Selwin Varghese

4/29/2010
Period 6

Plant Pigments and Photosynthesis

Abstract: In this lab, the student separated plant pigments using

chromatography and measured the rate of photosynthesis in
isolated chloroplasts using the dye DPIP. The student separated
pigments and calculated their Rf values and compared
photosynthetic rates at different light intensities or different
wavelengths of light using controlled experiments.
The student obtained chromatography paper, solvent, and
spinach leaves to study plant pigments. A coin was used to extract
the pigments from spinach cells. Once the solvent reached the top
of the paper, the solvent front was marked before its evaporation.
Then the distance from the pigment origin to the bottom of each
pigmented band was measured and compared with the distance
traveled by the solvent front to determine the Rf values. When
testing the effectiveness of the light reactions, the student used a
spectrophotometer. Five cuvettes were set up: three containing
boiled, unboiled, and no chloroplasts were exposed to light;
another contained unboiled chloroplasts kept in the dark; the last
one was a blank and did not contain DPIP. The cuvettes were then
incubated and the percentage of transmittance was calculated at
five minute intervals.
The student learned that chlorophyll a is the primary
photosynthetic pigment in plants. Also chlorophyll a molecules are
located at the reaction center of photosystems. Other chlorophyll a
and b molecules, along with carotenes and xanthophylls, capture
light energy and transfer it to chlorophyll a at the reaction center.
Carotenoids also protect the photosynthetic system from the
damaging effects of ultraviolet light.

Emphasis: Data collection and data analysis

Objectives: In this lab you will:

1. separate plant pigments using chromatography and
2. measure the rate of photosynthesis in isolated chloroplasts using
the dye DPIP.

The transfer of electrons during the light-dependent reactions of

photosynthesis reduces DPIP, changing it from blue to colorless.

Before doing this lab you should understand:

 how chromatography separates two or more compounds
that are initially present in a mixture;
 the process of photosynthesis;
  the function of plant pigments;
 the relationship between light wavelength and
photosynthetic rate; and
 the relationship between light intensity and photosynthetic
rate.

After doing this lab you should be able to:

 separate pigments and calculate their R values;
 describe a technique to determine photosynthetic rates;
 compare photosynthetic rates at different light intensities or
different wavelengths of light using controlled experiments;
and
 explain why the rate of photosynthesis varies under
different environmental conditions.

Problem: Can the student learn about the process of photosynthesis?

Data: Table 4.1

(Uncertainties - ± 0.1)

Distance Moved by Pigment Band (mm)

Band Number Distance (mm) Band Colour
1. 10 Olive green
2. 20 Bright green
3. 32 Yellow orange
4. 46 Yellow
Distant Solvent Front Moved – 80 mm

Table 4.2
0.400 = R for Carotene (yellow to yellow orange)
0.575 = R for Xanthophyll (yellow)
0.250 = R for Chlorophyll a (bright green to blue green)
0.125 = R for Chlorophyll b (yellow green to olive green)

Topics for Discussion

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

Solubility of the pigments, intermolecular bonds formed between

the pigment and the paper

2. Would you expect the R value of a pigment to be the same if a

different solvent were used? Explain.

A different solvent would change the Rf value of a pigment

 because the Rf value depends on the solubility of the pigment in a
solute.

3. What type of chlorophyll does the reaction center contain? What

are the roles of the other pigments?

Chlorophyll a; other pigments capture light and transfer it to

chlorophyll a at the reaction center. Carotenoids also protect the
photosynthetic system from UV light.

Table 4.4
Cuvette 0 5 10 15
2 Unboiled/Dark 68.4 69.5 68.5 68.0
3 Unboiled/Light 59.8 61.1 61.0 60.7
4 Boiled/Light 77.0 78.0 79.3 81.0
5 No Chloroplasts/Light 70.3 71.0 71.5 71.0

Analysis of Results

a. The independent variable: Time (minutes)

b. The dependent variable: Transmittance

Graph 4.1

(Uncertainties – ± 0.1 %)

Conclusion: To conclude, the student learned that light and chloroplasts are
required for light reactions to occur. The student discovered that
the boiled chloroplasts are required for light reactions to occur.
The student discovered that the boiled chloroplasts absorbed the
least amount of light as it had the highest percentage of light
 transmittance. The cuvettes containing the light exposed unboiled
chloroplasts absorbed the most amount of light as it had the lowest
light transmittance. The chloroplasts kept in the dark and the
cuvette containing no chloroplasts had low absorption rates as
well.

Discussion: Chromatography is a technique used to separate and

identify pigments and other molecules from cell extracts that
contain a complex mixture of molecules. This can be used to
identify the pigments that are used in the process of
photosynthesis. Photosynthesis is the process by which plants use
light energy to produce chemical energy in the form of food. This
is where plant pigments come into play because they are the reason
why the plant is able to absorb light. Chlorophyll a is one such
pigment. These pigments along with many others are contained in
organelles known as chloroplasts.
One of the problems encountered during the course of this
lab included human error when using the spectrophotometer. The
student made slight errors when setting the transmittance to the
required levels. On one occasion, the student accidentally
introduced light into a cuvette where the variable being tested was
the absence of light. This might have caused some error when
taking measurements of the percentage of transmittance. Also, the
student failed to realize that data had to be recorded precisely at
five minute intervals for fifteen minutes. Therefore, measurements
were made haphazardly at irregular time intervals. This resulted in
skewed data, which meant that the experiment had to be repeated
once more. During the first part of the lab, the student made an
error by allowing some part of the pigment to be in the solvent.
However, this does not seem to have affected the data
significantly, as it was only less than one fourth of the pigment that
lay in the solvent.
One of the real life applications for this lab would be to
create photosynthesis artificially. This is one of the most sought
after outcomes because if photosynthesis could be reproduced in a
laboratory, it would solve the energy crisis. This would be a cheap,
highly efficient source of renewable energy, one that could meet
the energy requirements of the world and reduce global warming at
the same time, by absorbing the carbon dioxide present in the
atmosphere.

Questions: Topics for Discussion:

1. What is the function of DPIP in this experiment?

DPIP is an electron acceptor

2. What molecule found in chloroplasts does DPIP 'replace' in this

experiment?

NADP is replaced by DPIP

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

Split water molecules are the source of electrons

4. What was measured with the spectrophotometer in this

experiment?

The transmittance of light is measured with the spectrophotometer

5. What is the effect of darkness on the reduction of DPIP?

Explain.

Darkness prevents the reduction of DPIP. Without light striking the

chloroplasts, the electrons cannot be boosted to high energy levels,
which are needed to reduce DPIP.

6. What is the effect of boiling the chloroplasts on the subsequent

reduction of DPIP? Explain.

Boiling the chloroplasts does not affect the reduction of DPIP

because the chloroplasts are denatured and no longer function.

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

When light struck the live chloroplasts, electrons were boosted to

high energy levels which reduced DPIP. As the DPIP was reduced,
there was an increase in the light transmittance. The chloroplasts in
the dark had no access to light; therefore the electrons were not
boosted to high energy levels, which caused the DPIP to remain
the same. Therefore the presence of light caused the difference in
the percentage of transmittance.

8. Identify the function of each of the cuvettes.

Cuvette 1: The control

Cuvette 2: Used to observe the rate of photosynthesis without light
Cuvette 3: Used to observe the rate of photosynthesis with light
Cuvette 4: Used to observe the rate of photosynthesis in boiled
chloroplasts
Cuvette 5: Used to observe the rate of photosynthesis without
chloroplasts
Citations: The AP Biology Development Committee, . AP Biology
Lab Manual. Revised Edition. US: 2001. 45-53.