Pigments in plants
Separation of chloroplast pigments
by paper chromatography
Refer to the Practical Manual
Unit 5: Cell Biology
Practical 16
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Aims:
1. To remove pigments from plant material.
2. To separate the pigments using paper
chromatography.
3. To identify the pigments by their colours
and relative positions on the
chromatograph.
4. To determine relative amounts of each
pigment.
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Theoretical basis:


(Textbook reference pp.361–367)
Various pigments found in the chloroplasts of plants are
used in the process of photosynthesis. The specific
pigments used depend to some extent upon the amount
of light normally present and the wavelengths of the light
that normally falls on the plant. Other pigments are not
used in photosynthesis. There are five pigments
commonly found in chloroplasts. Although leaves usually
appear green, the chlorophylls present may mask the
appearance of other pigments. Some leaves are red and
here a red pigment is masking the chlorophylls.
Variegated leaves, those which are green with white
areas, only photosynthesise in the green areas. The white
areas are devoid of chlorophylls.
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Theoretical basis:


(Textbook reference pp.361–7)
The chloroplast pigments can be separated and
identified by paper chromatography. Absorptive
paper containing a concentrated spot of
chloroplast extract is dipped into a suitable
solvent. The various pigments have different
sized molecules, with the result that as the
solvent ascends the absorptive paper it carries
the pigments with it at different rates, the smaller
molecules being more mobile than the larger
ones. In this way they can become separated
from each other and can be identified by their
different colours and positions.
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Requirements:
water
parsley or spinach
tweezers
mortar and pestle
solvent
boiling tube
metric ruler
ethyl alcohol
hot plate
test tube holder
glass rod
rubber stopper
scissors
chromatography paper
drawing pin
centrifuge & tube
Solvent = 1 part of 90% acetone to 9 parts of
petroleum ether (BP= 80 - 100 °C)
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Equipment
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Method: Part A

PREPARATION OF LEAF PIGMENTS
1. Quickly immerse leaves in boiling water.
2. Place killed leaves in mortar and pestle
and grind these up with a small amount
of alcohol.
3. Decant the dark green liquid produced
into a centrifuge tube and settle out using
a centrifuge.
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Quickly immerse leaves in boiling water


Immersing the
leaves in boiling
water kills the
leaves and so the
cell membranes
Do this process
VERY quickly
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Place killed leaves in mortar & pestle
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Add a small amount of alcohol
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Grind the leaves with the alcohol
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Decant dark green liquid into centrifuge tube
Centrifuge tube
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Settle out using the centrifuge
Lid
Speed control
Run
Brake
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Place tube in centrifuge
* Remember
its position
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Balance the centrifuge

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Remember the position of your tube e.g. 3a
Place the tubes opposite each other
If you have three tubes to do, fill a blank
tube with a similar amount of fluid to the
other tubes being spun and add to the
centrifuge
NEVER OPEN A CENTRIFUGE UNTIL YOU
ARE SURE THAT IT HAS STOPPED
SPINNING
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Method: Part B

PREPARING THE CHROMATOGRAPH
CHAMBER
1. Cut a length of chromatography paper of
sufficient length to almost reach the
bottom of a large test tube (width should
be less than that of the tube so that the
paper does not touch the sides of the
tube).
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Method: Part B

PREPARING THE CHROMATOGRAPH
CHAMBER
2. Rule a pencil line across the strip of paper
30 mm from one end. Make a cut from
each side of the line to the centre bottom
to form an arrow head. Fold the other
end through 90o and by means of a
drawing pin, attach it to the cork stopper.
The bottom tip of the strip should almost
reach the bottom of the tube when the
cork is inserted.
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Setup of chamber and appearance
of pigments
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Cut to form an arrow head
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Measure distance for line
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Rule pencil line 30 mm from end
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Fold other end 90o and attach to
cork stopper with drawing pin
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Fitting the paper in tube

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Tip of the strip should
almost touch the bottom of
tube with the cork in place
Paper should not touch the
sides of the tube as this
will upset the running of
the solvent and pigments
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Method: Part B
3. Place the chromatography paper on the
desk, dull side up. Add chloroplast
extract to the centre of the drawn line by
dipping the fine glass pipette into the
extract and then quickly touching it to the
centre spot on the paper. Allow the spot
to dry. Add more pigment to the same
spot. Repeat this procedure (allowing the
spot to dry before adding more pigment)
about 20 times, until there is a small spot
of concentrated pigment.
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Add chloroplast extract
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Finished pigment dot
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CAUTION


SOLVENT IS HIGHLY
FLAMMABLE
Make sure all flames are turned off
and the room is well ventilated
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Method: Part C
SEPARATING THE PIGMENTS
1. Add the solvent to a depth of 15 mm in the test
tube.
2. Carefully place the tube into the test tube
holder.
3. Place the chromatography paper into the tube.
Do not let the spot of extract touch the
solvent.
4. Do not shake or move the tube for at least 15
minutes. Remove the chromatograph from the
tube when the solvent has almost reached the
top of the paper.
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Add solvent to 15 mm depth
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Carefully place in test tube holder

then place
chromatography
paper into tube.
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Solvent front
Do not let the spot
of extract touch the
solvent.
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Leave the tube for 15+ minutes
Solvent front
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Remove the chromatograph
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Method: Part D
ANALYSIS OF THE CHROMATOGRAPH
1. Quickly measure the distance travelled by the
solvent. Record. (The solvent rapidly evaporates
in air.)
2. Examine the chromatograph for the presence of
different bands of colour. Each colour band will
be a different pigment. Some bands are very
faint.
3. Measure the distance from the pencil line to the
leading edge of each clearly detectable pigment.
4. Record.
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Mark distance solvent travelled
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
Mark and measure distance travelled by solvent
The solvent rapidly evaporates in air
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Look for different colours
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Mark and measure furthest distance travelled by
each different pigment
Record
Draw the outline of each of the pigment spots
These fade very quickly
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Close-up of strip
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Rf values
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The ratio of the rate of movement of the
pigment to solvent (the Rf value) can then
be calculated for each pigment
Rf = distance moved by the substance from the original position
distance moved by solvent from the same position
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

Use table to identify leaf pigments
Measure the width and depth of each of
the pigment bands
Estimate the relative quantities of each
pigment present
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Identify the leaf pigments
NAME
COLOUR
Rf
Carotene
yellow
0.95
Phaeophytin
yellow-grey
0.83
Xanthophyll
yellow-brown
0.71
Chlorophyll a
blue-green
0.65
Chlorophyll b
green
0.45
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Student results 1
Solvent
front
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Student results 2: Rf values
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Student results 3: areas
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Interpretation of results:

Write a discussion of your results including the
following points:
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The pigments present in the leaf.
Phaeophytin is a breakdown product of chlorophyll.
Suggest functions for the other pigments.
Chlorophyll a and b are easily broken down in cool
temperatures. Explain how leaf colour change can
come about in deciduous trees in autumn.
Possible differences in types and/or concentrations of
pigments present between plants with very dark green
leaves and those with light green leaves.
Some rainforest trees and ferns have young leaves that
are red in colour, as do the red algae that are found at
greater depths than the green and brown. Suggest a
scientific reason based on pigments that could explain
these observations.
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Red rainforest leaves
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