Honors
INVESTIGATING LEAVES
BACKGROUND:
Photosynthesis is an energy-producing reaction that occurs
in the leaves of plants. Photosynthesis is the main function of
leaves. The top surface of leaves allows light to penetrate to the
cells that carry out photosynthesis. The top surface of a leaf is
usually darker than the bottom surface. Dark surfaces are capable of
capturing more of the sun’s energy.
In most photosynthetic organisms, photosynthesis occurs in the
chloroplast. The size, shape, and number of chloroplasts will vary with the type
of organism. The structure; however, generally consists of stacked disks of
chlorophyll containing structures called grana that are surrounded by a
membrane.
A pigment is a substance that absorbs light at specific wavelengths;
chlorophyll is one of these pigments. Its green-yellow color is due to the
absorption of red, orange, blue, and violet wavelengths and the reflection of
green and yellow. This occurs when white light (containing all light wavelengths)
shines on the surface of a leaf. All of the wavelengths are absorbed except the
ones you see, which are being reflected. In addition to producing chlorophyll,
plants produce several other accessory pigments, in smaller quantities, that are
hidden by the vast amount of chlorophyll. They allow the plant to absorb energy
from the wavelengths of light that chlorophyll can not absorb effectively. The
pigments are carotene (orange), xanthophyll (yellow), chlorophyll a (blue –
green), chlorophyll b (yellow-green) and anthocyanin (red/violet).
In this lab you will use a technique called chromatography to separate the
various pigments that are found in plant leaves. Chromatography has long been
used to determine the composition of complex mixtures found in nature based on
the solubility and charge of the molecules that are being separated. You will then
record the Rf value (rate of flow) for each of the pigments present in the leaves.
The ratio of the distance moved by a pigment to the distance moved by the
solvent is a constant, called Rf. The Rf value is usually constant for any given set
of conditions and gives a characteristic pattern that one can readily recognize
and use to identify a substance. Each type of molecule has its own Rf value. It
should be emphasized that the Rf will vary according to the solvent used, so the
solvent should always be stated when recording the Rf value.
Objective: SWBAT separate and identify color pigments in leaves in order to
visualize the pigments that are present in plants
SWBAT record Rf values in order to quantitatively identify the
pigments present
Materials:
2 test tubes
Alcohol
Ruler
Coin
1 red poinsettia leaf
1 green poinsettia leaf
Safety:
1. Wear safety goggles
Test tube rack
2 Chromatography
paper strips
Honors
2. Use caution when handling alcohol
3. Follow all safety rules
Exploration: Leaf Chromatography
Procedures:
1. Make a PENCIL line about 2 ½ cm from the bottom edge of the
chromatography paper
2. Using the green leaf, make a dark green mark on the pencil line by
placing a leaf, top side down, onto the filter paper and rubbing the edge
of a coin back and forth over the leaf
3. Allow the green mark to dry
4. Label the top of the chromatography paper with PENCIL ONLY
5. Repeat steps 1 through 4 with the red leaf on the other piece of
chromatography paper
6. Slowly pour alcohol into the test tubes filling them each about 2 cm from
the bottom
7. Slowly lower the chromatography paper into the test tubes until the tip
of the filter paper is just touching the solvent (but not the sides of the
test tube)
*Note: the pigment dot must be above the level of the solvent
8. Fold the top of the paper over the edge of the test tube
9. Allow the paper to sit undisturbed for 15 minutes – 20 minutes or before
the solvent front migrates to the top of the test tube
10. Remove the strips from the test tubes, mark the top of the solvents
progression with a PENCIL line and let them dry
11. Study the colored streaks on the paper and record the colors observed
for the red and green leaves
12. Draw your observations and identify the pigments on the data sheet
13. Record the distance that the colors traveled up the chromatography
paper on the data sheet
14. Record the Rf value for each of the pigments on the data sheet
a. Carotene – 0.98
b. Chlorophyll a – 0.95
c. Chlorophyll b – 0.75
d. Xanthophyll – 0.44
e. Anthocyanin – 0.61
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Name:________________________________
Date:__________ Per:____
INVESTIGATING LEAVES
Data Sheet
Explanation:
Data:
GREEN
Chart 1:
Leaf
Green
poinsettia leaf
Red
poinsettia leaf
RED
Colors observed
Distance (cm)
Pigment
Present
Rf Value
Honors
Evaluation: Answer the following question in a complete sentence on a separate
sheet of paper
1. What is chromatography and why is it useful?
2. Why do the red leaves appear red and the green appear green?
3. Were there any colors visible on your chromatography that was not visible in
the leaves before the chromatography? Explain why or why not.
4. What did the red and green poinsettia leaves have in common?
5. What did the chromatography reveal that was different about the two leaves?
What do you think accounts for this difference?
6. The thickness and intensity of each band on the chromatography indicate the
relative amount of the pigment present in the leaf. Using this criteria, which
pigments were the most abundant and least abundant in the green leaf? What
about the red leaf?
7. Poinsettia leaves are green throughout the summer, but begin turning red in
the fall just like trees. Why do you think leaves change color during the fall?
8. Explain why Rf values may be helpful in analyzing the pigments present in
leaves.
9. If a biologist conducted an experiment and got the following Rf values: 0.94,
0.77, and 0.99 what pigments were probably in the leaf that she observed?