Unit 58 Laboratory Activity: Chromotography
Goal: To separate pigments in plant materials.
- Coffee Filters.
- Jars to hold solvents
- Isopropyl alcohol: 70% Rubbing alcohol
- Large soft green leaves (geranium leaves, large blades of grass, spinach leaves).
Do not handle the coffee filters 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.
- Cut strips of filter paper 1" wide and of a sufficient length to hang into solvent as described below.
- Mash the plant sample with a spoon until you have a pulpy mass (you can use a blender or chopper). Take the mass and rub it against each filter paper strip one inch from the bottom of the strip. You now have a dot of plant mass on the paper containing plant dyes. You can set the left-over mass aside to rerun the experiment if necessary.
- Put enough water at the bottom of one glass jar container to cover the bottom evenly about 1/4 inch deep. Put the same amount of rubbing alcohol in the second jar.
- Suspend the filter (I use paper clips or tape attached to a pencil balanced across the top of the jar) so that it will hang freely and not quite touch the bottom of each jar. The bottom edge of the paper should be in contact with the solvent.
- 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?
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). You can use the spinach bands to identify the banding in other plants.
Describe your procedure, the solvents you use, and the source of your pigments. Describe the bands which result as the solvent separates the pigments. How many pigments show up in your sample? Which solvent works best?
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