CERTIFICATE

This is to certify that Adarsh Kumar Mohapatra of Class XII, DAV Public
School, Chandrasekharpur with AISSCE Roll Number _________________
has satisfactorily completed the project in Biology on Study of Chlorophyll
Contents in Various Plants in partial fulfillment of the requirements as
prescribed by CBSE in the year 2015-2016.

Signature of the Candidate

Signature of Teacher in Charge

ACKNOWLEDGEMENT

I warmly acknowledge the continuous encouragement and timely suggestions

offered by our dear principal Dr. K C Satapathy. I extend my heartfelt gratitude
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. Manna Millian for her constant supervision,
providing necessary guidance and support in completing the project.
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

TABLE OF CONTENTS

S no
1
2
3
4
5
6
7

Topic
Introduction
Objective
Theory
Experiment
Procedure
Observations
Result

Page number
1
3
4
6
7
8
9

Bibliography

10

INTRODUCTION
Chlorophyll is a green photosynthetic pigment found in chloroplasts of
organisms like cyanobacteria, algae and plants. 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 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 nanometres ) of their red peak absorption maximum.
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.

OBJECTIVE
The objective of this experiment is to study the chlorophyll levels in different
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 various types of leaves 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, 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.

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.

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
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 Calcium Carbonate and crush it again. Filter the extract in a
funnel. This 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.
The lower acetone water layer is discarded.
To the remaining upper layer add 4ml 92% Methyl Alcohol. Shake the
funnel and let it separate into two layers. Upper layer contains petroleum
ether rich in Chlorophyll a and Carotenoids; lower is the methyl alcohol
layer rich in Chlorophyll b and Xanthophyll pigments.
To the upper layer add 1.5ml 30% Methyl Alcohol and KOH. Add water
and shake funnel.
Two layers are obtained. Upper layer has Chlorophyll a and lower layer
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.
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

S.No.

Type of Leaf

Weight Of Pigment
Chlorophyll- a

Chlorophyll-b

Carotene

Xanthophyll

1.

Spinach

3.4

0.6

4.6

4.82

2.
3.
4.
5.

Fenugreek
Bougainvillea
Mint

1.76
1.75
3.4
3.59

0.5
0.37
1.02
0.55

2.92
2.23
4.73
5.3

3.16
2.37
4.63
4.9

Cabbage

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 Bougainvillea. Cabbage had the most
Carotene and Bougainvillea had the least. Cabbage also had the greatest level of
Xanthophylls and Bougainvillea had the least.

BIBLIOGRAPHY
*www.wikipedia.org
* www.google.com
* www.howstuffworks.com
*Comprehensive Laboratory Manual in Biology, Laxmi Publications