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 in 1817,
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 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.

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 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 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.

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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 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.

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 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 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.

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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 II (PS II) that have their own
reaction centers P700 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 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.

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 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 II contain the majority of
chlorophyll b.
 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.
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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 greenplants, 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.
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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
 Winkarosea
 Banana leaves
 Separating funnel
 Measuring cylinder
 Beakers
 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

 The lower acetone water layer is discard.

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 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.

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 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.

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OBSERVATION TABLE:

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 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

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

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