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AP Biology
Lab #4
Adrienne Harreveld
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Introduction A: In paper chromatography the pigment will move up the paper by capillary action.
This occurs because of the attraction of the solvent molecules to the paper and the solvent
molecules to one another. Beta carotene is most abundant in carotene plants, is carried along near
the front because it is very soluble in the solvent being used because it forms no hydrogen bonds
with cellulose. Xanthophyll is different than carotene because it contains oxygen. Xanthophyll
will be found further from the solvent front because it is less soluble in the solvent and has been
slowed down by hydrogen bonding to cellulose (paper) (lab book). Chlorophyll's contain oxygen
and nitrogen and are bound more tightly to the paper than the other pigments. Chlorophyll | is a
primary photosyntheticpigment found in plants. One molecule of chlorophyll | is located at the
reaction center of the photo systems. Pigments will collect light energy and send it to the reaction
center. Carotenoids protect the photosynthetic systems from damaging effects of ultraviolet light.

Introduction B: Shorter wavelengths of energy have greater amounts of energy. When light is
absorbed by pigments, electrons become excited and reach a new energy level. This energy is
used to produce ATP and to reduce NADP+ to NADPH. ATP and NADH are used to combine a
CO2 into organic molecules. This is called carbon fixation. In this experiment a dye-reduction
will be used. In this experiment DPIP will be substituted for NADP+.

Question A: Will the pigments move up the chromatography paper through capillary action and
will the pigments separate as they ascend?

Hypothesis A: Capillary action will cause the pigments to move up the chromatography paper
and the pigments will separate.

Question B: What controls the rate of photosynthesis?

Hypothesis B: Temperature and the light a plant receives control the rate of photosynthesis. If a
light reaction were to occur, then light and chloroplasts will be required. In this experiment DPIP
will be substituted for NADP. When light strikes the chloroplasts electrons boosted to high
energy levels will reduce the DPIP. This will change from blue to colorless.

Procedure:

1.‘ Obtain a 50-ml graduated cylinder that has 1 cm. of solvent in the bottom. The cylinder is
tightly stopped because this solvent is volatile, be careful to keep the stopper on as much
as possible.
2.‘ Cut a piece of filter paper that will be long enough to reach the solvent. Cut one end of
the filter paper to a point. Draw a pencil line about 2 cm above the point.
3.‘ Use a nickel to extract the pigments from spinach leaf cells. Place a small section of leaf
on top of the pencil line. Use the ribbed end of the coin to crush the cells. Be sure that
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the pigment line is on top of the pencil line. Repeat this 8-10 times making sure that a
new portion of the leaf is used each time.
4.‘ Place the chromatography paper in the cylinder so that the pointed end is barely
immersed in the solvent. Don¶t allow the pigment to be in the solvent!
5.‘ Stopper the cylinder. When the solvent is about 1 cm from the top of the paper remove
the paper and immediately mark the location of the solvent before it evaporates.
6.‘ Mark the bottom of each pigment band. Measure the distance each pigment migrated
from the bottom of the pigment origin to the bottom of the separated pigment band.
Record the distance that each front including the solvent front, moved. You should
observe about 4-5 pigment bands.

Procedure B:

1.‘ Turn the spectrophotometer to warm up the instrument and set the wavelength 605nm.
2.‘ Have your teacher demonstrate how to prepare a chloroplast suspension from spinach
leaves (in a blender)
3.‘ Set up an incubation area that includes a light a water flask, and a test tube rack. The
water in the flask acts as a heat sink by absorbing most of the light¶s infrared radiation
while having little effect on the light¶s visible radiation
4.‘ Your teacher will provide you with two beakers, one containing a solution of boiled
chloroplasts, the other with unboiled chloroplasts. Keep the beakers on ice.
5.‘ At the top rim label the curvettes 1,2,3,4,5 repectively. Wipe the outside of each curvette.
Cover the walls of curvette two with foil and make a foil cap to cover the top. Light
should not be admitted into curvette two.
6.‘ To each curvette add a mL of phosphate buffer. To curvette 1, add 4ml of distilled H2O.
To curvettes 2,3,4 add 3ml of distilled H2O and 1 ml of DPIP. To curvette 5 add 3 ml
plus three drops of distilled water and 1 mL of DPIP.
7.‘ Bring the spectrophotometer to zeroby adjusting the amplifier control knob until the
meter reads 0% transmittance. Add 3 drops of unboiled chloroplasts to curvette 1. Cover
the top with Parafilm and invert to mix. Insert curvette 1 into the sample holder and
adjust the instrument to 100% transmittance by adjusting the light-control knob. Curvette
1 is the blank to be used to recalibrate the instrument between readings. In other words,
you will measure the light transmitted through each of the other tubes as a percentage of
the light transmitted through this tube. For each reading, make sure that the curvettes are
inserted into the sample holder so that they face the same way as in the previous reading.
8.‘ Obtain the unboiled chloroplast suspension, stir to mix, and transfer 3 drops to curvette 2.
Immediately cover and mix curvette 2. Then remove it from the foil sleeve and insert it
into the spectrophotometer¶s sample holder, read the transmittance and record the time
reading. Replace curvette two in the foil sleeve and place it in the incubation test tube
rack. Turn on the flood light. Take and record additional readings at 5, 10, and 15
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minutes. Mix the curvette¶s contents just prior to each reading. Remember to use curvette
one occasionally to check and adjust the spectrophotometer to 100% transmittance.
9.‘ Obtain the unboiled chloroplast suspension, mix, and transfer 3 drops to curvette 3.
Immediately cover and mix curvette 3. Insert into the sample holder, read the %
transmittance. Place curvette three in the incubation test tube rack next to curvette two.
Take and record additional readings at 5, 10, and 15 minutes. Mix the curvette¶s contents
just prior to each reading. Remember to use curvette one occasionally to check and adjust
the spectrophotometer to 100% transmittance.
10.‘Obtain the boiled chloroplast suspension, mix, and transfer 3 drops to curvette 4.
Immediately cover and mix curvette 4. Insert into the sample holder, read the %
transmittance. Place curvette four in the incubation test tube rack next to curvette two
and three. Take and record additional readings at 5, 10, and 15 minutes. Mix the
curvette¶s contents just prior to each reading. Remember to use curvette one occasionally
to check and adjust tÀe spectrophotometer to 100% transmittance.
11.‘Cover and mix the contents of curvette 5. Insert into the sample holder, read the %
transmittance. Place curvette four in the incubation test tube rack next to curvette two
and three. Take and record additional readings at 5, 10, and 15 minutes. Mix the
curvette¶s contents just prior to each reading. Remember to use curvette one occasionally
to check and adjust tÀe spectrophotometer to 100% transmittance.
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Data A: Chlorophyll

Band number Distance mm Band Color

1 10 cream
2 20 Light yellow
3 35 cream
4 42 yellow
5 50 orange
6 52 Dark green
7 53 Blue green

Distance solvent front moved: about 60mm.

Rf- .8 for Carotene

Rf- .7 for Xanthophyll
Rf- .88 for Chlorophyll a
Rf- for Chlorphyll b
For leaf

Band number Distance mm Band color

1 4 green
2 15 clear
3 25 Light yellow
4 50 Light green
Distance solvent front moved: 55 mm.

Data B :

Transmittance Percentage% :

Curvette 0 5 10 15
2Unboiled/Dark 45.0% 45.0% 45.0% 45.0%
3 Unboiled/Light 30.0% 30.1% 30.0% 31.1%
4 Boiled/Light 29.0% 29.2% 29.2% 29.3%
5 No Chlroplasts/ 48.0% 48.1% 48.2% 48.0%
Light
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Percentage of Transmittance over time

D

D  Unboiled/Light
4D
4 Boiled/ Light
D

D No chlorplasts/
Light

D
 Unboiled/ Dark
D
D 
D


The independent variable is time.

The dependent variable is percent transmittance.

Analysis A:

1. What factors are involved in the separation of the pigments?

The solubility, size of particles, and their attraction to the paper are all involved in the separation.

2. Would you expect the Rf value of the pigment to be the same if a different solvent were used?

Explain.

No, because the pigments would have different solubilities which would change the Rf values.
For example chlorophyll b is only soluble to fatty solutions.

3. What type of chlorophyll does the reaction center contain? What are the roles of the other
pigments?

The reaction center contains chlorophyll a. The other pigments collect different light waves and
transfer

the energy to chlorophyll a.

Analysis B:

4. What is the purpose of DPIP in this experiment?

 
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DPIP acts as the electron acceptor in this experiment.

5. What molecule found in chloroplasts does DPIP "replace" in this experiment?

DPIP substitutes for the NADP+ molecules.

6. What is the source of the electrons that will reduce DPIP?

The electrons come from the photolysis of water.

7. What was measured with the spectrophotometer in this experiment?

The spectrophotometer will measure the percentage of light transmitted through the cuvette due
to DPIP

reduction.

8. What is the effect of darkness on the reduction of DPIP? Explain.

When it is dark, no reaction will occur.

9. What is the effect of boiling the chloroplasts on the subsequent reduction of DPIP? Explain.

Boiling denatures the protein molecules/enzymes and stops the reduction.

10. What reasons can you give for the difference in the percent transmittance between the live
chloroplasts that were incubated in the light and those that were kept in the dark?

In the dark cuvette, there was no light energy available, so there was no flow of electrons and no
photolysis of water, while in the lighted cuvette these processes were allowed to continue.
Energy is necessary for these reactions to occur. The photolysis of water is powered by energy
found in light that excites electrons giving energy to make NADPH and ATP.

Variables section A: There was no control in this section. Some independent variables were how
much pigment was on the chromatography paper, the age of the paper the amount of solvent.

Variables section B: Curvette one was the control. The different chloroplast mixtures was the
independent variable. Time was an independent variable. The Spectrophotrometer may not have
been fully accurate.

Conclusion: In the first section, we discovered that different pigments found in chloroplasts are
all involved in gathering energy from sunlight. The spectrum of color displayed on the filter
paper showed the pigments and the solubility of each. The results of the chromatography were
not that accurate. We were not able to see a full spectrum of colors and sometimes the bands
would jump from clear to a pigment. This could have been because we did not have enough
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pigment, the paper was old or we used too much solvent. If we all used leaves with a lot of
pigment and were able to extract it better. It is possible that we would have been able to see the
full spectrum. The purpose of the second section was to observe the DPIP go from a blue color to
a clear color. This would indicate that photosynthesis was occurring and at what rate it was
occurring. The cuvette with the unboiled chloroplasts that had been exposed to light showed the
biggest change in % transmittance, which indicates that the amount of light available has a very
big effect on the rate at which the light reactions of photosynthesis occur .The reason why the
third curvette showed the largest change in transmittance was because it was exposed to light,
and because it wasn¶t boiled, enzymes in the chloroplasts were not denatured causing the process
of photosynthesis to occur more quickly.
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Works Cited

"AP Biology Lab Four: Plant Pigments and Photosynthesis." G
. Web. D Dec. DD.

<http://www.scribd.com/doc/7847D7/AP-Biology-Lab-Four-Plant-Pigments-and-

Photosynthesis?autodown=pdf>.

Campbell, Neil A.  . San Francisco: Pearson, Benjamin Cummings, DD. Print.