A Study of the Effect of Light Intensity on the Photosynthesis of Hydrilla verticellata

sprigs and of the Separation of Photosynthesis Pigments by Paper Chromatography

Mark Louie Alvarez

Botany 1
Professor Janice Polizon
April 24, 2015
INTRODUCTION

Plants having the vascular plant level of organization produce leaves that
function for the vital process of photosynthesis. These leaves vary greatly both in its
internal and external features that are highly adapted to accommodate the whole
photosynthetic process. Wong (n.d.) enumerates the anatomical structure of the leaves
responsible for their ability to facilitate photosynthesis. First, their thinness, flatness, and
maximum surface area to volume ratio make them susceptible to abundant supply of
sunlight. Second, they have tiny openings called stomata within the surface to facilitate
aeration or gas exchange. Third, the vast intercellular spaces within them allow for an
internal atmosphere that governs effective cell- to cell contact. Fourth, the walls of the
cells of the leaves contain saturated amount of water to dissolve and transport carbon
dioxide, a raw material for photosynthesis. Fifth, their veinlets or small veins that contain
vascular bundles conduct water and mineral salts from the roots in the xylem [water
conducting plant tissue] and delivers the food manufactured in the phloem [food
processing plant tissue]. Sixth, the waxy and transparent cuticle layer present on their
upper and lower epidermis provides protection from dessication and infection. Lastly,
and the most important photosynthetic structure present in the leaves, is the chloroplast
that is mostly found on the upper mesophyll layer [and contains the green pigment
chlorophyll, the pigment associated with the capturing of light necessary for
photosynthesis].
Chloroplasts of higher plants are typically discoid or ellipsoidal in shape,
bounded by the double membrane chloroplast envelope, and composed of
membranes that are of lipoprotein composition. A complex system of membranes
(lamellae) are embedded in a granular matrix (stroma). The pairing of lamellae form sac
like structures called thylakoids, which are stacked at intervals to form highly ordered
structures called grana [These structures are the site of the two processes of
photosynthesis, the light reactions and the carbon fixation reactions.]. (Devlin &
Barker, 1971). The chloroplasts, as mentioned earlier, contains the pigment chlorophyll,
but it can also have other pigments, called accessory pigments, that provide the leaf its
different colors. According to Hall and Rao (1994), All photosynthetic organisms contain
one or more pigments capable of absorbing visible radiation which will initiate the
photochemical reactions of photosynthesis. These pigments can be extracted from most
1

leaves From the alcoholic extract, individual pigments can be separated by

chromatography Three major classes of pigments found in plants and algae are
chlorophylls, the carotenoids, and the phycobilins The two previous are called the
accessory photosynthetic pigments since the quanta absorbed by these pigments can
be transferred to chlorophyll Chromatography is used to separate mixtures of
substances into their components. All forms of chromatography works on the same
principle [polarity and molecular weight of compound]. (Clark, 2007). Moreover,
Schooley (1996) further elucidated that there are actually different kinds of chlorophyll,
being chlorophyll a and chlorophyll b, and the pigments carotene and xanthophyll.
Although it was a convention for most people to think that only chlorophyll is contained
in green leaves, German chemist Richard Willstatter proved that carotene and
xanthophyll are also present.
The term photosynthesis literally means building up or assembly by light.
Photosynthesis illustrates the complex process of the synthesis of inorganic compounds
[anabolic catabolism] from inorganic raw materials with the aid of radiant energy
Plants, being autotrophic in nature meaning that they can manufacture food of their
own, serve as the fuel for most animals for the latter cannot directly utilize the energy
coming from the sun. This has placed photosynthesis in the center of the maintenance
of all life forms The conversion of carbon dioxide and water into carbohydrates [triose
phosphate] and oxygen is the major chemical pathway of photosynthesis. The reaction
below represents the summary of the process:

Photosynthesis is therefore regarded as the conversion of radiant energy into chemical

energy of plant tissues (Hall & Rao, 1994). Wong (n.d.) abridges the photosynthetic
process, describing the two successive steps the light reactions and the dark
reactions [light independent reactions or Calvin cycle]. The light reactions occur in the
grana of the chloroplasts and involve the excitation of pigments by light,... conversion of
light energy, and mechanism composing of ATP synthesis by phosphorylation On
the other hand, the dark reactions initiate at the chlorophyll lacking stroma and

encompasses carbon dioxide fixation, the reduction phase, and the regeneration of the
carbon dioxide acceptor.
The significance of photosynthesis, as stated by Hubbard (2012), is that it allows
the organisms, plants, algae and bacteria capable of generating the process, to use the
energy from the sun to convert CO2,

one of the major air pollutants, into more

practical materials, which are organic substances. These organisms, therefore,

effortlessly contribute to the lessening of air pollution, maintaining a clean and fresh air
in the surroundings. In the photosynthetic process, they produce oxygen that is
necessary for the functioning of several biological progressions in both animals and
humans, as it is significantly present in the air we breathe. Most importantly, the
absence of photosynthesis equates for a very brief sustenance of life.
Conferring to Lambers, there are several factors that affect the rate of
photosynthesis. These are light intensity, temperature, carbon dioxide concentration,
amount of water and minerals, and internal factors like adaptation range, activity of
enzymes, and utilization of sucrose in correlation to carbon dioxide supply (2015).
However, in order to fully understand these factors, having knowledge of the rate of
photosynthesis is a prerequisite. Only blue and red wavelengths from the visible
spectrum drives the photosynthetic process (Schooley, 1997). The bubble counting
method is a procedure wherein the photosynthetic rate of a plant is dependent on the
light intensities that the specimen are exposed into. The bubbles given off by the plant
are the signals for oxygen evolution.
The study aims to determine the effect of the differences in light intensity to the
photosynthetic rate of Hydrilla verticillata sprigs and to separate and observe the
photosynthetic pigments of chlorophyll extracts using paper chromatography.

REVIEW OF RELATED LITERATURE

The specimen used for the study is the Hydrilla verticellata plant.
Langeland (1996) states that the Hydrilla was discovered in the United States on1960
and native to the tropical Asian regions. It is very well adapted in freshwater
environments because of its highly modified characteristics. He further discusses
different physiognomies and features of the plant and here are some with other
unnecessary details excluded:
1. It is highly polymorphic, and its appearance depends on its living environment.
It grows submerged in water with the roots suspended at the bottom.
2. In terms of the presence of reproductive structures, it can be imperfect
monoecious or imperfect dioecious, with both antheridium and archegonium flowers
singly froma [sic] spathe.
3. It produces hibernacula, turions in leaf and tubers terminally on rhizomes,
which are very compact dormant buds that are produced in leaf axils and falls from the
plant when they mature.
4. It can establish and then display native aquatic plants such as pondweeds
(Potamogeton sp.) and eelgrass (Vallisneria americana Michaux).
5. It is able to grow under wide range of water chemistry conditions.
6. It is adapted to use low light levels for photosynthesis.
7. It has an efficient way of self reproduction and maintenance during hostile
conditions.
On the effect of light intensity on photosynthesis, the Diploma Programme
Experimental Sciences Teacher Support Material (2003) accounts for designing an
experiment to investigate the influence of light on photosynthesis using a sample of
Elodea. For the planning phase, the research question would be established, a
hypothesis would be formulated, proper variables (independent and dependent) would
4

be identified and selected, materials and apparatus appropriate for the study would be
chosen, a method for variable control would be designed, and a method for sufficient
data collection would be premeditated. Afterwards, the variables for the study were to
be identified and categorized into what would be kept constant or altered. Light would
be the variable (independent) variable and the availability of carbon dioxide would be
constant as well as temperature and other external factors like water and wind. The light
intensity would have to be controlled, so the most efficient way to determine the effect
would be to employ an illuminating material that could be moved from a near to far
distance. The experiment have to be performed at an enclosed room with windows
closed to avoid stray lights and air. The plant should be submerged in water containing
sodium bicarbonate, with replacement at every light intensity change, to ensure CO 2
availability. An external water bath could be placed between the plant and the light
source to maintain a constant temperature. For the actual set up, the Elodea would be
suspended in a test tube in a water bath (make sure no oxygen would escape), and
bubbles would generate as it photosynthesizes. Then, these bubbles would be counted
to measure the photosynthetic rate. The water displacement could also be directly
observed or the bubbles could be captured by means of a syringe through bulb
accumulation and capillary tube drawing. At least two to three trials should be made to
arrive upon an average bubble count. The size of the bubbles per trial should be the
same. It would have been efficient to start the experiment with the light source 10 cm
away from the set up and the bubbles to be counted for about 30 minutes. Afterwards,
the water should be replaced and the light source should be moved by an additional 10
cm. The process could be repeated a number of times until one meter of light source

distance have been reached. As a predicting conclusion, the most number of bubbles
produced should have been at the high light intensity range. If the light would be
intensified furthermore, it may not anymore influence the rate when a certain plateau
has been reached with the temperature to become a limiting factor. If the distance of
the source away from the plant would be doubled, the light intensity would have
decreased four times.
There were several works regarding the extraction of photosynthetic pigments in
the article An improved method for extraction and separation of photosynthetic
pigments (2003) on the Journal of Biological Education. In the article, it was explained
that in 1997, reverse paper chromatography was proposed by Neil Reese for separating
the pigments. However, due to the high demands of cost for laboratory equipment, the
method failed to be introduced into the secondary level laboratory classes even though
it provides conspicuous results. Thus, it was concluded that PC or paper
chromatography and TLC or thin layer chromatography were the appropriate methods
for the latter school level. Moreover, these methods were proposed as such because
the solvents (PE mixture and acetone) were of low volatility and were safer than the use
of diethyl ether even if the latter yields very conspicuous results. The cost that the
exercise entails was also reduced due to the less amount of organic solvents required
As the separation of pigments by TLC is clearer than that by PC, many kinds of TLC
plates were tested. Of the thin layer material, silica gel resulted in better separation
than cellulose powder Among the silica gel TLC plastic plates provided, Mercks TLC
plate is the best one because thin layer silica gel does not come off easily when the
plate is cut into small strips. It was further implied that the laboratory exercise on the
6

extraction and separation of photosynthetic pigments from terrestrial plant leaves and
algal fronds might be useful to allow the students to realize both the unity and diversity
of plants.
METHODOLOGY
For the determination of the effect of light intensity, Hydrilla verticellata sprigs
were used as experimental specimen. There were three set ups (ordinary room light
intensity, high light intensity and low light intensity), all done with the following
procedures, but with different light intensities. First, the sprigs were placed in a large
glass funnel such that their freshly cut ends were towards the stem of the funnel. Next,
the funnel was inverted into a 500 mL beaker that was filled with water. Finally, a
test tube containing water was carefully inserted over the funnel stem. The first set up
was placed under high light condition using an illumination lamp, the other under
ordinary room light condition (for control), and the last under low light condition wherein
the room lighting was dimmed. Afterwards, the funnel escaping bubbles, representing
oxygen released, given off by the sprigs were counted and were recorded per unit time.
Observations were made three times for each set up. These were made by dividing
the class into three groups, each with a specific set up to focus with.
For the separation of photosynthetic pigments by paper chromatography, 2 x 9
cm strip of chromatogram paper with a hole at the bottom was obtained from the
instructor. A narrow band of the chlorophyll extract was then applied about 1 cm from
the edge of the opposite end of the paper using a Pasteur pipette. The point for the
application was beforehand noted using a pencil. This was allowed to dry and then
another was added on the same narrow band. Drying was further allowed.
7

Subsequently, a 15 mL of chromatogram solvent, composed of 14 parts of ethyl alcohol,

3 parts benzene and 1 part petroleum ether, was placed on a 250 mL beaker. A glass
tubing was inserted in the hole of the chromatogram solvent and was suspended over
the solvent, with the lower end of the strip totally immersed. The strip was carefully
positioned in a way that it did not touch the sides of the beaker. Then, a 1000 mL
beaker lined with carbon paper was used to cover the set up to prevent light and wind
from affecting the capillary movement of the solvent through the strip. After 15 minutes,
the cover was removed and the four bands of photosynthetic pigments were observed
and noted.

RESULTS AND DISCUSSION

A. Effect of Light Intensity on Photosynthesis
The aim of the first part of the experiment was to conclude on the resulting
rate of photosynthesis under different light intensities. After the conduction, the following
results were yielded.
Table 1. The number of oxygen bubbles per minute given off by photosynthesizing
Hydrilla sprigs in high and low intensities.
Observation
Control
Low Light Intensity High Light Intensity
Number
(per minute)
(per minute)
(per minute)
1
60
2
120
2
30
1
240
3
180
2
300
Average
90
1.67
220
Table 1 above shows the estimated total and average number of bubbles per
minute evolved in each set up. On the first observation, 60, 2, and 120 bubbles
8

evolved per minute; on the second observation, 30, 1, and 240 bubbles evolved per
minute and; on the third observation, 180, 2, and 300 bubbles evolved per minute, all in
the control, low light intensity, and high light intensity set ups respectively. The
average number of bubbles evolved per minute in the control was 90, in the low light
intensity was 1.67, and in the high light intensity was 220.
The total number of bubbles evolved for each observation was highest in the high
light intensity, followed by the control, and then in the low light intensity. This suggests
that as the light intensity is increased, the number of bubbles evolved from the Hydrilla
sprigs increases as well. However, decreasing the light intensity significantly reduces
the number of bubbles evolved. These bubbles that evolve represent the rate of
photosynthesis of the sprigs under the several light conditions, as these are the oxygen
gas that are produced from the process. Plants obtain light energy from photons that
are absorbed by the pigments in the photosystems. This energy is then utilized as the
initiating factor for photosynthesis. Low light intensities will prevent water from
undergoing the process of photolysis or the splitting into hydrogen and oxygen atoms,
and therefore will not result in any oxygen gas evolution. Heightened light intensities
energize numerous electrons in the reaction center of the photosystem, and so more
water is split into hydrogen and oxygen to replace the high energy electron. This will
cause the release of higher concentration of oxygen gas, as synonymous to the
production of many bubbles.
Moreover, the Brilliant Biology Students (2015) webpage states that light can be
a limiting factor when the light intensity is too insignificant to promote the maximum
action of the light dependent reactions. It does not usually limit the photosynthetic
9

process except for forest plants and plants that are under shady environments. Too
strong light intensities may result to chlorophyll bleaching and will eventually inhibit
photosynthesis. Nevertheless, plants having quite exposure to such settings usually
have adaptation features, such as the thick, waxy cuticle and hairy leaves, to prevent
photosynthetic delays due to the previously stated action of chlorophyll.
As explicated by the Royal Society of Chemistry (n.d.), the rate of the light
dependent reactions in the process of photosynthesis tends to hasten when the light
intensity increases at low light environments, showing a straight line or direct
proportionality. The more photons of light that fall on a leaf, the greater the number of
chlorophyll molecules that are ionized [sic] and the more ATP and NADPH are
generated. Since light dependent reactions use light energy, they are unaffected by
temperature changes. However, further increase in the light intensity may attribute the
photosynthetic rate into other factors, such as the destruction of the chlorophyll that
intensely slows down the rate Light consisting of a great proportion of energy
concentrated on wavelengths between 680nm and 700nm (efficient energy absorption
wavelengths of photosystem II and I respectively) will yield significant photosynthetic
frequencies.

B. Separation of Photosynthetic Pigments by Paper Chromatography

10

The concluding part of the experiment was intended to separate the different
photosynthetic pigments using paper chromatography through chlorophyll extract and
chromatograph solvent. The results of the experiment are illustrated below.
Solvent used: Chromatographic solvent composed of 14 ethyl: 3 benzene: 2
petroleum ether

51

Color Observed

Pigment

yellow

xanthophyll

yellow green

chlorophyll B

green

chlorophyll A

brown

carotene

44
Figure 1. Photosynthetic pigments in the leaf chloroplast
ORIGIN

25
36

11

Figure 1 depicts the observable layers of color changes representing pigments in

the chromatogram paper after applying a narrow band of chlorophyll extract at the
bottom of the paper and immersing it in the chromatogram solvent. At 25 cm in height,
the brown carotene emerged just close to the origin. The green chlorophyll appeared
from 36 cm. Moreover, the yellow green chlorophyll b was sighted up to the 44cm
height. Lastly, the yellow xanthophyll appeared at the topmost from 51 cm.
The chromatogram solvent used in the experiment is polar, since it contains a
great proportion of alcohol, ethyl that is polar in itself. The band of chlorophyll extract in
the chromatogram paper actually separates and the pigment with the highest polarity,
which is xanthophyll, settles at the top. According to Clark, the use of paper
chromatography involves two phases the mobile phase and stationary phase. The
mobile phase, a liquid or a gas, flows through the stationary phase, (a solid or liquid
supported on a solid), carrying the components of the mixture with it The stationary
phase is the chromatogram paper itself, which is uniformly absorbent, while the mobile
phase is the chromatogram solvent (2007). Thus, the chromatogram solvent (mobile)
moves along the chromatogram paper (stationary), bringing the components of the
pigment extract from the bottom of the paper through the top, with the less polar
pigment being easily left away from the lower layers. Therefore, the arrangement of
pigments in terms of increasing polarity is: carotene, chlorophyll a, chlorophyll b, and
xanthophyll.

CONCLUSION

12

Photosynthesis is a biological process by which carbon dioxide and water are

transformed into carbohydrates (triose phosphate) and oxygen through its two chief
stages, the light reactions and the Calvin cycle, with the aid of sunlight. It primarily
occurs in the chloroplast present in leaves, with the first stage initiating at the
thylakoids/grana and the second stage at the stroma. The chloroplasts pigments mainly
the green chlorophyll, captures red and blue light radiating from the sun and utilizes this
energy into chemical energy necessary for the photosynthetic process. There are
several factors affecting its rate, and a major one is the level of light intensity. Moreover,
the pigments of the plants can be studied and separated through the method of paper
chromatography.
Light is a vital source of energy that plants need to start photosynthesis.
Therefore, its strength has a major effect on the photosynthetic process itself.
Increasing the light intensity yields an increase in photosynthetic rate, as observed
through the significant number of bubbles formed, meaning the evolution of oxygen gas.
However, continuous increase in illumination may cause other factors to blend in like
chlorophyll damage commencing the downfall in rate. On the contrary, decreasing the
light intensity causes the rate of photosynthesis to slow down as seen in the very few
bubbles formation, for minimal photons are being utilized by the pigments in the
chloroplast.
These pigments in the chloroplast can be observed and studied by
separating them from each other. To do this, the method of chromatography should be
considered. Chromatography is the separation of the components of a mixture or
substance through the use of substances that can act as a mobile or stationary phase.
13

The stationary phase (chromatogram paper) with the chlorophyll extract is passed
through by the polar mobile phase (chromatogram solvent), causing the different
colored pigments to be displaced throughout the paper with the most polar at the top
and the least at the bottom.

LITERATURE CITED
An improved method for extraction and separation of photosynthetic pigments. (2003).
Journal of Biological Education, 186-189.
Clark,

J. (2007). Paper Chromatography. Retrieved April 18,

http://www.chemguide.co.uk/analysis/chromatography/paper.html

2015,

from

Devlin, R. M., & Barker, A. V. (1971). Photosynthesis. In Photosynthesis. New York: Van
Nostrand Reinhold Company.
EFFECT OF LIGHT INTENSITY ON THE RATE OF PHOTOSYNTHESIS. (2015).
Retrieved
from
Brilliant
Biology
Student:
http://brilliantbiologystudent.weebly.com/effect-of-light-intensity.html
Hall, D. O., & Rao, K. K. (1994). Photosynthesis Fifth Edition. Great Britain: Cambridge
University Press.
Hubbard, B. (2012, November 19). The Power of Photosynthesis. Retrieved from Helix:
https://helix.northwestern.edu/article/power-photosynthesis
Lambers, H. (2015, March 17). Photosynthesis. Retrieved from Encyclopedia
Britannica: http://www.britannica.com/EBchecked/topic/458172/photosynthesis
Langeland, K. A. (1996). Hydrilla verticillata (L.F.) Royle (Hydrocharitacae), "The Perfect
Aquatic Weed". Florida: University of Florida, Institute of Food and Agricultural
Sciences.
Material, Diploma Programme Experimental Sciences Teacher Support. (2003).
Investigating the Effect of Light Intensity on Photosynthesis. (M. D. Support, Ed.)
Retrieved from IBO: http://blog.canacad.ac.jp/bio/BiologyIBHL1/files/242900.pdf

14

Rate of photosynthesis: limiting factors. (n.d.). Retrieved from Royal Society of

Chemistry:
http://www.rsc.org/learnchemistry/content/filerepository/CMP/00/001/068/Rate%20of%20photosynthesis
%20limiting%20factors.pdf
Schooley, J. (1997). Introduction to Botany. U.S.A.: International Thomson Publishing
Inc.
Wong,

D.
(n.d.).
Photosynthesis.
Retrieved
from
http://www2.hkedcity.net/sch_files/a/bch/bch-wtw/public_html/notes/Photo-02.pdf

15