INTRODUCTION

Chlorophyll is a green photosynthetic pigment

found in chloroplasts of organisms like
cyanobacteria, algae and plants. Its name is
derived fromthe 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
vitalrole 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 lightmost strongly in the
blue portion of the electromagnetic
spectrum,followed by the red portion. However,
it is a poor absorber of green andnear 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 primaryfunctions : to absorb light,

and to transfer that light energy by

resonanceenergy 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 ( innanometres ) of their
red peak absorption maximum. The identity,
function and spectral properties of the types of
chlorophyll in each photo system are distinct,
and determined by each other and he 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 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 ear
this atmosphere. Photo system 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.

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.

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

datain 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 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 color of leaves we see is not due to a single

pigment but due to four pigments namely
chlorophyll a,
chlorophyll b,
xanthophylls
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 centersP700 and P680 respectively
.Within each PS I and PS II their are
photochemical light harvesting systems present
which are made up of many pigment molecules
bounded to proteins .Chlorophyll a is essential
for most photosynthetic organisms to
release chemical energy but is not the only
pigment that can be used for photosynthesis
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 chlorin ring, whose four nitrogen atoms
surround a central magnesium atom, and has
severa lother attached side chains and
a hydrocarbon tail. This photosynthetic
pigmentis essential for photosynthesis in
eukaryotes, cyanobacteria& prochlorophytes be
cause 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 polarsolvents 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 ofchlorophyll b.

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 nonphotochemical 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
Cabbage
Bougainvillea 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 ina
Buchner funnel. The filtrate is called acetone
extract and it is richin 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.

 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.
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.
Upper layer contains chlorophyll b and lower
contains xanthophyll.
Collect the samples, weigh them and note
the amount of chlorophyll pigments present
in them.

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
Chlorophylla, 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.

OBSERVATION TABLE

Chlorophlly Chlorophyll Carotene Xanthophyll

A
Bl
fenugreek

0.6

4.6

4.82

Bougainvillea 1.76

0.5

2.92

3.16

spinach

1.75

0.37

2.23

2.37

cabbage

3.4

1.02

4.73

4.63

mint

3.59

0.55

5.3

4.9

Type of leaf

3.4

Weight of pigments