THE CHLOROPHYLL IN

FIVE DIFFERENT SPECIES

A Project Report
OF Submitted
PLANTS by
NISHANT MISHRA
CBSE GRADE XII

ROLLNO.-3057

DAV PUBLIC
SCHOOL
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 CERTIFICATE
This is to certify that NISHANT MISHRA of grade
XII, DAV PUBLIC SCHOOL KALINGA
NAGAR,BHUBANESWAR with school register
number 3057 has satisfactorily completed the project
in Biology on “COMPARATIVE STUDY OF THE
CHLOROPHYLL IN FIVE DIFFERENT SPECIES OF
PLANTS”, in partial fulfillment of the requirements
as prescribed by CBSE in the year 2018-2019.

Signature of candidate Signature of Subj.Teacher Signature of Examiner

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ACKNOWLEDGEMEN
T
I warmly acknowledge the continuous encouragement and
timely suggestions offered by our dear principal Mr.Bipin
Kumar Sahoo. I extend my hearty thanks for giving me the
opportunity to make use of the facilities available in the
campus to carry out the project successfully I am highly
indebted to Mrs. Jeesha Satpathy for the constant supervision,
providing necessary information and support in completing
the project. I would like to express my gratitude towards them
for their kind co-operation and encouragement. Finally, I
extend my gratitude to one and all who are directly
or indirectly involved in the successful completion of this
project work.

Signature of the Candidate

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 TABLE OF CONTENTS
SL NO. TOPIC PG NO.

1 Introduction 5

2 Objective 8

3 Scope and limitat 9

ions
4 Theory 10

5 Experiment 16

6 Procedure 18

7 Observations 22

8 Result 23

9 24 4
 INTRODUCTION
Chlorophyll is a green photosynthetic pigment found in chloroplasts of organisms like cyanobacteria,
algae and plants. Its name is derived from the Greek words chloros, meaning „green‟ and
phyllon meaning „leaf". First isolated by Joseph BienaimeCaventou and Pierre Joseph Pelletier
in1817, chlorophyll is an extremely important biomolecule, 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 known as chlorophyll 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 a poor absorber of green and near green portions
of spectrum, hence green color 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 2
and photosystem I, which have their own distinct reaction centre chlorophylls, named P680 and
P700, respectively. These pigments are named after the wavelength ( nanometers ) of their red peak 5
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, aspecific redox reaction in
which the chlorophyll donates an electron into 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 2, the electron that reduces P680+ ultimately comes
from the oxidation of water into O2 and+ 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
earth’s atmosphere. Photosystem I typically works in series with photosystem 2; 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 2. 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 issued to shuttle H+ ions across the thylakoid
membrane, setting up achemiosmotic potential used mainly to produce ATP chemical energy; and
those electrons reduce NADP+ to NADPH, a universal reductantused to reduce CO2 into sugars as
well as for other biosynthetic reductions 6
 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 porphyrin pigments such as heme. At the centre of the
chlorine ring id=s 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 anew
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 total synthesis of the molecule. In
1967, Ian Fleming completed the last remaining stereo chemical elucidation, and7
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 presenting leaves
like, chlorophyll a, chlorophyll b, carotenoids, 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.
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 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 plant’s “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. 9
 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 chloroplast’s 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 color 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.
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 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 2 (PS 2) that have their own reaction centersP700 and
P680 respectively. Within each PS I and PS 2 their are photochemical light harvesting systems
present which are made up of many pigment molecules bounded to proteins.
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 Chlorophyll a
structure

 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 2 (PS 2) that have their own reaction
centersP700 and P680 respectively. Within each PS I and PS 2 their 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 12
centre. It absorbs energy from wavelengths of violet and red light.
 The molecular structure of chlorophyll a consists of a chlorin 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 2 contain the majority of chlorophyll b.

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 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.

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CAROTENES
 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

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 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
WINKAROSEA
BANANA LEAVES
SEPARATING FUNNEL
MEASURING CYLINDER
BAKERS
VIALS

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 CHEMICALS REQUIRED Acetone
Diethyl ether
Petroleum ether
Methyl alcohol
Calcium carbonate
Potassium hydroxide
Distilled water

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 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 the 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. FIG(1)
 The lower acetone water layer is discard.
 To the upper remaining layer add 4ml 92% methyl alcohol. Shake the
funnel and let it separate into two layers. Upper layer contains petrol and
ether rich in chlorophyll a and carotenoids; lower is the methyl alcohol layer
rich in chlorophyll b and xanthophyll pigments. (FIG 2)
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 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 1ml at a time. Two layers are
obtained. The upper layer is the diethyl ether layer and lower
contains methyl alcohol.
 Discard lower layer.

 To the upper layer add 1.5ml 30% methyl alcohol-KOH.

Shake funnel and add water.
 Two layers are obtained.
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 Upper layer contains chlorophyll b and lower contains
xanthophyll.
 Collect the samples, weigh them and note the amount of
chlorophyll pigments present in them. FIG(4)

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 OBSERVATION TABLE
SL NO. TYPE OF WEIGHT OF PIGMENT
LEAF
CHL.A CHL.B CAROTENE XANTHOP
HYLL

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 2.23 2.37

4 MINT 3.4 1.02 4.73 4.63

5 CABBAGE 3.59 0.55 5.3 4.9 22

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

NISHNTMISHRA

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