Photosynthesis, you might remember, is the process in which plants convert light

energy from the sun to chemical food energy. Brightly colored pigments in leaves are
essential to the first steps of light absorption, with chlorophyll being the most important
pigment. There are two main types of chlorophyll: chlorophyll A, which is bluish-green,
and chlorophyll B, which is yellowish-green. During most of the growing season,
leaves contain more chlorophyll than any other pigment, making them appear green. In
the fall, however, chlorophyll begins to break down, and the other pigments, which have
been there all along, are finally revealed: we see pretty yellow and orange fall leaves.
The yellow pigments in leaves are called xanthophylls, and the orange pigments are
called carotenoids (carotenoids are easy to remember because they are orange like a
carrot!).
If you want to see the hidden yellow and orange pigments in green leaves for yourself,
you can use a simple but powerful technique called chromatography, which is the
separation of a mixture by passing it through a medium (in our case, filter paper)
through which different parts of the mixture will move at different rates. Chromatography
works because different substances in a mixture have different degrees of
solubility. Solubility refers to how much of a particular substance can dissolve in a
particular solvent. For instance, you can dissolve lots of sugar in water, but not quite as
much salt. In chromatography, the least soluble substances fall out of the filter paper
column first, while the most soluble one travel the furthest up the paper.

LEAF CHROMATOGRAPHY
Chromatography- the separation of a mixture by passing it in solution or suspension
through a medium in which the components move at different rates.

Problem
How Can the Different Pigments in Leaves Be Revealed?

Materials
 Soft green leaves (if no leaves are available, fresh spinach works fine, too)
 Scissors
 Mortar and pestle or food processor
 Glass or ceramic cup
 Isopropyl alcohol (or acetone in nail polish remover, but be careful—acetone
creates more fumes)
 Flat coffee filters
 Tape
 Pencil or straw
 Procedure
1. Use the scissors to cut up enough leaves to make a ¼ cup of pieces.
2. Use the food processor or mortar and pestle to grind the leaves into a fine pulp.
3. Pour the pulpy leaves into the cup. What color do you see?
4. Pour enough isopropyl alcohol over the leaves to cover them. Stir. Why can’t you
use water as solvent?
5. Cut out strip of coffee filter paper about 6 inches long and 1 inch wide.
6. Attach the strip to the pencil with a piece of tape. Make sure the bottom of the
strip hangs straight.
7. Adjust the length of the filter paper strip so that the bottom just touches the green
liquid in the cup.
8. Checking every couple minutes, wait for the band of solvent to migrate to the top
of the filter paper. Different colored bands should become evident along the strip.
9. Study your strip, and try to identify the pigments.

Results
Your results will vary depending on the types of leaves that you chose and how careful
your leaf chromatography technique was. You might not see all the pigments. An
orange-colored band is likely to be near the top. Below that, you should see a yellowish
band, a blue-green band, and a greenish-yellowish band, respectively.

Why?
When you ground up your leaves, the pulp was probably green because of the
overwhelming amount of chlorophyll. You were instructed not to use water as a solvent
because the pigments in leaves are not very soluble in it: they are much more soluble in
solvents like alcohol or nail polish remover. The orange colored band, made of the
pigment called carotenoids. is the most soluble in alcohol, so it traveled the farthest.
The yellow xanthophylls are the next most soluble, followed by the blue-green
chlorophyll A. The least soluble pigment is the yellow green chlorophyll B. You may be
wondering why plants have more than one pigment. One reason is that these differently
colored pigments absorb different colors of light: by having many pigments, plants can
use more of the light energy shining down them.

Going Further
Investigate what colors of light plants grow best in. Buy a green, blue and red light bulb.
See what color of light seem to be the best for growth.