“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

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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 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

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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.

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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

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hypothesis would be formulated, proper variables (independent and dependent) would

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 CO2

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

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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, Merck’s TLC

plate is the best one because thin – layer silica gel does not come off easily when the

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plate is cut into small strips….” It was further implied that “the laboratory exercise on the

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 ¾ 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

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another was added on the same narrow band. Drying was further allowed.

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 Number

Control (per minute)

Low Light Intensity (per minute)

High Light Intensity (per minute)

1 60 2 1202 30 1 2403 180 2 300

Average 90 1.67 220

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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

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.

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Moreover, the Brilliant Biology Student’s (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

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.

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ORIGIN

25

36

44

51

B. Separation of Photosynthetic Pigments by Paper Chromatography

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

Color Observed Pigment

yellow xanthophyll

yellow green chlorophyll B

green chlorophyll A

brown carotene

Figure 1. Photosynthetic pigments in the leaf chloroplast

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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

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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 chloroplast’s 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.

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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, 2015, from http://www.chemguide.co.uk/analysis/chromatography/paper.html

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

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Rate of photosynthesis: limiting factors. (n.d.). Retrieved from Royal Society of Chemistry: http://www.rsc.org/learn-chemistry/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

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