Separation of Photosynthetic Pigments by Paper Chromatography


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Separation of Photosynthetic Pigments by Paper Chromatography

Introduction

Chlorophyll is in fact only one pigment in a group of closely related
pigments commonly found in photosynthesising plants called
photosynthetic pigments. This can be demonstrated by extracting the
pigments from leaves with acetone and separating them by means of
paper chromatography. With a bit of luck five pigments can be
identified: chlorophyll a (blue-green), chlorophyll b (yellow-green),
xanthophylls (yellow), carotene (orange) and phaeophytin (grey, it is
a breakdown product of chlorophyll).

Absorptive paper with a concentrated spot of leaf extract is used in
this experiment. When dipping in a suitable solvent, the pigments
ascend the absorptive paper at different rates because they have
different solubilities in the solvent. In this way they become
separated from one another and can be identified by their different
colours and positions.

Requirements:

l Large test tube (24 * 150 mm);

l Stopper to fit test tube;

l Pin;

l A small glass tube to transfer pigment solution;

l Chromatography paper or filter paper;

l Rack of test tube;

l Pigment solution;

l Solvent (5 cm3).

Procedure:

l A strip of absorptive paper has been prepared. It has such a length
that it almost reaches the bottom of a large test tube and such a
width that the edges do not the sides of the tube;

l Draw a pencil line across the strip of paper 30 mm from one end. The
paper has been folded at the other end through 90 degrees and attached
to the stopper using a pin. Take care not to let the lower end of the
paper touch the bottom of the tube or edges touch the sides;

l Remove the paper from the boiling tube and use the small glass tube
provided, place a drop of the pigment solution at the centre of the
pencil line. Dry the spot under the heat from a hairdryer or let it
dry naturally. Place a second small drop on the first.

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Repeat this
process for about 15 minutes and a small area of concentrated pigment
has been set up. N.B. the smaller and more concentrated the spot is,
the better;

l While preparing the pigment spot, pour a mixture of propanone and
petroleum ether into the boiling tube to a depth of no more than 15
mm. Seal the tube with a stopper for about 10 minutes so that the
inside of the boiling becomes saturated with vapour;

l Suspend the strip of paper in the boiling tube. The lower end of the
paper should dip into the solvent but the pigment spot should not be
immersed;

l The solvent will ascend rapidly carrying the pigments and in about
10 minutes the pigments can be separated. When the solvent is about 20
mm from the top of the paper remove the strip, rule a pencil line to
mark the solvent front and dry the paper;

l Detectable pigments can be identified by their colours and the Bf
values;

l Measure the distance from the pencil line to the leading edge of
each clear pigment and work out the Rf value for each one using this
formulae:

Rf = a / b

Where a = distance moved by substance from its original position;

b = distance moved by solvent from the same position.

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

Table 1

Colours and Rf values of the pigments found in a typical leaf

(Rf values for propane/ ether mixture).

Result and calculation:

b = 8.4 cm

For the first pigment: a = 8.1 cm Rf = 8.1 / 8.4 = 0.965

For the second pigment: a = 7.2 cm Rf = 7.2 / 8.4 = 0.857

For the third pigment: a = 5.9 cm Rf = 5.9 / 8.4 = 0.70

For the fourth pigment: a = 5.1 cm Rf = 5.1 / 8.4 = 0.61

For the fifth pigment: a = 3.8 cm Rf = 3.8 / 8.4 = 0.452

The colours the different pigments displayed are not distinguishable
from each other but we can compare the Rf values to find out each
pigment's identity.

Compare the results with the typical Rf values in Table 1, we can
found that all of the pigments have been identified: pigment one is
Carotene; pigment two is phaeophytin; pigment three is xanthophyll;
pigment four is chlorophyll a and pigment five is chlorophyll b.

Evaluation:

The experiment can be improved if the following actions can be taken
into account:

l A hair dryer was used to dry the chlorophyll. I don't recommend
using it because when over heated, the pigments can be denatured thus
their colours and solubilities can be affected. Instead I would
recommend letting the pigments dry naturally;

l In order to obtain more favourable results we should let the solvent
run as long distance as possible. We can remove the paper strip when
the solvent almost reaches the top of the paper instead of taking the
paper out too early;

l An extra technique can be employed to make the pigments more
concentrated thus enhance the result. Pour some of the pigment
solution onto a watch glass and leave it for about half an hour. Water
will evaporate during that time and leave the pigment more
concentrated;

l I would recommend drawing a pigment line rather than a pigment spot.
When separated, pigments will appear as bands rather than smears so
the calculations can be made easier;

l The chlorophyll pigment was taken out from the aluminium paper long
before it was used. Light could have affected the nature of
chlorophyll thus led to inaccurate results. I would recommend not
taking out the pigment until the last minute when it is needed in
order to minimise the effect of light;

l An alternative technique (thin layer chromatography) mentioned in
the preparation paper can be employed: instead of absorptive paper, a
glass of plastic slide (or aluminium sheet) coated with silica gel is
used. The gel is spotted and then the slide or aluminium sheet is
placed vertically in a beaker or Coplin jar containing solvent at the
bottom;

l Further experiment can employ pigments from different plants.
Chlorophyll a is of universal occurrence in all photosynthesising
plants but we can compare the different pigments in different plants.
Because different pigments absorb light of different wavelengths, by
comparing the occurrence of different pigments we can deduce the
plants' habitat;

l We can do further experiment to determine the absorption spectrum of
each pigment. We can make separate solution of each pigment and use a
colorimeter to find out what wavelength of light each one absorbs.
Then we can find out what wavelength of light a plant uses most to
carry out photosynthesises.


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