COMPARITIVE STUDY OF THE

CHLOROPHYLL CONTENT IN
FIVE DIFFERENT SPECIES OF
PLANT

AIM:
To compare and study the chlorophyll content in different plant
species.

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 Bienaime Caventou 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, bacterio chlorophyll and bacterio viridin. 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 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 earth’s 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 id=s 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.

OBESITY:

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.

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.
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.

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.
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.

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.
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.
 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
 OBSERVATION:

S TYPE OF LEAF WEIGHT OF PIGMENT

NO
CHLOROPHYLL CHLOROPHYLL CAROTENE XANTHOPHYLL

A B
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

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.

REFERENCE:
* www.wikipedia.org

* www.google.com

* www.howstuffworks.com

* www.letsmakesciencefun.com