INDEX

Sr.No Topic Page No

1 Introduction 2-3
2 Objective 4
3 Scope and Limitation 5
4 Theory 6-7
5 Experiment 8
6 Procedure 9
7 Observations 10
8 Result 11
9 Bibliography 12
 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 avital 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
function: 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- 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 forming 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 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 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 LIMITATION

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 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.

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 center. It absorbs energy from wavelengths of
violet and
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 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 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 non-
photochemical agent to deal with excited chlorophyll. Carotenes an orange
photosynthetic pigment important for photosynthesis. Carotenes are all coloured to
the human eye.
 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 or Fenugreek leaves
 Cabbage
 Bougainvillia
 Separating funnel
 Measuring cylinder
 Beakers
 Vials

Chemicals required:
 Acetone
 Diethyl ether
 Petroleum ether
 Methyl alcohol
 Calcium carbonate
 Potassium hydroxide
 Distilled water
 PROCEDURE

1) 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.
2) Take 4ml of the acetone extract and add petroleum ether. Shake the funnel
gently.
3) Add water and shake again. Two layers will be formed. Upper containing
petroleum ether will contain chlorophyll a and carotene.
4) The lower acetone water layer is discard.
5) 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.
6) Discard lower layer.
7) To the upper layer add 1.5ml 30% methyl alcohol-KOH. Shake the funnel
and add water.
8) Two layers are obtained.
9) Upper layer contains chlorophyll b and lower contains xanthophyll.
10) Collect the samples, weigh them and note the amount of chlorophyll
pigments present in them.

OBSERVATION TABLE
RESULTS
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.howstuffworks.com
 www.scribd.com
 www.letsmakesciencefun.com