The effect of light intensity on the amount of chlorophyll in Cicer arietinum

The effect of light intensity on the amount of chlorophyll in Cicer arietinum

Extended Essay

Biology (SL)

The effect of light intensity on the amount of chlorophyll in Cicer


Word count: 4 413 words


Abstract 2

Introduction .. 3

Hypothesis 3


Description .... 8

Results .. 10

Discussion .... 14

Conclusion .... 14

Evaluation of the method .. 15

Bibliography . 16


Plants, growing on the shaded area has less concentrated green color

and are much longer and thinner than plants growing on the sun areas as

they are dark green, short and thick. Research question was: How does the

amount of chlorophyll-a and chlorophyll-b, gram per gram of plant, depends

on the light intensity in which plants are placed?

Hypothesis suggests that there are several inner and outer factors

that affect the amount of chlorophylls a and b in plants and that with the

increase of light intensity the amount of chlorophyll will also increase

until light intensity exceeds the value when the amount of destructed

chlorophylls is greater than formatted thus decreasing the total amount of

chlorophylls in a plant.

The seeds of Cicer arietinum were divided into seven groups and

placed into various places with different values of light intensities.

Light intensities were measured with digital colorimeter. After three weeks

length was measured. Then plants were cut and quickly dried. Their biomass

was also measured. Three plants from each group were grinded and the

ethanol extract of pigments was prepared. The amount of chlorophylls was

measured using method of titration and different formulas.

The investigation showed that plants growing on the lowest light

intensity equal 0 lux contained no chlorophyll and had the longest length.

The amount of chlorophyll quickly increased and length decreased with the

increase of light intensity from 0 lux to 1200 lux. The amount of

chlorophyll in plants unpredictably decreased during light intensity equal

to 142 lux and than continued increasing and didnt start decreasing

reaching very high value (1200 lux).

The sudden decrease happened due to mighty existence of some inner

genetical damages of seeds which prevented them from normal chlorophyll

synthesis and predicted decrease didnt decrease because extremely high

light intensity was not exceeded.

Word count: 300 words

I. Introduction.

This theme seemed to be attractive for me because I could see that

results of my investigation could find application in real life.

While walking in the forest in summer I saw lots of plants of

different shades of green color: some of them were dark green, some were

light green and some even very-very light green with yellow shades, hence I

became very interested in this situation and wanted to know why it happens

to be so. I also saw that those plants that were growing on sunny parts of

forest, where trees were not very high, had dark green color and those,

that were growing in shady parts of the same forest had very light green

color. They also had difference in their length and thickness those, that

were growing on light were very short, but thick and strong, and those,

growing in shady regions were very thin and fragile.

Hence I became very interested in finding scientifical description of

my observations.

The aim of my project is to find out how does the changes in light

intensity affect balance of chlorophyll in Cicer arietinum.

II. Hypothesis.

There are several factors that affect the development of chlorophyll

in plants.[1]

Inner factors. The most important one is genetical potential of a

plant, because sometimes happen mutations that follow in inability of

chlorophyll formation. But most of the times it happens that the process of

chlorophyll synthesis is broken only partly, revealing in absence of

chlorophyll only in several parts of the plant or in general low rate of

chlorophyll. Therefore plants obtain yellowish color. Lots of genes

participate in the process of chlorophyll synthesis, therefore different

anomalies are widely represented. Development of chloroplasts depends on

nuclear and plastid DNA and also on cytoplasmatic and chloroplastic


Full provision of carbohydrates seem to be essential for chlorophyll

formation, and those plants that suffer from deficit of soluble

carbohydrates may not become green even if all other conditions are

perfect. Such leaves, placed into sugar solution normally start to form

chlorophyll. Very often it happens that different viruses prevent

chlorophyll formation, causing yellow color of leaves.

Outside factors. The most important outside factors, affecting the

formation of chlorophyll are: light intensity, temperature, pH of soil,

provision of minerals, water and oxygen. Synthesis of chlorophyll is very

sensitive to all the factors, disturbing metabolic processes in plants.

Light. Light is very important for the chlorophyll formation, though some

plants are able to produce chlorophyll in absolute darkness. Relatively low

light intensity is rather effective for initialization and speeding of

chlorophyll development. Green plants grown in darkness have yellow color

and contain protochlorophyll predecessor of chlorophyll , which needs

lite to restore until chlorophyll . Very high light intensity causes the

destruction of chlorophyll. Hence chlorophyll is synthesized and destructed

both at the same time. In the condition of very high light intensity

balance is set during lower chlorophyll concentration, than in condition of

low light intensity.

Temperature. Chlorophyll synthesis seems to happen during rather broad

temperature intervals. Lots of plants of synthesize

chlorophyll from very low temperatures till very high temperatures in the

mid of the summer. Many pine trees loose some chlorophyll during winters

and therefore loose some of their green color. It may happen because the

destruction of chlorophyll exceeds its formation during very low


Provision with minerals. One of the most common reason for shortage of

chlorophyll is absence of some important chemical elements. Shortage of

nitrogen is the most common reason for lack of chlorophyll in old leaves.

Another one is shortage of ferrum, mostly in young leaves and plants. And

ferrum is important element for chlorophyll synthesis. And magnesium is a

component of chlorophyll therefore its shortage causes lack of production

of chlorophyll.

Water. Relatively low water stress lowers speed of chlorophyll synthesis

and high dehydration of plants tissues not only disturbs synthesis of

chlorophyll, but even causes destruction of already existing molecules.

Oxygen. With the absence of oxygen plants do not produce

chlorophyll even on high light intensity. This shows that aerobic

respiration is essential for chlorophyll synthesis.

Chlorophyll.[2] The synthesis of chlorophyll is induced by light.

With light, a gene can be transcripted and translated in a protein.

The plants are naturally blocked in the conversion of protochlorophyllide

to chlorophyllide. In normal plants these results in accumulation of a

small amount of protochlorophyllide which is attached to holochrome

protein. In vivo at least two types of protochlorophyllide holochrome are

present. One, absorbing maximally at approximately 650 nm, is immediately

convertible to chlorophyllide on exposure to light. If ALA is given to

plant tissue in the dark, it feeds through all the way to

protochlorophyllide, but no further. This is because POR, the enzyme that

converts protochlorophyllide to chlorophyllide, needs light to carry out

its reaction. POR is a very actively researched enzyme worldwide and a lot

is known about the chemistry and molecular biology of its operation and

regulation. Much less is known about how POR works in natural leaf


ALA Portoporphyrine



Chlorophyll b Chlorophyll a

Chlorophyll[3] is a green compound found in leaves and green stems of

plants. Initially, it was assumed that chlorophyll was a single compound

but in 1864 Stokes showed by spectroscopy that chlorophyll was a mixture.

If dried leaves are powdered and digested with ethanol, after concentration

of the solvent, 'crystalline' chlorophyll is obtained, but if ether or

aqueous acetone is used instead of ethanol, the product is 'amorphous'


In 1912, Willstatter et al. (1) showed that chlorophyll was a mixture

of two compounds, chlorophyll-a and chlorophyll-b:


Chlorophyll-a (C55H72MgN4O5, mol. wt.: 893.49). The methyl group marked

with an asterisk is replaced by an aldehyde in chlorophyll-b (C55H70MgN4O6,

mol. wt.: 906.51).

The two components were separated by shaking a light petroleum

solution of chlorophyll with aqueous methanol: chlorophyll-a remains in the

light petroleum but chlorophyll-b is transferred into the aqueous methanol.

Cholorophyll-a is a bluish-black solid and cholorophyll-b is a dark green

solid, both giving a green solution in organic solutions. In natural

chlorophyll there is a ratio of 3 to 1 (of a to b) of the two components.

The intense green colour of chlorophyll is due to its strong

absorbencies in the red and blue regions of the spectrum, shown in fig. 1.

(2) Because of these absorbencies the light it reflects and transmits

appears green.


Fig. 1 - The uv/visible adsorption spectrum for chlorophyll.

Due to the green colour of chlorophyll, it has many uses as dyes and

pigments. It is used in colouring soaps, oils, waxes and confectionary.

Chlorophyll's most important use, however, is in nature, in

photosynthesis. It is capable of channelling the energy of sunlight into

chemical energy through the process of photosynthesis. In this process the

energy absorbed by chlorophyll transforms carbon dioxide and water into

carbohydrates and oxygen:

CO2 + H2O [pic](CH2O) + O2

Note: CH2O is the empirical formula of carbohydrates.

The chemical energy stored by photosynthesis in carbohydrates drives

biochemical reactions in nearly all living organisms.

In the photosynthetic reaction electrons are transferred from water to

carbon dioxide, that is carbon dioxide is reduced by water. Chlorophyll

assists this transfer as when chlorophyll absorbs light energy, an electron

in chlorophyll is excited from a lower energy state to a higher energy

state. In this higher energy state, this electron is more readily

transferred to another molecule. This starts a chain of electron-transfer

steps, which ends with an electron being transferred to carbon dioxide.

Meanwhile, the chlorophyll which gave up an electron can accept an electron

from another molecule. This is the end of a process which starts with the

removal of an electron from water. Thus, chlorophyll is at the centre of

the photosynthetic oxidation-reduction reaction between carbon dioxide and


Treatment of cholorophyll-a with acid removes the magnesium ion

replacing it with two hydrogen atoms giving an olive-brown solid,

phaeophytin-a. Hydrolysis of this (reverse of esterification) splits off

phytol and gives phaeophorbide-a. Similar compounds are obtained if

chlorophyll-b is used.


Chlorophyll can also be reacted with a base which yields a series of

phyllins, magnesium porphyrin compounds. Treatment of phyllins with acid

gives porphyrins.


Now knowing all these factors affecting the synthesis and destruction

of chlorophyll I propose that the amount of chlorophyll in plant depends on

light intensity in the following way: with the increase of light intensity

the amount of chlorophyll increases, but then it starts decreasing because

light intensity exceed the point when there is more chlorophyll destructed

than formed.




. Light intensity, lux


. pH of soil

. water supply, ml

. temperature, to C


. length, cm

. amount of chlorophyll in gram of a plant, gram per gram

III. Method.


. seeds of Cicer arietinum

. 28 plastic pots

. water

. scissors

. ruler (20 cm ( 0.05 cm)

. CaCO3

. soil (adopted for home plants)

. digital luxmeter (( 0.05 lux)

. test tubes

. H2SO4 (0.01 M solution)

. Pipette (5 cm3 ( 0.05 cm3)

. mortar and pestle

. burette

. ethanol (C2H5OH), 98%

. beakers

Firstly I went to the shop and bought germinated seeds of Cicer

arietinum. Then sorted seeds and chose the strongest ones. After that I

prepared soil for them and put them in it.

As the aim of this project is to investigate the dependence of mass of

chlorophyll in plants during different light intensities it was needed to

create those various conditions. Pots with seeds were placed into the

following places: in the wardrobe with doors (light intensity is o lux),

under the sink (light intensity is 20,5 lux), in the shell of bookcase

(light intensity is 27,5 lux), above the bookcase (light intensity is 89,5

lux), above the extractor (light intensity is 142 lux), beyond the curtains

(light intensity is 680 lux) and on the open sun (light intensity is 1220

lux). Light intensity was measured with the help of digital luxmeter. It

was measured four times each day: morning, midday, afternoon, evening.

During those four periods four measurements were done and the maximum value

was taken into consideration and written down. Those measurements lasted

for three weeks of the experiment as the whole time of the experiment was

three weeks. The luxmeters sensitive part was placed on the plants (so it

was just lying on them) in order to measure light intensity flowing

directly on plant bodies, then two minutes were left in order to get

stabilized value of light intensity and the same procedure was repeated.

All those actions were done in order to get more accurate results of light


Growing plants were provided with the same amount of water (15 ml, once a

day in the morning) and they were situated in the same room temperature

(20o C), pH of soil was definitely the same because all the plants were put

in the same soil (special soil for room flowers).

After three weeks past the length of plants was measured with the help of

ruler. Firstly the plants were not cut, so their length had to be measured

while they were in the pots. The ruler was placed into the pot and plants

were carefully stretched on it. The action was repeated three times and

only maximum value was taken into consideration. After that plants were

cut. Then those already cut plants were put into the dark place and quickly



I have chosen three plants from each light intensity group and measured

their weight. . In order to obtain the pigments, three plants were cut into

small pieces and placed in a mortar. Calcium carbonate was then added,

together with a little ethanol (2 cm3). The leaf was grinded using a pestle

until no large pieces of leaf tissue were left, and the remaining ethanol

was poured into the mortar (3 cm3). Then 1 ml of obtained solution was

placed into the test tube and this 1 ml of solution was then titrated

against 0.01 M solution of sulfuric acid, through the use of a pipette. The

titration was complete when the green solution turned dark olive-green[4].

This solution obtained from the first action was stored as the etalon for

the following ones. The settled olive-green coloring meant that all

chlorophyll had reacted with H2SO4. So the process of titration was

repeated 7 times for all light intensity groups.

The solution is titrated until the dark olive-green color because it is

known that when the reaction between chlorophyll and sulfuric acid happens,

chlorophyll turns into phaeophetin which has grey color (see table 1),

therefore when the solution is olive-green, than the reaction has

succeeded. But while searching in the internet and books I found out that

there are several opinions about the color of phaeophytin in the book

written by Viktorov it is ssaid to have grey color, but in the internet

link http://www.ch.ic.ac.uk/local/projects/steer/chloro.htm it is said to

have brown olive-green color. Also I made chromatography in order to

investigate the color of phaeophytin and the result was that it has grey

color. It can be proposed that olive-green color is obtained because grey

phaeophetyn is mixed with other plant pigments.

So titration is one of the visual methods that can be used in order to

find the mass of chlorophyll in plants.

All the measurements and even chromatography were done three times and

the mean value was taken, for chromatography grey color was confirmed.

Table 1. Plant pigments.

|Name of the pigment |Color of the pigment |

|Chlorophylls ( a and b ) |Green |

|Carotene |Orange |

|Xanitophyll |Yellow |

|Phaeophytin-a |OLIVE BROUN or GREY |

IV. Results.

Table 2. Raw data.

|Number of |Light intensity (lux) |

|plant | |

|0 |0,273 |0,041 |84,98 |41,89 |0,0000 |

|20,5 |0,579 |0,056 |90,33 |41,76 |0,0496 |

|27,5 |0,332 |0,033 |90,06 |36,33 |0,1462 |

|89,5 |0,181 |0,018 |90,06 |19,81 |0,1769 |

|142 |0,511 |0,047 |90,80 |41,33 |0,0697 |

|680 |0,338 |0,043 |87,28 |29,33 |0,1557 |

|1220 |0,301 |0,034 |88,70 |18,64 |0,1939 |


Calculation of amount of chlorophyll in plants basing on the results of


H2 SO4 + C56 O5 N4 Mg => C56 O5 N4 H + MgSO4

Concentration of H2SO4 is 0,01 M

C concentration

V volume

n quantity of substancy

m mass

Mr molar mass

For light intensity equal to 20,5 lux.

n = V (in dm3) ? C

2 ? 10-3 ? 0,01 = 2 ? 10-5

n = m / Mr => m = n ? Mr

m = 2 ? 10-5 ? 832 = 1,664 ? 10-2 grams

mass of plant mass of chlorophyll

1,68 grams - 0,08335 grams of


1 gram - x grams of


Hence there are 0,0496 grams of chlorophyll.


Table 5. The correlation between mean length of plants and mean dry


| | | | | | | |

| | | | | | | |


Table 6. The correlation between mean length and mass of chlorophyll per 1

g of plant.

Site |Mean length, cm |Rank (R1) |Mass of chl. In 1 g |Rank (R2) |D (R1-

R2) |D^2 | |1 |41,89 |1 |0,0000 |7 |-6 |36 | |2 |41,76 |2 |0,0496 |6 |-4

|16 | |3 |36,33 |4 |0,1462 |4 |0 |0 | |4 |19,81 |6 |0,1769 |2 |4 |16 | |5

|41,33 |3 |0,0697 |5 |-2 |4 | |6 |29,33 |5 |0,1557 |3 |2 |4 | |7 |18,64 |7

|0,1939 |1 |6 |36 | | | | | | | | | |

Rs = -1

| | | | | | | | | | | | | | | |


Table 7. The correlation between mean dry biomass and mass of chlorophyll

per 1 g of plant.

Site |Mean dry biomass, g |Rank (R1) |Mass of chl. In 1 g |Rank (R2) |D

(R1-R2) |D^2 | |1 |0,041 |4 |0,0000 |7 |-3 |9 | |2 |0,056 |1 |0,0496 |6 |-5

|25 | |3 |0,033 |6 |0,1462 |4 |2 |4 | |4 |0,018 |7 |0,1769 |2 |5 |25 | |5

|0,047 |2 |0,0697 |5 |-3 |9 | |6 |0,043 |3 |0,1557 |3 |0 |0 | |7 |0,034 |5

|0,1939 |1 |4 |16 | | | | | | | | | | | | | | | | | |Rs = -0,57 | | | | | |

| |

| | | | | | | | | | | | | | | | | | | | | | | |


V. Discussion.

Several tendencies can be clearly seen.

For the first, with the increase of light intensity mean length of

plants is decreasing, but there are exceptions. For light intensity 142 lux

the value of mean length is approximately equal to the values of length for

light intensities 0 lux and 20,5 lux. If exclude this data it is also seen

that for light intensity equal to 680 lux mean length is also slightly

falling out from the main tendency decreasing from 19.81 cm.

The second tendency is increase of mass of chlorophyll per 1 gram of

plant biomass with the increase of light intensity. But the values of mass

of chlorophyll of those plants under light intensities 142 lux and 680 lux

are falling out from the main tendency. The first and the second ones are

too small approximately equal to the value corresponding to 20.5 lux

light intensity and to 89.5 lux respectively. This may happen because not

all the seeds of Cicer arietnum were of the same quality, because it is

impossible to guarantee that more than 250 seeds in one box have the same

high quality. At the mean time it was expected that starting from the light

intensity more than 680 lux the amount of chlorophyll in plants will

decrease, because the value of destructed chlorophyll with be bigger than

the value of newly formatted. But the experiments showed that the amount of

chlorophyll was constantly increasing even when the light intensity level

exceeded the point 1220 lux. This could happen because light intensity

equal to 1220 lux is not so extremely high that the amount of total

chlorophyll in plants will start decreasing.

Also it is clearly seen that there are no correlations between light

intensity and values of wet and dry biomass.

Basing on these arguments the sudden decrease of the amount of

chlorophyll in plants placed on light intensity equal to 142 lux was likely

to be insignificant and could not be considered as a trend.

But it is impossible to forget such important factor as plant hormones

that affect the growth and development of plants. There are five generally

accepted types of hormones that influence plant growth and development.

They are: auxin, cytokinin, gibberellins, abscic acid, and ethylene. It is

not one hormone that directly influences by sheer quantity. The balance and

ratios of hormones present is what helps to influence plant reactions. The

hormonal balance possibly regulates enzymatic reactions in the plant by

amplifying them.

VI. Conclusion.

Due to results of my investigation it is seen that my hypothesis

didnt confirm fully (for example, comparing the diagram 1 and diagram 7),

because I proposed that when light intensities will be very high, mass of

chlorophyll in plant will start decreasing and due to my observations it

didnt happen. I should say that the only reason I can suggest is that I

havent investigated such extremely high light intensities, so that

chlorophyll start destructing. But if we will not pay attention to that

fact the other part of my hypothesis was confirmed and mass of chlorophyll

in plants increased with the increase of light intensity. Furthermore I

didnt estimate amount of plant hormones and so didnt estimate their

influence on results.

Questions for further investigation:

1. Investigating very high light intensities.

2. Implementation of colorimetric analysis.

3. Paying attention to estimation of plant hormones level.

Those questions should be further investigated in order to get clearer

picture and more accurate results of the dependence of the amount of

chlorophyll in plants on the light intensity, knowing the fact that the

amount of chlorophyll has a tendency to decrease at extremely high light

intensities. So this statement needs an experimental confirmation and as in

this investigation conditions with extremely light intensity were not

created in further investigations they have to be created.

Implementation of colorimetric analysis is also very important thing,

because it gives much more accurate results comparing with the titration

method. The colorimetric method suggests that as different pigments absorb

different parts of light spectrum differently, the absorbance of a pigments

mixture is a sum of individual absorption spectra. Therefore the quantity

of each individual pigment in a mixture can be calculated using absorbance

of the certain colors and molecular coefficients of each pigment. This was

proposed by D. A. Sims, and J. A. Gamon (California State University,

USA)[5] with the reference on Lichtenthaler (1987).

VII. Evaluation.

There are several results in my work, that are falling out from the

main tendencies. It may seem that such results may occur due to different

percentage of water in plants, but when I was calculating mass of

chlorophyll in 1 gram of plant I was using only values of mean dry biomass

so it couldnt affect my results. (see table 3)

At the same time such differences in the percentage of water are

easily explained. The rate of evaporation of water from plants, which were

put under 1220 lux light intensity was much higher than of those put under

20.5 lux light intensity, therefore percentage of water in the soil may

vary, though I provided all the plants with the same volume of water at the

same periods of time.

One more reason that could be proposed is the reason connected with

the pH of water with which flowers were provided. It was not measured but

the thing that could have happened is that it had somehow changed the pH of

soil in which seeds were placed and therefore changed the amount of

synthesized chlorophyll.

Titration is not a perfect way of obtaining results. This happens

because the method is based on visual abilities of a person he has to

decide whether the color he obtained is dark olive-green or not so dark

olive-green. Such a situation concerns lots of mistakes due to different

optical abilities of each person, even some humans are not able to

distinguish those colors, because of the disease called Daltonism.

Even those who do not suffer from this disease can also make mistakes

in such experiment. It is known that people who suffer from Myopia can

hardly see objects that are far from them, but dont have problems with

objects that are near, but it is also important to take into consideration

the fact that their ability to distinguish colors is also lower comparing

with humans with normal eyesight.

There also exist the so called human factor, which also affects the

investigation. Man cant be absolutely objective, because sometimes it is

too hard for a person to falsify his own theory or hypothesis, so one can

ignore results that are not suitable for his statements and select only

those that are suitable, which will also affect the investigation not in

good way.

So as humans eye is not a perfect instrument and humans are not

perfectly objective there should be other methods of investigating the

amount of chlorophyll in plant.

Moreover titration method doesnt distinguish between chlorophylls-a

and chlorophyll-b, phaeophytin-a and phaeophytin-b, as their colors differ,

this giving not very accurate results. Also due to this limiting factor it

is impossible to know whether the whole amount of chlorophyll reacted with

the sulfuric acid and again it adds an uncertainty to the results.

Furthermore the saturation of color depends on the extent of dilution and

it is nearly impossible to say if the solution was diluted till the same

color or not, because it is very difficult to distinguish between different

shades of olive green color.


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[5] http://vcsars.calstatela.edu/esa_posters/ds/dan_esa99.html 05/05/2004


Chlorophyll, gram per gram of plant.

Light intensity, lux

Diagram 1. The predicted change of amount of chlorophyll in leaves of

depending on light intensity

0,57<0,79, therefore there is no significant correlation between mean

length of plants and mean dry biomass.




There is negative correlation between mean length of plants and mass of

chlorophyll per 1 g of plant

0,57<0,79, therefore there is no significant correlation between mean dry

biomass and mass of chlorophyll per D[pic]D[pic]1 g of plant