Signature of the Principal Signature of the External Examiner

ACKNOWLEDGEMENT

I warmly acknowledge the continuous encouragement and timely

suggestions offered by our dear Chairman Mr. D Muniraju, and Mr.

Ashish. I extend my hearty thanks for giving me the opportunity to make

use of the facilities available in the campus to carry out the project

successfully.

I am highly indebted to Mrs. Deepthi for the constant supervision,

providing necessary information and support in completing the project. I

would like to express my gratitude towards her for her kind co-operation

and encouragement.

Finally, I extend my gratitude to one and all who are directly or indirectly

involved in the successful completion of this project work.

Signature of the Candidate

TABLE OF CONTENTS

S no Topic Page number

1 Introduction 1

2 Objective 4

3 Scope and limitations 5

4 Theory 6

5 Experiment 11

6 Procedure 13

7 Observations 17

8 Result 18

9 Bibliography 19

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.

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 electrons 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, carotenoids, 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.

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.

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

S

N

O

TYPE OF

LEAF

WEIGHT OF PIGMENT

CHLOROPHYLL A CHLOROPHYLL

B CAROTENE XANTHOPHYLL

1 SPINACH 3.4 0.6 4.6 4.82

2 FENUGRE

EK

1.76 0.5 2.92 3.16

3 BOUGAIN

VILLA

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