INTRODUCTION:
Chlorophyll often hides the other
pigments present in leaves. In Autumn, chlorophyll breaks
down, allowing xanthophyll and carotene, and newly made
anthocyanin, to show their colors.
The mix of pigments in a leaf may be separated into bands
of color by the technique of paper chromatography.
Chromatography involves the separation of mixtures into
individual components. Chromatography means "color
writing." With this technique the components of a mixture
in a liquid medium are separated. The separation takes
place by absorption and capillarity. The paper holds the
substances by absorption; capillarity pulls the substances
up the paper at different rates. Pigments are separated
on the paper and show up as colored streaks. The pattern
of separated components on the paper is called a chromatogram.
PRELAB PREPARATION:
Gather leaves from several different
plants. CAUTION: Avoid poisonous plants. Autumn
leaves from deciduous trees are especially interesting.
Sort the leaves by kind (maple, etc.) and color. Review
a diagram of a plant cell . Find the grana and
the chloroplasts of the cell.
MATERIALS:
Safety goggles Chromatography solvent
(92 parts Petroleum ether to 8 parts acetone)
Chromatography paper (or filter paper) about 1 cm x 15
cm
Ethyl alcohol
Fresh spinach
Test tube
Test tube rack
Scissors and Ruler
Fresh leaves of plants
Glass stirring rod
Paper clip
Cork (to fit test tube)
Mortar and pestle
Sand (optional)
10-ml Graduated cylinder
PROCEDURE:
Leaves should be grouped by kind
(maple, etc.) and color. Work with a spinach leaf and
with one or more other types. CAUTION: Chromatography
solvents are flammable and toxic. Have no open flames;
maintain good ventilation; avoid inhaling fumes.
1. Cut a strip of filter paper or chromatography
paper so that it just fits inside a 15-cm (or larger)
test tube. Cut a point at one end. Draw a faint pencil
line as shown in figure 1. Bend a paper clip and attach
it to a cork stopper. Attach the paper strip so that
it hangs inside the tube, as shown. The sides of the
strip should not touch the glass.
2. Tear a spinach leaf into pieces about the
size of a postagestamp. Put them into a mortar along
with a pinch or two of sand to help with grinding. Add
about 5 ml ethyl alcohol to the leaf pieces.
Crush leaves with the pestle, using a circular motion,
until the mixture is finely ground. The liquid in which
the leaf pigments are now for paper chromatography dissolved
is called the pigment extract.
3. Use a glass rod to touch a drop of the pigment
extract to the center of the pencil line on the paper
strip. Let it dry. Repeat as many as 20 times, to build
up the pigment spot. NOTE: You must let the dot
dry after each drop is added. The drying keeps the pigment
dot from spreading out too much.
4. Pour 5 ml chromatography
solvent into the test tube. Fit the paper and cork assembly
inside. Adjust it so that the paper point just touches
the solvent (but not the sides of the tube). The pigment
dot must be above the level of the solvent. Watch the
solvent rise up the paper, carrying and separating the
pigments as it goes. At the instant the solvent
reaches the top, remove the paper and let it dry. Observe
the bands of pigment. The order, from the top, should
be carotenes (orange), xanthophylls (yellow),
chlorophyll a (yellow-green), chlorophyll b
(blue-green), and anthocyanin (red). Identify and
label the pigment bands on the dry strip. Write the species
of leaf on the strip as well.
Record the species, external color, and chromatogram pigments
in the DATA TABLE of your report sheet.
5. Each pigment has an Rf
value, the speed at which it moves over the paper compared
with the speed of the solvent.
