This document describes procedures for isolating chloroplasts from plant leaves and characterizing the photosynthetic pigments contained within them. The key steps are: 1. Isolating intact chloroplasts from spinach or lettuce leaves through cell disruption and differential centrifugation. 2. Separating the photosynthetic pigments within the chloroplasts using paper chromatography and determining their Rf values. 3. Eluting the separated pigment bands and analyzing their absorbance spectra from 350-700nm using a spectrophotometer to identify the specific pigments. The procedures allow for studying the structure and function of chloroplasts as well as characterizing their light-absorbing pigments like chlorophylls and

1. ISOLATION OF
CHLOROPLASTS AND
CHARACTERIZATION OF
PHOTOSYNTHETIC PIGMENT
CMB Lab. Activity 4
Ms. O.Hara

2. Studying subcellular components
◦ Composition – the native structure of the cell organelles
◦ Function – the activity of their components
◦ Morphological
◦ Biochemical where component structures
◦ Biophysical difffer
◦ Physiological

3. Studying subcellular components
◦ usually initiated by cell disruption
◦ To receive a mixture of organelles which is very convenient for further
separation.

4. Cell fractionation
◦ The resulting homogenate is composed of the broken cells with
subcellular components released in a buffered medium.
◦ Debris (unseparated and unbroken cells) must be separated by
standing the homogenate for some time or by filtration.
◦ Sediment or the residue is discarded
◦ Supernate or the filtrate is subjected to CENTRIFUGATION.

5. Centrifugation
◦ a process that involves the use of the
centrifugal force for the separation of
mixtures used in laboratory, increasing the
effective gravitational force on a test tube
so as to more rapidly and completely cause
the precipitate (pellet) to gather on the
bottom of the tube. The solution
(supernatant) is then either decanted or
used in other step.
The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in RPM or g.

6. Centrifugation
◦ used for the isolation and purification of organelles and
macromolecules.
◦ when a particle is subjected to centrifugal force by spinning a
cellular extract at extremely rapid rates in a laboratory
centrifuge, the rate of movement of the particle through a
specific solution depends on its size and density, as well as
the solution’s density and viscosity.

7. Types of Centrifuges
• Low-speed centrifuge
- table centrifuge
- typical maximum speed is 6000rpm.
- room temp.
- cell, nucleus, etc. (easily precipitated material)
• High-speed centrifuge
- max. speed 20000-25000rpm (60000 X g)
- temp. control
- cell, nucleus, organelle, etc.
• Ultracentrifuge
- max. speed 9800km/s2 (1000000 X g)
- temp. control and vacuum system
- cell, nucleus, organelle, components of memb., polysome, macromolecule, etc.

8. Types of centrifugation
◦ Differential centrifugation is a procedure in which the
homogenate is subjected to repeated centrifugations each time
increasing the centrifugal force.

9. Types of centrifugation
◦ Density gradient centrifugation is a procedure for separating
particles in which a sample is placed in a preformed gradient such
as sucrose. Upon centrifugation, the particles are “banded” in the
gradient and can be collected as a pure fraction.
Example:

10. Paper Chromatography
◦ Paper chromatography is a technique that can separate compounds
on paper by a solvent system that carries the mixture of pigments up
the paper by capillary action.
◦ Two phases are formed:
◦ the mobile phase - solvent
◦ stationary - chromatography paper
◦ separation is caused by a difference in
distribution of the components between
the stationary phase and the mobile phase

11. Spectrophotometry
◦ Spectrophotometer - an instrument which measures the absorption or
transmission of light by a solution at any wavelength(s) selected by the
experimenter.

12. Part 1: Isolation of Chloroplast
◦ http://vlab.amrita.edu/?sub=3&brch=187&sim=878&cnt=2
◦ https://youtu.be/6JJBvh-NQZA

13. Isolation of Chloroplasts from
a plant sample
Leaves of spinach, lettuce
are commonly used for the
isolation of chloroplasts.
Intact chloroplasts are the
best source for studying
the processes like carbon
assimilation, electron flow
and phosphorylation.

14. Estimation of chlorophyll concentration
1. Add 10 ul of chloroplast suspension to
990ul of 80% acetone solution and mix
gently.
2. Centrifuge at 3000xg for 2 minutes.
3. Take 100ul of the supernatant and
transfer into a cuvette and measure the
absorbance at 650 nm. Use 100 ul of
80% acetone as blank.
4. Take duplicate OD 650 values.
5. Take the average of the two values and
estimate the mg/ml chlorophyll
concentration using the following
formula:
Where A 650 is the absorbance at 650 nm,
100 is the dilution factor and 36 is the
extinction coefficient of chlorophyll.
http://cbi-au.vlabs.ac.in/cell-biology-1/Isolation_of_Chloroplast/experiment.html
A 650 x 100/36 = mg/ml chlorophyll.

15. Worksheet 4a – Isolation of Chloroplasts
◦ A. Schematic Diagram
◦ B. Results
◦ Microscopy.
◦ chlorophyll concentration (mg/ml) of the chloroplast isolates
◦ Answers to guide questions

16. Part 2. Photosynthetic Pigments
◦ https://www.youtube.com/watch?v=FQqVqVZgKjA

17. • With a capillary tube, the mixture is
streaked on the chromatography
paper: enough sample is applied so
that there will be an adequate amount
for subsequent extraction and
spectrophotometric analysis.

18. B. Separation of Photosynthetic
Pigments
◦ chromatography strip (punctured on top)
◦ Crude chlorophyll suspension applied on
the marked area (1.5 cm from the bottom)
◦ Lower the bottom edge with the extract
towards the developing solution.
◦ Allow to develop

19. Characterization of photosynthetic Pigments by
Rf value
◦ Rf – Reference front
1. Remove the developed chromatogram (separated
pigments) from the developing chamber.
2. Quickly before it dries, mark where the solvent stopped
with a pencil. This is called the solvent front.
3. Also mark where each pigment stopped moving.
4. Measure the distance traveled by the pigment from the
origin and distance travelled by the solvent (developing
solution) from the origin.
Rf = distance pigment migrated
distance solvent front migrated
What is the Rf value for carotene calculated from
the chromatogram?

20. Characterization of photosynthetic Pigments by
absorbance spectrum
◦ The light absorbing photosynthetic pigments do not absorb
all wavelengths of light equally.
◦ Plant pigments have specific wavelength absorbance
patterns known as the absorbance spectrum.
◦ This will help determine which light waves are absorbed by
the leaf pigments.

21. Developed chromatogram
Yellow-orange
Bluish green
Yellow green
Yellow

22. C. Elution of pigments
◦ Cut bands from strips
◦ Group according to color
◦ Put 1 group of colored band in a tube
containing 4 ml of pure acetone.
◦ Shake for 5 -10 mins.
◦ Partially purified pigments ELUENTS

23. D. Spectrophotometry
◦ Place the blank containing pure acetone in a
cuvette.
◦ Decant the eluents in individual cuvettes.
◦ Read the absorbance of each sample from 350
to 700 nm at 25 nm interval.
◦ Plot absorbance against wavelength.
◦ Compare the obtained spectra with the known
spectra of the colored pigments found in
reference books.
◦ Identify the pigments separated.

24. Absorbance Readings:Spinach leaf extract
Wavelength
(nm)
ABSORBANCE
Pigment 1 Pigment 2 Pigment 3 Pigment 4
360 0.032 0.065 0.015 0.009
380 0.033 0.085 0.017 0.011
400 0.035 0.091 0.025 0.018
420 0.047 0.113 0.035 0.026
440 0.064 0.106 0.042 0.033
460 0.067 0.021 0.038 0.037
480 0.037 0.015 0.036 0.034
500 0.006 0.008 0.007 0.015
520 0.005 0.008 0.002 0.004
540 0.007 0.009 0.001 0.002
560 0.006 0.011 0.002 0.0
580 0.009 0.017 0.003 0.001
600 0.011 0.019 0.001 0.001
620 0.010 0.026 0.0 0.0
640 0.018 0.029 0.001 0.0
660 0.030 0.083 0.0 0.0
680 0.009 0.034 0.001 0.0
700 0.001 0.003 0.0 0.0

25. Reference for Absorption spectra of plant photosynthetic
pigments in acetone (Bilodeau et al. 2019)
• Take note of the peaks
or highest absorption
values per pigment

26. ◦ A. Schematic diagram
◦ B. Results
◦ Give the Rf value of each separated pigment based on the chromatogram result.
◦ Make a line graph that shows the absorbance of each pigment at 360-700 nm. Use color
pencils to represent each pigment.
◦ Compare the obtained spectra with the known spectra of the colored pigments found in
reference books. What are the possible identities of the unknown pigments?
◦ Answers to guide questions
Worksheet 4b