Answer in lab notebook!!
CHLOROPLAST PIGMENT ANALYSIS
When you look at chloroplasts under a microscope or examine a plant leaf, the only color which
appears to be present is a green pigment called chlorophyll. However, there are other
pigments in a leaf. Yellow and orange pigments, not normally seen, are usually present within
chloroplasts.
Purpose: In this investigation, you will:
a. Separate pigments from one another by using a technique called chromatography;
b. Identify pigments by their colors and positions on the chromatograms;
c. Determine relative amounts of each pigment.
Materials:
Strip of filter paper at least 15 cm.long
scissors
Plastic vial
glass pipette
Spinach solution
solvent
Procedure:
1. Obtain a strip of filter paper at least 15 cm long. Use scissors to taper the bottom of
one end of the paper to a point.
2. Cut 2 small notches about 2 cm from the bottom as shown.
3. Use a coin to extract the pigments from spinach leaf cells. Place a leaf over the
tapered end of the paper strip.
4. Use the edge of the coin to crush the cells by rolling it over the leaf at the same
distance from the end as the notches (about 2 cm. from the tapered end).
5. Repeat this process 4-5 times in the same place to transfer enough pigment, moving
the leaf each time.
6. Add 1 ml of solvent to the plastic vial.
7. Carefully place the vial into the test tube rack. Then place the filter paper strip into
the vial. It is important that the pointed tip dip into the solvent without the
chlorophyll solution touching the solvent. Place the cap on the vial.
8. DO NOT move or shake the vial for at least 15 minutes. Remove the paper
chromatogram from the test tube when the level of solvent almost reaches the top
of the paper strip.
9. Examine the chromatogram for the presence of different bands of color. Each color
band is a different pigment.
Analysis
1. Draw your filter paper strip at the conclusion of the experiment. Color the bands of
color as seen.
2. Is chlorophyll composed of one or several pigments? What proof do you have?
3. What is the value of chromatography?
4. Examine your chromatogram strip. Each color band is a different pigment. Listed in
order from top to bottom on an ideal chromatogram are:
Carotene- orange in color
Xanthophyll- yellow in color
Chlorophyll a – bright green in color
Chlorophyll b – a dull or khaki green in color
NOTE: Usually the two chlorophylls are very close to one another.
a. How many different pigments can be seen on your chromatogram?
b. Which ones are they?
5. Using your chromatogram, which pigments are present in the smallest amounts in the
leaf?
6. Which pigments are present in the greatest amounts in the leaf?
Use your text, if necessary, to answer the following questions.
7. What is the role of chlorophyll a?
8. What is the role of carotene and xanthophyll?
9. How might the distribution of leaf pigments differ in a leaf that is dark green in color
versus one which is light green in color?
10. Many leaves change color in the autumn. How is it possible for this color change to
happen?