LAB: Chemistry Alternate Lab: Chromatography

Goal: To separate pigments in plant materials.

Materials

• Filter paper. Lab grade filter paper is expensive, but good substitutes are coffee filters, some vacuum cleaner filters, or paper towels.
• GLASS beakers, jars or shallow dishes to hold solvents.
• Solvents: at least two of the following:
  • Water
  • Isopropyl alcohol: 70% Rubbing alcohol
  • Isopropyl alcohol: 99% Rubbing alcohol
  • Ethyl alcohol
  • Acetone (you can use finger nail polish remover, but it's more expensive)
  • Paint thinner

    WARNING: All of the above, except water, are flammable and volatile.

    • Use small amounts to minimize risks.
    • Be sure to set your experiment up away from sources of flame or electrical sparks.
    • Work in a well-ventilated room.

• Sources of pigment: green leaves and leaves turning color, but still pliant.
• Spinach leaf (as control: it has four pigment colors).
• Scissors or sharp knife
• Mortar and pestle or dish and spoon (stainless steel, not aluminum) OR food processor
• Pencil

Procedure

Plant preparation

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

• Spoon and hard-sided dish or Mortar and pestle
  • Cut the leaf material into small pieces or chop into small bits about 1/8" across.
  • Using a spoon in a bowl, or a mortar and pestle, mash the bits until you have a relatively uniform, pulpy mass with some liquid escaping.
• Food processor
  • Place your leaves in the food processor and chop them with a bottom-blade until roughly pureed. It is not necessary to get the mass completely smooth.

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.

• You may suspend a long narrow strip of paper so that it will dangle down into your beaker or jar and the end will just barely be immersed in the solvent, so that it will soak it upwards.
• Cut a disk or cone of paper (or use a coffee filter) so that it will sit in your beaker with the point touching the bottom. Mark where you will place the pigment material near the center (low) point.
• Cut your filter paper into a rectangle which, when rolled into a cylinder shape and stapled so that it won't unroll, will fit inside your jar or beaker without touching the sides and will stand on its own without falling over. Mark where you will put the pigment spots near the bottom edge (which will be the base of the cylinder in the solvent).

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.

• Isolate a small amount of the prepared leaf sample in a test tube or small dish. Add a small amount of the solvent you will use to the sample. Use an eyedropper or non-porous tip to transfer the source solution to the appropriate spots on the filter paper. Let the spots dry, then repeat at least four times, so that each spot has had 4 applications of the solution.
• Place the sample on the filter paper at the first spot, and using the pencil lead (not too sharp: you don't want to tear the source), rub the sample with the pencil tip 10-12 times against the paper. Move the source to the next spot, and repeat rubbing (using a different area of the source), until you have 4-6 good spots. Repeat the whole process 4-6 times, so that each spot has multiple applications of the source material.

Procedure

1. 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.
2. 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.
3. 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.
4. 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?
5. Measure the distance the pigment moved from source to band.
6. Measure the distance the solvent moved from starting level to ending level.
7. 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.

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