The Effect of Light Intensity on the Rate of Photosynthesis and the Utilization of Paper

Chromatography on the Separation of Photosynthetic Pigments1

Mari Fei C. Lagmay

Botany 1 Sec. B-1L

September 16, 2013

1
A scientific paper submitted in partial fulfillment of the requirements in Botany I under Mr. Mark
Anthony F. Rabena, 1st sem., 2013-2014
 ABSTRACT

The effect of light intensity on the rate of photosynthesis

was determined using the bubble-counting method. Freshly-
cut Hydrilla sprigs were placed in the stem of a funnel. The
funnel was placed in an inverted manner inside a beaker filled
with water. A test tube filled with water was placed over the
funnel in an inverted manner. This set-up was set under
ordinary room light condition (control), under strong light
condition (high light intensity), and to a darker part of the
room (low light intensity) where the experiment was done.
The control had given-off an average of 29.67 bubbles per
minute, while an average of 172.33 bubbles per minute for the
high light intensity and none for the low light intensity. Thus,
light intensity affects photosynthesis. Photosynthetic pigments
were separated using paper chromatography. A narrow band
of chlorophyll extract has been applied twice to a
chromatogram. The chromatogram was then suspended and its
end was immersed to chromatogram solvent. The whole set-up
was covered with a larger beaker and then observed for 10-15
minutes. It showed that the pigments seen in the experiment
made by the Henderson State University were chlorophyll,
xanthophyll and carotene.

INTRODUCTION

Light is a form of energy, and when it falls on the chloroplasts in the leaf, it is trapped by

chlorophyll, which then makes energy available for chemical reactions in the plant. As the

amount of light falling on the plant increases, the more energy chlorophyll can trap, so more

energy is available for chemical reactions, so more photosynthesis can take place in a given time.

According to Tamayo-Zafaralla, et. al., (2007), photosynthesis is a light-driven chemical

reaction occurring in all green cells, whereby carbon dioxide and water react to form sugars and
other organic compounds. It is the plants’ process of making food. All organisms benefit from

this reaction because of oxygen production that is an agent in respiration.

The Hydrilla, the plant used in the experiment, is a submersed plant that can be found in

all types of water bodies. Hydrilla stems are slender and its small leaves are strap-like and

pointed. They grow in whorls of four to eight around the stem. The leaf margins are distinctly

saw-toothed.

The bubble-counting method was used in the experiment to observe the rate of oxygen

that is released through bubbles.

Pigments are chemical compounds which reflect only certain wavelengths of visible light.

More important than their reflection of light is the ability of pigments to absorb certain

wavelengths. Because they interact with light to absorb only certain wavelengths, pigments are

useful to plants and other autotrophs, organisms which make their own food using

photosynthesis. Different pigments appear different colors because they have differing abilities

to absorb and reflect various colors of light.

Paper chromatography is one method for testing the purity of compounds and identifying

substances. This method was used in the experiment because it is easy to do.

The study aimed to determine the effect of light intensity on the rate of photosynthesis

and to identify the photosynthetic pigments separated by paper chromatography. The specific

objectives were:

1. To test the rate of oxygen released through bubbles in every type of light intensity

exposed to the set-up

 2. To explain the possible mechanisms behind the observed effect of light intensity on

photosynthesis

3. To separate pigments through paper chromatography

MATERIALS AND METHODS

In determining the effect of light intensity on the rate of photosynthesis, the bubble-

counting method was utilized. A 500 ml beaker filled with 3/4 water and freshly-cut Hydrilla

sprigs were prepared. The sprigs were placed under and opposite the stem of a glass funnel. The

funnel with the sprigs were soaked or put inside the beaker with water. An inverted test tube

filled with water was put onto the stem of the funnel.

The set-up was placed under normal room light condition, which serves as the control in

the experiment. To observe the rate of oxygen, bubbles should therefore appear and should be

counted. The same set-up was imitated but under strong light, which has a high light intensity.

Observations were also made.

The same set-up was transferred to a darker and dimmer part of the same room utilized.

Also, observations were again made. A graph describing how the method or set-up was done

follows.

In the separation of photosynthetic pigments through paper chromatography, several

steps were done by the Henderson State University in Arkansas. A 2x9 cm strip of

chromatogram with one hole at one hand was obtained. A narrow band of chlorophyll extract

was put at 1cm from the edge of the opposite end of the chromatogram using a Pasteur pipette.
Drying follows, then another application of the extract was done with the same narrow band.

Drying was done again.

Fifteen (15) ml of chromatogram solvent, comprising 14 parts ethyl alcohol, 3 parts

benzene, and 1 part of petroleum ether, was placed in a 250-ml beaker. Then, a glass tubing was

inserted through the hole of the chromatogram. The chromatogram was suspended then to the

solvent and the lower end of it was immersed. Afterwards, the set-up was covered with an

inverted 1000 ml beaker to prevent light and wind from affecting the capillary movement of the

solvent through the strip. After 10-15 minutes, the beaker was removed and the chromatogram

was then observed.

Fig. 1. Bubble-counting method set-up

 Fig. 2. Paper chromatography set-up

RESULTS AND DISCUSSIONS

As seen in the table, the bubbles being released differ depending on the intensity of the

light given off to the set-up. It is also seen in the table that as the observation repeats, the more

bubbles is being released by the set-ups with medium and high intensities of light. As observed,

the set-up didn’t release any bubbles during the set-up with a low light intensity was done.

Photosynthesis is a process that engages light in it. But as what the experiment through the table

showed, it is seen that photosynthesis requires a higher intensity of light for it to process.

Paper chromatography is a procedure used to separate substances in a mixture. This

mixture is usually a solution of liquid plant pigments containing different kinds of chlorophylls

and other colored photosynthetic pigments. (retrieved from

http://regentsprep.org/regents/biology/units/laboratory/instrumentation.cfm)

The paper is contact with a solvent solution at its bottom. This solvent moves through the

paper due to capillary action and dissolves the mixture spot. Some parts of the solvent mixture

to be separated have a greater attraction for the chromatography paper, so they move a lesser
distance, while other parts of the solvent mixture have a lesser attraction, so they move a greater

distance up the paper.

The testing was based on the results of the Henderson State University when they did the

experiment. The results on the chromatogram are seen in figure 3.

The resulting pigments seen in the chromatogram are chlorophyll, xanthophyll, and

carotene. Chlorophyll was noted by chlorophyll a and b in the image, and the green stain.

Chlorophyll is the primary pigment of green plants. Xanthophyll and Carotene were noted with

yellow stains on the chromatogram. These are both subclasses of carotenoids, plant pigments

known to have antioxidant properties. The "loading line" is the location of the original pigment

line painted on the paper.

Table 1. The number of oxygen bubbles per minute given off by photosynthesizing

Hydrilla sprigs in high and low intensities. (Tamayo- Zafaralla, et. al., 2007)

Medium Light
Observation number Low Light Intensity High Light Intensity
Intensity (Control)

1 19 0 100

2 33 0 120

3 37 0 297

Average 29.67 0 173

 Fig. 3. The results of the chromatography done by HSU

SUMMARY AND CONCLUSION

The effect of light intensity on the rate of photosynthesis is determined by utilizing the

bubble-counting method, where a Hydrilla was enclosed in a funnel within a beaker with water,

and an inverted test tube being put onto the stem of the funnel itself. Several observations were

made. Through this, a table was made to summarize the result of the whole experiment. The

result showed increasing rate on the release of bubble for the high and medium light intensity,

while none resulted from the low light intensity.

 Therefore, light intensity greatly affects the rate of photosynthesis. However,

photosynthesis requires a medium to high light intensity to make it function.

As for the separation of photosynthetic pigments, paper chromatography is the method or

technique used. After doing the whole set-up, observations were made. The pigments were

determined. Chlorophyll, noted by the green stain was seen, together with xanthophyll and

carotene which are types of carotenoids, giving the yellowish stains.

LITERATURE CITED

Zafaralla, M.T., et.al. 2007. Explorations in Plant Science. Philippines: Photosynthesis and

Transpiration. p. 63

Hydrilla: retrieved from http://plants.ifas.ufl.edu/node/183

Speer, B. R., 1997. Photosynthetic pigments

Paper chromatography: retrieved from

http://regentsprep.org/regents/biology/units/laboratory/instrumentation.cfm

Experiment on paper chromatography: Henderson State University. Retrieved from

http://www.hsu.edu/pictures.aspx?id=1653