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Chemistry Core/AP

Chromotography

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Laboratory Exercise

Goal: To separate pigments in plant materials.

Materials

Procedure

Plant preparation

Use one of the following methods to prepare both your plant sample and the spinach control materials.

Filter paper preparation

Remember that you will need two setups for each type of solvent you use: one for your test pigment and one for your spinach control. If you have 3 solvents, you will need 6 setups.

Do not handle the filter paper any more than necessary, and be sure that your hands are clean and dry before working with it. Skin oils will interfere with the separation rates. Use one of the procedures below to trim position the filter papers, add the plant material to its filter paper, then place the papers in the solvents you have selected. The point is to have the solvent move slowly enough to separate the different pigments, so however you set your filter paper up, be sure that the pigment is moving against the gravitational field (that is, UPWARDS) to slow it down, and that you have at least 6 cm for the solvent to move from spot to end of filter.

Decide which method best suits your filter paper and other equipment and where you will apply the solvent. You will need 4-6 pigment spots for each filter paper.

Mark the filter paper with the solvent.

Whichever filter paper method you use, you will need to transfer 4 to 6 spots of the plant material to the filter paper about 1/2" (1 cm) from what will become the bottom edge of your paper strip, cone, or cylinder — the edge in the solvent.

Use one of these methods to add the pigment material to your filter paper.

Procedure
  • Put enough solvent at the bottom of each glass container to cover the bottom evenly about .5 cm deep. You should not need a lot.
  • Place the filter paper in the solvent. Be sure that the pigment spots are above the level of the solvent! Note the starting position of the solvent with respect to the paper.
  • Watch the solvent climb up the paper until it is about 1 cm from the top. Note the time, and calculate how long it took to reach this point.
  • Note the positions at which the colors separate out and are deposited on the filter paper. Is there more than one? What color is each one?
  • Measure the distance the pigment moved from source to band.
  • Measure the distance the solvent moved from starting level to ending level.
  • Calculate the reference front: distance pigment moved/distance solvent front moved.

    The banding is characteristic of the pigments, and can be used to identify them in different plants. In spinach, the pigments are carotene (yellow/yellow orange), xanthophyll (yellow), chlorophyll a (bright green to blue green), and chlorophyll b (yellow green to olive green). Use the spinach bands to identify the banding in other plants.

    Data to be recorded:

    You should record the following data as you perform the lab. This data should be included in your results:

    1. Sources of plant pigmentation (minimum: two; at least one of which must be spinach).
    2. Solvent used (minimum: two. Water will give the least successful results).
    3. Distance between sample source line and each major color band (describe band colors).
    4. Identification of bands in non-spinach source, using spinach as the referent.

    Please describe IN DETAIL your procedure, the materials you used, and your results. Indicate how you could improve the experiment to make it more accurate.