    11. Mark the final distance of the mobile phase and dry the filter.

    12. Measure the distance of each spot from the starting point.

    1. Each spot is a separate analyte.
    2. Some spots separate into multiple analytes.
    3. Measure EACH one.

    13. Measure the distance from the starting point to the final point that the solvent reached.

    14. Calculate Rf values and tabulate results.

    Table of Reference Standards


    Table of Unknowns



    1. Compare and average the RF values of each analyte across the entire class.

    2. Did you predict the number of spots that would appear from each analyte (reference or candy)?

    3. Assuming all the dye molecules are of the same mass, what influenced the migration patterns of each spot?

    4. Were the colors used in the candy the same as the references?

    5. What does it mean if the candy color didn’t match anything from the food colors from the cake decorating set used as references?

    This page titled 2.4: Chromatography is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Bio-OER.

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