Photosynthesis

John Benedict C. Palo, Kyra Christine A. Rios, Rochelle Anne R. Tan, Nathea Agatha C. Valdez
Submitted to: Maam Elena Ragragio
Submitted: September 20, 2012

ABSTRACT
This activity explores the different factors and processes that affect the photosynthetic rate of plants. Chlorophyll, light,
and carbon dioxide are the important things in photosynthesis since chlorophyll is the necessary green pigment, light
provides the energy needed, and carbon dioxide combines with ribulose biphosphate (RuBP) to make its food (simple
sugars). Because plants only use the hydrogen in the water it absorbs, oxygen is released back to the atmosphere; hence
oxygen liberation. Chloroplast pigment separation is important in determining which wavelengths plant need from the
sun in order to drive photosynthesis.

1. Introduction
Photosynthesis is an important process that benefits
all living things. If it were to suddenly stop because of an
unexpected reason, a chain of unfortunate events is to
happen; therefore, we must look closer to further
understand what and how factors affect photosynthesis.
The group hypothesized several things. First, the
amount of chlorophyll, light and CO2 present and
absorbed by the plant is directly proportional to its
photosynthetic rate. Second, the oxygen released by the
plant is the oxygen from H2O. Lastly, the pigment nearest
the top of the paper is the most soluble.
By the end of this activity, this group is expected to:
determine the role of chlorophyll, light, and carbon
dioxide in photosynthesis, observe oxygen liberation
during photosynthesis, and demonstrate how chloroplast
pigments are separated and to identify these pigments.

2. Methodology
2.1. The
Role
Photosynthesis

of

Chlorophyll

in

A variegated leaf was sketched that showed its

pattern of green and white areas. The leaf was then
boiled in water for 10 minutes then immersed in a test
tube with 95% ethyl alcohol. Afterwards, it was placed in
a hot water bath until pigments were extracted then it
was washed with water and tested for presence of starch
using IKI solution.

2.2. The Role of Light in Photosynthesis

After a potted plant was placed in the dark for 48
hours, random leaves were chosen to wrap portions of it
with carbon paper. The plant was then exposed to light

for 5 hours and tested for starch as previously done in

2.1.

2.3. The Role of CO2 in Photosynthesis

Three test tubes labeled A, B, and C were filled
with previously boiled then cooled distilled or tap water.
Hydrilla sprigs were placed invertedly into tubes A and B
and a pinch of NaHCO3 was added to tubes B and C. The
3 tubes were then exposed to bright light then the group
determined whether bubble evolution occurs in A and/or
B. After the hydrilla sprigs were removed, 5 drops of
phenol red were added to each then gently shook (phenol
red changes to yellow at low pH). The color intensities of
the solutions in the test tubes were then compared.

2.4. Oxygen Liberation in Photosynthesis

Once the 1L beaker was filled with tap
water, air was gently blown into it using a plastic straw
for 3-5 minutes to enrich CO 2. Afterwards, the ends of
the hydrilla sprigs were cut under water (make sure the
cuts face the tube of the funnel) and a thistle funnel was
inserted over those. A test tube filled with water was
place upside down over the tube of the funnel which was
partially dipped in the water of the beaker. The emerging
bubbles coming out from the cut ends were then
observed and the nature of the gas was determined by
placing a lighted match in the test tube.

2.5. Separation of Chloroplast Pigments

The leaf extract was prepared by getting 15 to 20
mature hibiscus leaves, cutting it into small pieces, then
putting it in a vial, and setting it aside.
The filter paper was prepared by fitting a piece of
paper into a clean, dry test tube and setting this as the

Photosynthesis

mold. With this, cut the filter paper into strip, all the
while making sure that only the edges will be held since
fingerprints on the surface of the paper may affect
chromatogram. When the filter paper can slide in and out
of the tube without touching the side, a 2 cm mark from
the top and a 1cm mark from the base of the strip was
made using a pencil. A point was then cut at the base of
the strip. Mark the outside of the test tube to show proper
solvent level. Remove the paper strip from the dry test
tube and lat it on the table. Meanwhile, solvent was
poured into the dry test tube up to the marked line. The
cork was replaced without the paper strip and kept
inside.
The chromatogram was prepared by dipping
capillary tube or a fine-tip dropper into the vial which
contained the extract. The drop that came out of the
capillary tube was placed onto the center of the 1 cm
mark at the base of the tip and dry by waving paper in
the air. This process was repeated until the spot became
fully saturated with the green extract (18 times). The
green spot was as small as possible and the drops were
added only after each drop had completely dried up. The
end result was a paper with a small, dark green spot in
the center of the 1 cm mark with the spot not reaching
the sides of the strip.
After the spot has completely dried up, the cork was
removed from the tube and the paper strip was placed on
the hook and inserted into the test tube containing the
solvent mixture of 95 parts petroleum ether and 5 parts
acetone with the solvent level below the 1 cm mark.
Minor adjustments were made with the paper clip. The
cork was replaced with the strip and the flow of the
solvent was observed until the 2 cm mark. The paper was
then removed from the tube and left to dry. After which,
the chromatogram showed the different pigments present
in the specimen.

3. Results
3.1.
The Role
Photosynthesis

of

Chlorophyll

in

Starch was present in the whole leaf.

3.2. The Role of Light in Photosynthesis

Starch was only present in the areas uncovered by

carbon paper.

3.3. The Role of CO2 in Photosynthesis

Bubbles were formed in both test tubes A and B
although tube B produced more.

Table 1. Formation of Bubbles

Test Tube
A
B
C

Color Intensity
+
++
+++

3.4. Oxygen Liberation in Photosynthesis

Bubbles emerged from the cut ends.

3.5. Separation of Chloroplast Pigments

From top to bottom, the pigments were carotene
(orange), xantophyll (yellow), chlorophyll a (blue green),
and chlorophyll b (light green).

4. Discussion
4.1. The
Role
Photosynthesis

of

Chlorophyll

in

The leaf was boiled so as to burst the chloroplast,

making it easier to extract chlorophyll.
The green areas are photosynthetic whereas the
white ones are non-photosynthetic. Based on the
experiment, only those areas with chlorophyll (green
areas) can undergo photosynthesis.

4.2. The Role of Light in Photosynthesis

It was necessary so as to use up the plants starch
reserves. Only the parts that were uncovered showed
positive reaction to the starch test. It can be concluded
therefore that light is necessary for photosynthesis since
only the uncovered parts were able to produce starch.

4.3. The Role of CO2 in Photosynthesis

Test tube C had the most color intensity followed
by B, then A upon addition of phenol red. Bubbles were
formed in tubes A and B since the NaHCO 3 supplied
more CO2, hence tube B produced more bubbles since it
had both a sprig and NaHCO 3. Boiled water was used so
as to eliminate dissolved air.

4.4. Oxygen Liberation in Photosynthesis

The tube was placed upside down so as to probably,
the sprig shall catch all the water test tubes got to bring.
The bubbles indicate carbon dioxide mixing with water,
and releasing oxygen.

4.5. Separation of Chloroplast Pigments

Photosynthesis

The colors present were orange which represents

carotene, yellow which represents xantophyll, blue green
for chlorophyll a, and light green for chlorophyll b. The
least soluble is chlorophyll b since this is at the bottom
and therefore took the longest amount of time travelling
up the paper.

REFERENCES
[1]

Committee on Biology 21, Biology 21 General

Botany Laboratory Manual Vol. 2008-2009,
Exercise 13: Respiration

[2]

James D. Mauseth, Botany: An Introduction to

Plant Biology, Jones and Bartlett, Fourth Edition,
pp. 244-246