Name ________________

Photosynthesis Lab (AP Lab #4)

Exercise 4A: Plant Pigment Chromotography

Paper chromatography is a useful technique for separating and identifying pigments and other molecules
from cell extracts that contain a complex mixture of molecules. The solvent moves up the paper by capillary
action, which occurs as a result of the attraction of solvent molecules to the paper and the attraction of solvent
molecules to one another. As the solvent moves up the paper, it carries along any substances dissolved in it. The
pigments are carried along at different rates because they are not equally soluble in the solvent and because they
are attracted, to different degrees, to the fibers in the paper through the formation of intermolecular bonds, such
as hydrogen bonds.

Procedure
1. Obtain a piece of filter paper. Draw a straight line 1 inch from the bottom using a black overhead pen.
2. Fill a glass bottle with just enough water to cover the bottom of a jar.
3. Place the paper down in the jar with the black line toward the bottom. The water level should be lower
than the black line. If the black line gets wet initially, the experiment will not work!
5. Allow the water line to move about ¾ of the way up the paper.
6. Draw and color your results below.
7. Repeat steps 1-6, this time using a spinach leaf. Make sure the leaf is completely dry.
8. To transfer green pigment onto your filter paper, use two rulers. Stretch the rulers across the width of
the paper, leaving a small space in between them. Place the spinach leaf over the top of the space and
use a coin to rub pigment onto the paper. Rub back and forth from one side of the paper to the other,
careful not to cause the paper to tear.
9. This time, place the paper into a jar containing chromatography solvent instead of water.
10. Once the spinach pigments have been properly separated, color in the results below. Mark the bottom of
each pigment band and measure the distance each pigment migrated from the pigment origin to the bottom of
the separated pigment band. Record this distance (including the solvent front) in Table 4.1.

Black Pen Spinach

Table 4.1

Distance Moved by Pigment Band

Band Number Distance (mm) Band Color
1
2
3
4
Questions
1. How many different pigments/shades are visible? On the marker there are six different shades, and with the
spinach there are four different shades

2. One of the pigments found in plants is called chlorophyll b. Chlorophyll b is highly attracted to the cellulose
in the paper, but of all 4 pigments, is least soluble in the solvent. Label this pigment on your diagram. Another
pigment is known as chlorophyll a. This pigment is less attracted to the paper than chlorophyll b, but more
soluble in the solvent. Label this pigment on your diagram. Another pigment found in green plants is called
xanthophyll. This pigment is more soluble in the solvent than chlorophyll a and has a weaker attraction to the
paper. Label this pigment on your diagram. The last pigment might be slightly hard to see and is known as
carotene. This is the primary pigment found in carrots. It has the weakest attraction to the paper, but has the
greatest solubility with the solvent. Label this pigment on your diagram.

Part 4B (The light-dependent Reaction)

Light is a part of a continuum of radiation or energy waves. Shorter wavelengths of energy have greater
amounts of energy. For example, high-energy ultraviolet rays can harm living tissues. Wavelengths of light
within the visible part of the light spectrum power photosynthesis.
When light is absorbed by leaf pigments, electrons within each photosystem (Pigment proteins) are
boosted to a higher energy level and this energy is used to produce ATP and reduce NADP+ to NADPH. This
process is known as the light-dependent reaction. ATP and NADPH are then used to fuel the dark or light-
independent reaction. During this reaction, carbon fixation occurs, which is when CO2 is "fixed" into organic
molecules (glucose, amino acids, nucleic acids, lipids).

Purpose: To determine how light affects the light reaction.

To determine how extreme heat affects the light reaction.

Hypothesis: 1)If Elodea is exposed to room light vs. a flood lamp, then the greatest amount of oxygen bubbles
will be observed ________________________________.
2)If the oxygen production of boiled Elodea vs. unboiled Elodea is compared, then the greatest
amount of oxygen bubbles will be observed in the _________________ Elodea.

Procedure: 1. Fill two test tubes 3/4 full with water.

2. Place 1 sprig of unboiled Elodea into a test tube. Place 1 sprig of boiled Elodea into the
second test tube with the cut ends down.
3. Expose each test tube to room light letting them equilibrate for 1 min.
4. Count the number of oxygen bubbles that rise to the top every minute for 5 minutes.
You may need to gently tap the test tube on the counter occasionally to dislodge bubbles.
5. Expose each test tube to a flood lamp letting them equilibrate for 1 min.
6. Count the number of oxygen bubbles that rise to the top every minute for 5 minutes.
Table 1.

Light Source Classroom Flood Lamp

Time (min) Boiled Unboiled Boiled Unboiled
1 0 1 0 5
2 0 2 0 10
3 0 3 0 15
4 0 4 0 20
5 0 5 0 25

Graph: Independent Variable: _____________________________________________________

Dependent Variable: ______________________________________________________
Graph Title: _____________________________________________________________
________________________________________________________________________

Conclusion:

1. Was your hypothesis supported? Explain.

2. Overall, what happened? Why?

3. Explain how we could tell the light reaction was occurring. Could we tell whether the dark reaction was
occurring? Explain.
4. What three products were being produced during the light-dependent reaction?

5. What was the direct source of oxygen bubbles we observed being produced during the experiment?

6. List two ways we could test to determine whether the light-independent (dark) reaction was occurring.

7. How did the results compare in the boiled vs. the unboiled chloroplasts in the Sunlight?
Explain why these results were different.

8. List the two variables in this experiment. Why were we able to have two variables, yet still have a
"controlled" experiment?

9. List any other controls for the experiment. Were there any uncontrolled variables? If yes, identify them.

10. Fill in the reactants and products for the light and dark reactions below:
Light-dependent reaction:

___________ + __________ + __________  ___________ + ___________ + ___________

Light-independent reaction:

__________ + ___________ + __________  ____________ + ___________ + ___________

11. All four of the pigments found in a spinach leaf are actually found in photosystems I and II in the thylakoid
membranes of the chloroplast. Xanthophyll and carotene are known as the carotenoids and are clustered
together with cholorophyll b. Describe the role of these three pigments in the light reaction.

12. What role does chlorophyll a play in the light reaction?