Name: ____________________________________ Period: ___________
L3 Biology Lab Activity: Separating Plant Pigments
Problem: What pigments are present in chlorophyll?
Most plant leaves appear green, but actually contain a variety of different colored pigments: blue-green chlorophyll a,
yellow-green chlorophyll b, yellow-orange carotenes, yellow xanthophylls, and red and blue anthocyanins. These
pigments function as absorbers of different wavelengths of light, providing energy to the plant that can be used during
The process of chromatography separates compounds from one another because of their differences in solubility.
Basically, the more soluble a substance is, the more easily it will move through a medium. In paper chromatography,
the physical act of moving through the paper separates the pigments into distinct bands of color that appear at different
points on the paper. The paper with the separated bands of color is referred to as a chromatogram.
Green plants synthesize their own food by using carbon dioxide and water to produce sugars. This process requires
energy from a strong light source such as sunlight. Sunlight is a source of radiant energy that is absorbed by the
pigments in chloroplasts of leaves of green plants. The radiant energy is converted to chemical energy during
photosynthesis. The chemical energy can be used by the plant (and other organisms that eat plants) during cellular
The sunlight used for photosynthesis and most artificial light sources like light bulbs consists of white light. White
light can be separated by a prism (or a rain drop) into the colors of the spectrum. Each color seen represents a different
wavelength of light and a slightly different energy level. Not all wavelengths of light are absorbed by plants for
photosynthesis. Wavelengths in the middle of the visible spectrum, mainly greens and yellows, are reflected by the
pigments, giving them the green color seen by the human eye. Wavelengths of light in the red and blue-violet areas of
the visible spectrum are absorbed by plants and that energy is used for photosynthesis. Plant pigments other than
chlorophyll are called accessory pigments and help absorb some wavelengths of light that are not absorbed by
chlorophyll, providing a little more energy for photosynthesis.
Students gather and use: Teacher will set-up:
Scissors, ruler, pencil (NOT pen) Large test tube
Chromatography paper (13-14cm strip) Cork stopper with paper clip
Spinach leaf Chromatography solvent (95% acetone/5% ether)
A coin (quarter works well)
1. Be sure your hands are clean and dry before handling the chromatography strip. Cut the •
chromatography paper so that it is 13-14 cm long with a point on one end as shown. Using a pencil,
draw a light line horizontally across the strip about 2 cm from the tip of the point.
2. Place the spinach leaf on the strip, covering the end where the pencil line was drawn. Press down and
roll the edge of a coin (or the round part of scissors handle) across the leaf to extract some pigment.
Move the leaf slightly to get a fresh section and press and roll the coin to get another layer of pigment.
Try to get all the pigment in the same spot creating a thin dark line of chlorophyll.
3. Using a pencil, poke a small hole about 1 cm from the top of the strip (the end opposite the point). Write
your group member names at the top of the strip.
4. Bring the strip to your teacher so it can be placed in the chromatography solvent.
5. After about 10-15 minutes your teacher will return your chromatogram to you. Observe the pigments
separated and their locations on the strip. Pigments that are most soluble will move farthest up the strip.
Analysis and Conclusions:
1. How many pigments were separated? (refer to the possible plant pigments listed in the background
information to help determine what to look for)
2. Name each pigment, listing them from most soluble to least soluble.
3. All terrestrial green plants require chlorophyll to do photosynthesis and survive. Explain how both Japanese
and Norway maple trees (having only reddish, maroon leaves) can carry out photosynthesis.
4. Many trees have green leaves in the summer and red or yellow leaves in the autumn. Why were these red and
yellow colors not seen during the summer, but appear “magically” during the fall?