INDEX
S.NO TOPIC PAGE NUMBER
1 Introduction 1-3
2 Objective 4
3 Scope and limitations 5
4 Theory 6-8
5 Experiment 9
6 Procedure 11-12
7 Observations 13
8 Result 13
9 Bibliography 14
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,
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 (innanometres ) 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.
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.
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.
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.
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.
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
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 TABLE
TYPE OF
S NO WEIGHT OF PIGMENT
LEAF
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 BOUGAINVILLEA 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.
BIBLIOGRAPHY
* www.wikipedia.org
* www.google.com
* www.howstuffworks.com
* www.letsmakesciencefun.com
