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Experiment No 4
Isolation of Chlorophyll Content
from spinach leaves
Overview
Introduction
1
Experiment
2
Material and Chemicals
3
Procedure
4
5
Objective
Introduction
Chlorophyll
 Chlorophyll is vital for photosynthesis, which allows plants to
absorb energy from light.
 Good and bad absorbance from the electromagnetic spectrum.
 First isolated by Joseph Bienaime Caventou
and Pierre Joseph Pelletier in 1817.
 Chlorophyll molecules are embedded in
the thylakoid membranes of chloroplasts.
Two primary functions of Chlorophyll
 Absorbance of light.
 Transfer of that light energy by resonance
energy transfer.
 Photo system units and their reaction centers.
 Why green and not black?
 Chlorophyll is a chlorin pigment, which is
structurally similar to a pigment named as Heme.
 The general structure of chlorophyll a was elucidated
by Hans Fischer in 1940.
Structure of Chlorophyll a and Chlorophyll b
Source of Chlorophyll and Chlorosis
In plants chlorophyll is synthesized from succinylCoA and glycine.
Chlorosis is a condition in which leaves produce insufficient
chlorophyll, turning them yellow.
Chlorosis can be caused by a nutrient deficiency of iron, called
iron chlorosis or by a shortage of magnesium or nitrogen.
Experiment
Apparatus and Chemicals
ice bath
 Mortar
 Pestle
 Beaker
 Graduated cylinder
 Funnel
 Cheesecloth
 Centrifuge
 Centrifuge Tubes
 Funnel
 Sucrose

Layout and protocol
of the Experiment
Preparation of an ice bath
Maintaining of chilled
condition throughout
chloroplast isolation
Removal of petiole and midrib
Measuring Weight and cutting of leaves
Addition of 50 ml of cold, 0.5M
sucrose and Grinding
Use of Cheesecloth in extraction process
Process of
Suspension and re
suspension
Protocol
 Prepare an ice bath to keep mortar and pestle, 100mL beaker and
graduated cylinders chilled throughout chloroplast isolation.
 Remove the petiole and midrib from several spinach leaves, then
weigh out 10g of leaf material.
 Cut the leaf fragments into small pieces in the mortar then add
50 ml of cold 0.5M sucrose. Grind the leaves for 2 minutes to
prepare a homogenate.
 Line a funnel with four layers of cheesecloth. Pour the
homogenate through the cheesecloth layers and collect the
filtrate in a chilled 100 ml beaker.
Transfer
the green filtrate to two chilled 15 ml centrifuge tubes.
Balance the tubes.
Centrifuge
the filtrate at 500 rpm for 5 min.
Transfer
the supernatant to two chilled centrifuge tubes and
balance the tubes (use a Pasteur pipette to transfer the
supernatant to ensure the pellet is not disturbed).
Centrifuge
the samples at 1500 rpm for 7 min. While the
samples are being centrifuged, take time to clean your work area
and any glassware that is no longer needed.
Carefully
discard the supernatant using a Pasteur pipette and
gently resuspend the chloroplast pellet in 10 ml of cold 0.5M
sucrose. Keep the chloroplast suspension on ice.
Chlorophyll content meter measuring chlorophyll content by
absorption
Advantages of Chlorophyll in food industry
 Chlorophyll is registered as a food additive (colorant), and its E
number is E140.
 Chefs use chlorophyll to color a variety of foods and beverages
green, such as pasta.
 Healing properties of chlorophyll
Thank You