Name:________________________________________

Period:________

Objective: Do different colored leaves from the same tree contain the same pigments?
Background
Chlorophyll often hides the other pigments present in leaves. In Autumn, chlorophyll breaks down,
allowing xanthophyll and carotene, and made anthocyanin, to show their colors.
The mix of pigments in a leaf may be separated into band of color by the technique of paper
chromatography, shown in this exercise. 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 separate components on the paper is called a chromatogram.
Before Lab
Review the diagram of the chloroplast in Chapter 6 of your text. Find the grana.
1. What is the function of the grana?
2. Where are plant pigments located?
3. How does location of a pigment relate to its function?
4. What is the purpose of chromatography as a laboratory tool?
Materials
For Group of 3-4
Fresh leaves of plants and trees
Strip of filter paper
Coin
Scissors
Test tube (2)
Paper clip (2)

Cork (to fit test tube) (2)

Safety goggles
10 ml graduated cylinder
10 ml Chromatography solvent
Test tube rack
Rulers

Procedure
CAUTION: Chromatography solvents are flammable and toxic. Have no open flames,
maintain good ventilation, avoid inhaling fumes.
A. TRY TO MINIMIZE TOUCHING THE
STRIP. IT MAY INTERFERE WITH THE
CHROMATOGRAPHY. Cut a point at one
end of the strip of filter paper. Measure 2

cm from the bottom of the paper strip

and draw a faint line as shown in the
picture. 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.

A. Take a leaf, lay it over the pencil line on the strip of filter paper.
Deposit pigment from the leaf by rolling the quarter over the leaf
about 15 times to make a heavy green line. Do this gently so
that the filter paper isnt torn.

5. Why is it important that the pigment line be heavy and dark?

B. Pour 5 ml of 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 line 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 and use a pencil to mark the top of the solvent front. DO THIS QUICKLY
BECAUSE THE SOLVENT EVAPORATES.
C. Observe the bands of pigment. The order, from the top, should be carotenes (C)(orange),
xanthophylls(X) (yellow), chlorophyll b(Cb) (yellow green), chlorophyll a(Ca) (blue-green), and
anthocyanin (A) (red). Identify and label the pigment band on the dry strip. Write
your names of the strip as well. Record the colors and pigment names on the data
chart.
Leaf

External Color

Colors from
Top(list)

Pigment
Names(list)

Rf Value

Green Leaf

Colored leaf

D. Repeat steps A through C for the second leaf type assigned to you.
Each pigment has an Rf values, the speed at which it moves over the paper compared with the
speed of the solvent.
Rf= Distance moved by the pigment
Distance moved by the solvent
E. Measure the distance in cm from the starting point to the center of each pigment band. Then
measure the entire distance traveled by the solvent. Do this for both chromatograms you
made.
F.

Calculate the Rf value as a decimal for each pigment, on both chromatograms. Record the R f
values in the data chart.

G. Compare your results for both leaves with those of other students.
6. Are the Rf values for all chromatograms constant for each pigment type?
Analysis
1. Based on the class data, which kind of leaf showed the most pigments? For this leaf, which
pigments were most visible in the chromatogram? Are these the pigments responsible for the
leafs original color? Which pigments were least visible in the chromatograms? Are these
pigments obvious in the leafs original color?

2. Did the leaves contain any pigments that surprised you?

3. What is the purpose for each of these pigments? (how are they used by the plant?)
4. Describe why the leaves change color in autumn. What is the biological purpose for this
process?