BIOLOGY INVESTIGATORY PROJECT

ON COMPARATIVE STUDY OF
CHLOROPHYLL CONTENT IN
DIFFERENT SPECIES OF PLANTS
 CERTIFICATE

This is to certify that DIVYENDU SEKHAR SAHOO, a student of class XII-A has
successfully completed the research on the below mentioned project under the
guidance of Dr. Manna Milan (subject teacher) during the year 2016-17 in partial
fulfillment of biology practical examination conducted my DAV Public School.

Signature of biology teacher Signature of the examiner

 ACKNOWLEDGEMENT

In the accomplishment of this project successfully, many people have best owned
upon me their blessings and the heart pledged support, this time I am utilizing to
thank all the people support who have been concerned with the project.

Then I would like to thank my biology teacher dr. Manna Milian, whose valuable
guidance has been the ones that helped me patch this project and make it full
proof success with suggestions and his instructions has served as the major
contributor towards the completion of the project.

Then I would thank my parents and friends who have helped me with their
valuable guidance have been helpful in various phases of the completion of the
project.

Last of not the least I would like to thank my classmates who have helped me a
lot. I cant forget to offer my sincere thanks to my classmates who helped me to
carry out this project work successfully and for their valuable advice and support,
which I received from them time to time.
 INDEX

CONTENTS PAGE NO

1. INTRODUCTION 1-3

2. OBJECTIVE 4

3. SCOPE AND LIMITATIONS 5

4. THEORY 6-8

5. EXPERIMENT- AIM 9

6. PROCEDURE 10-11

7. OBSERVATION 12

8. RESULT 13

9. REFERENCES AND BIBLIOGRAPHY 14

 INTRODUCTION
Chlorophyll is a green photosynthetic pigment found in chloroplasts of organisms
like cyanobacteria, algae and plants. Its name is derived from Greek words
chloros, meaning green and phylln meaning leaf. First isolated by Joseph
Bienaime Caventou and Pierre Joseph Pelletier in 1817, chlorophyll is an
extremely important molecule, playing a vital role in nature. Chlorophyll is critical
in photosynthesis, where the green pigment plays the role of absorbing energy for
plants to use.

There are at least seven types of chlorophyll know as a, b, c, d, e,

bacteriochlorophyll and bacterioviridin. Chlorophyll absorbs light most strongly in
the blue portion of the electromagnetic spectrum, followed by the red portion.
However, it is absorber of green and near green portions of spectrum, hence
green colour of chlorophyll containing tissues.

Chlorophyll molecules are specifically arranged in and around photosystems that

are embedded in thylakoid membranes of chloroplasts. In these complexes, the
vast majority of chlorophyll serves two primary functions: to absorb light, and to
transfer that light energy by resonance energy transfer to a specific chlorophyll
pair in the reaction centre of the photosystems.

The two currently accepted photosystem units are photosystem II and

photosystem I, which have their own distinct reaction centre chlorophylls, named
P680 and P700, respectively. These pigments are named after the wavelength (in
nanometers) of their red peak absorption maximum. The identity, function and
spectral properties of the types of chlorophyll in each photosystem are distinct,
and determined by each other and the protein structure surrounding them.
Once extracted from the protein into a solvent (like acetone or methanol), these
chlorophyll pigments can be separated in simple paper chromatography
experiment and, based on the number of polar groups between chlorophyll a and
chlorophyll b, will separate out on the paper.

The function of reaction centre chlorophyll is to use the energy absorbed by, and
transferred to it from other chlorophyll pigments in the photosystems, so that the
reaction centre undergoes a charge separation, a specific redox reaction in which
the chlorophyll donates an electron into a series of molecular intermediates
called an electron transport chain. The charged reaction centre chlorophyll
(P680+) is then reduced back to its ground state by accepting an electron. In
photosystem II, the electron that reduces P680+ ultimately comes from the
oxidation of water into O2 and H+ through several intermediates. This reaction is
how photosynthetic organisms such as plants produce O2 gas, and is the source
for practically all the O2 in earths atmosphere. Photosystem I typically works in
series with photosystem II; thus the P700+ of photosystem I is usually reduced via
many intermediates in the transfer reactions in the thylakoid membrane by
electros ultimately from photosystem II. Electron transfer reactions in the
thylakoid membranes are complex, however, the source of electron used to
reduce P700+ can vary.

The electron flow produced by the reaction centre chlorophyll pigments is used to
shuttle H+ ions across the thylakoid membrane, setting up a chemiosmotic
potential used mainly to produce ATP chemical energy; and those electrons
reduce NADP+ to NADPH, a universal reductant used to reduce CO2 into sugars as
well as for other biosynthetic reductions.
Reaction centre chlorophyll protein complexes are capable of directly absorbing
light and performing charge separation events without other chlorophyll
pigments, but the absorption cross section (the likelihood of absorbing a photon
under a given light intensity) is small. Thus, the remaining chlorophylls in the
photosystem and antenna pigment protein complexes associated with the
photosystems all cooperatively absorb and funnel light energy to the reaction
centre. Besides chlorophyll a, there are other pigments called accessory pigments,
which occur in these pigment- protein antenna complexes.

Chlorophyll is a chlorine pigment, which is structurally similar to and produced

through the same metabolic pathway as other porphyrion pigments such as
heme. At the centre of the chlorine ring is a magnesium ion. At time of discovery
in 1900s, this was the first time this element was detected in a living tissue. The
chlorine ring can have several different side chains, usually including a long phytol
chain. There are a few different forms that occur naturally but most widely
distributed form in terrestrial plants is chlorophyll A. after initial work done by
German chemist Richard Willstatter spanning from 1905-1915, general structure
of chlorophyll a was elucidated by Hans Fischer in 1940. By 1960, when most of
stereochemistry of chlorophyll a was known, Robert published a total synthesis of
the molecule. In 1967, Ian Fleming completed the last remaining stereo chemical
elucidation, and in 1990 Woodward and co-authors published an updated
synthesis.
 OBJECTIVE

The objective of this experiment is to study the chlorophyll levels indifferent plant
species. In this experiment I seek to use chromatography to separate the
various pigments present in the leaves of various plants. Through this, we can
measure the amount of each pigment present in each type of leaf and hence,
understand the chlorophyll content in the assorted plants. We extract the
pigments from various leaves, and with the addition of various chemicals
methodically, we separate the various pigments present in leaves like, chlorophyll
a, chlorophyll b, carotenioids, and xanthophylls. We then measure the quantity of
each, and put all the data in a table to compare the levels of various pigments in
various plants.

In this manner, we also perform an internal study where we compare pigment

levels in yellow and green leaves of the same plants to understand the pigment
difference when senescence takes place and leaf yellowing takes place.
 SCOPE AND LIMITATIONS

This project also helps us in understanding the importance of chlorophyll for

animals as well as in human diet.

Chlorophyll is known to be the plants blood, in other words the principle

physiology of plant life. Chlorophyll is so important to plants because it performs
metabolic functions such as respiration and growth.

Just as significantly, chlorophyll supplies our bodies with the much needed,
micronutrient magnesium which is essential to how our body produces energy.
Many health specialists use chlorophyll as a tonic for the blood due to its richness
in nutrients.
 THEORY
Chlorophyll is a green pigment found in cyanobacteria and chloroplasts of algae
and plants. It is a critical biomolecule in the process of photosynthesis, which
allows plants to absorb energy from light. It is present in the chloroplasts
thylakoid membrane. Within the chloroplast, there is a membranous system of
grana, stroma lamellae and fluid stroma. The membrane system is responsible for
trapping light energy and for synthesis of ATP and NADPH.

The colour of leaves we see is not due to a single pigment but due to
four pigments namely chlorophyll a, chlorophyll b, xanthophylls and carotene.

Although Chlorophyll a is the chief pigment associated with photosynthesis, other

thylakoid pigments like chlorophyll b, xanthophylls and carotenes are the
accessory pigments. They absorb light and transfer the energy to chlorophyll a.
The function of the vast majority of chlorophyll is to absorb light and transfer that
light energy to a specific chlorophyll pair in the reaction centre of the
photosystems.

There are two photosystem unit present photosystem I (PS I) and photosystem II
(PS II) that have their own reaction centers P700 and P680 respectively.
Within each PS I and PS II there are photochemical light harvesting systems
present which are made up of many pigment molecules bounded to proteins.

Chlorophyll a

Chlorophyll a is essential for most photosynthetic organisms to release chemical

energy but is not the only pigment that can be used for photosynthesis. One
molecule of chlorophyll a forms the reaction centre. It absorbs energy from
wavelengths of violet and red light.

The molecular structure of chlorophyll a consists of a chlorine ring, whose four

nitrogen atoms surround a central magnesium atom, and has several other
attached side chains and a hydrocarbon tail.

This photosynthetic pigment is essential for photosynthesis in

eukaryotes, cyanobacteria and pro chlorophytes because of its role as primary
electron donor in the electron transport chain.
Chlorophyll b

Chlorophyll b helps in photosynthesis by absorbing light energy. It is more soluble

than chlorophyll a in polar solvents because of its carbonyl group. Its color is
yellow, and it primarily absorbs blue light. In land plants, the light harvesting
antennae around photosystem II contain the majority of chlorophyll b.

Xanthophylls

Xanthophylls (originally phylloxanthins) are yellow pigments that form one of

two major divisions of the carotenoid group. Their molecular structure is similar
to carotenes, which form the other major carotenoid group division, but
xanthophylls contain oxygen atoms, while carotenes are purely hydrocarbons
with no oxygen

Like other carotenoids, xanthophylls are found in highest quantity in the leaves of
most green plants, where they act to modulate light energy and perhaps serve as
a non-photochemical agent to deal with excited chlorophyll.

Carotenes

Carotene is an orange photosynthetic pigment important

for photosynthesis. Carotenes are all coloured to the human eye. Carotenes
contribute to photosynthesis by transmitting the light energy they absorb
to chlorophyll. They also protect plant tissues by helping to absorb the energy
from singlet oxygen an excited form of the oxygen molecule O2 which is formed
during photosynthesis.
 EXPERIMENT
Chlorophyll content in various plant species
Aim:
To compare and study the chlorophyll content in different plant species.

Requirements:

Fresh leaves of spinach

Mint
Methi leaves
Bougainvilla
Cabbage leaves
Separating funnel
Measuring cylinder
Beakers
Vials

Chemicals required:

Acetone
Diethyl ether
Petroleum ether
Methyl alcohol
Calcium carbonate
Potassium hydroxide
Distilled water
 PROCEDURE

Take 10g of fresh leaves in pestle and crush it with 4ml 80%acetone. Add a
little CaCO3 and again crush it. Filter the extract in a Buchner funnel. The
filtrate is called acetone extract and it is rich in chlorophyll and carotenoids.

Take 4ml of acetone extract and add petroleum ether. Shake funnel gently.

Add water and shake again. Two layers will be formed. Upper containing
petroleum ether will contain chlorophyll a and carotene.

The lower acetone water layer is discarded.

To the upper remaining layer add 4ml 92% methyl alcohol. Shake the funnel
and let it separate into layers. Upper layer contains petrol and ether rich in
chlorophyll a and carotenoids; lower is the methyl alcohol layer rich in
chlorophyll b and xanthophylls pigments.

To the upper layer add 1.5ml 30% methyl alcohol and KOH. Add water and
shake funnel.

Two layers are obtained. Upper has chlorophyll a and lower has carotene.
 To the lower methyl alcohol layer add 5ml diethyl ether and shake. Add
water slowly 1 ml at a time. Two layers are obtained. The upper layer is the
diethyl ether layer and lower contains methyl alcohol.

Discard the lower layer

To the upper layer add 1.5ml 30% methyl alcohol KOH. Shake funnel and
add water.

Two layers are obtained. Upper layer contains chlorophyll b and lower layer
contains xanthophyll.

Collect the samples, weigh them and note the amount of chlorophyll
pigments present in them.
 OBSERVATION TABLE

SL NO. TYPE OF LEAF TAKEN WEIGHT OF PIGMENT

CHLOROPHYLL A CHLOROPHYLL B CAROTENE XANTHOPHYLL

1. SPINACH 3.4 0.6 4.6 4.82

2. FENUGREEK 1.76 0.5 2.92 3.16

3. BOUGAINVILLA 1.75 0.37 2.33 2.37

4. MINT 3.4 1.02 4.73 4.63

5. CABBAGE 3.59 0.55 5.3 4.9

 RESULT

Each type of leaf has various levels of pigments based on its genetic constitution,
exposure to light, age, season, wind, precipitation, photosynthetic rate,
respiration rate, and protein level.

Out of the five leaves tested, cabbage had the highest level of Chlorophyll a, and
Bougainvillea the lowest. The highest level of Chlorophyll b was present in mint
while the lowest level was present in mint while the lowest level was present in
Bougainvillea. Cabbage had the most Carotene and Bougainvillea had the least.
Cabbage also had the greatest level of Xanthophylls and Bougainvillea had the
least.

As seen clearly, chlorophyll value decreases with leaf senescence.

REFERENCES
Comprehensive Laboratory Manual In Biology- XII
Biology Text for Class XII - NCERT

BIBLIOGRAPHY
www.wikipedia.org
www.google.com
www.howstuffworks.com
www.letsmakesciencefun.com