Chromatography of photosynthetic pigments
Learning outcomes
Introduction
These learning outcomes aim to cover subject knowledge requirements for this part of A-level Biology in
the specifications from OCR, AQA, Edexcel and Eduqas. You may need to adapt this information for your
specification.
This information can be used to produce learning resources, revision materials, quizzes etc.
Students should be able to:
1. State the precise location of the pigments involved in photosynthesis within the leaf
The pigments involved in photosynthesis are embedded in the thylakoid membrane in chloroplasts.
Chloroplasts have an internal membrane system of flattened sacs called thylakoids, stacked together to
form grana, with grana joined by lamellae (extended sacs). These thylakoids, grana and lamellae are made
of this thylakoid membrane.
2. Name 5 photosynthetic pigments (or types of pigment) found in leaves
Chlorophyll a
Chlorophyll b
Carotenes (a category of carotenoids)
Xanthophylls (a category of carotenoids)
Pheophytins (a and b)
(Note: these molecules are not explicitly named in the specifications but are useful for interpreting the
chromatograms.)
3. Outline the role of the leaf pigments in photosynthesis
These photosynthetic pigments are involved in the “capturing” of light energy and its conversion to chemical
energy.
The primary photosynthetic pigment is chlorophyll which uses energy from light to excite electrons. The
carotenoids are accessory pigments that “channel” more captured light energy to the chlorophyll. The
pheophytins are electron carriers that are part of the electron transport chain where the excited electrons
are passed from one molecule to the next in a series of chemical reactions.
4. Explain how the terms “light harvesting systems”, “photosystems” and “reaction centres”
are related
The capturing of light energy to excite electrons is performed by a complex arrangement of molecules and
so rather than use the name of a particular molecule these phrases are often used.
Photosystem is the term given to the whole arrangement of molecules. In green plants there are two
different photosystems (termed photosystem I and photosystem II) each composed of a different set of
molecules.
Science & Plants for Schools: www.saps.org.uk
Chromatography of leaf pigments – Learning outcomes: p. 1
Each photosystem has a reaction centre where the light energy is used to excite electrons that are then
passed down the electron transport chain. Surrounding the reaction centre is a light harvesting system that
“channels” more light energy to the reaction centre than the reaction centre can capture on its own.
(Note: not all specifications use these terms – but an awareness that there are many different molecules
involved in the capturing of light energy is important for the context of this chromatography)
5. Explain why many plants have a variety of photosynthetic pigments
Different photosynthetic pigments absorb light best at different wavelengths. By having more photosynthetic
pigments plants can absorb light energy over a wider range of wavelengths. This allows plants to capture
more energy for the same sunlight intensity.
6. Describe how to conduct chromatography to separate pigments from a leaf and explain the
importance of each step
Step
Importance
1) Tear up a sample of plant leaves into small
To break open plant cells and chloroplasts.
pieces, place in a mortar with a pinch of sand,
and grind with a pestle
2) Add solvent and continue to grind with a
To allow the photosynthetic pigments to
pestle
dissolve in the solvent.
3) Gently draw a pencil line 1cm from the base
The pencil line marks the starting position of
of the chromatography paper and repeatedly
the pigments. It is in pencil so that the line
spot a small quantity of the leaf extract onto
doesn’t move with the solvent and won’t mask
the centre of the line, allowing the spot to dry the results of the chromatography. The spot is
between each application
made with small quantities through repeated
spotting and drying in order to build up a
concentrated, but still small, spot.
4) Suspend the chromatography plate/paper
The solvent is use to draw the photosynthetic
from a bung in the glass vial with 1cm depth
pigments up through the chromatography
of solvent in the bottom. Ensure that the
plate. The spot needs to be above the surface
chromatography plate dips into the solvent
of the solvent so that the pigments travel up
but the spot of leaf extract remains above the the plate and don’t just dissolve out into the
surface of the solvent
solvent in the vial.
5) Allow the solvent to run up the
Identification of pigments by chromatography
chromatography plate until the solvent front
relies on identifying how far each pigment has
is near the bung then remove the plate, mark
travelled as a proportion of how far the solvent
the location of the solvent front, and allow to
itself has travelled. The solvent evaporates
dry
quickly and so the extent to which it has
travelled up the plate needs to be marked
whilst it can be seen. Drying ensures that the
movement of solvent and pigments stops.
6) The resultant chromatography plate is called
To allow the distance each pigment has
a chromatogram which can then be
travelled to be measured and for its Rf value to
photographed or the location of each
be calculated.
separated pigment can be marked
7. List 3 characteristics of the solute (in this case a pigment) that influences how far it travels
during chromatography, and for each describe the effect it has
Factor
Explanation
Solubility in the solvent (mobile/liquid
The more soluble the pigment is in the solvent the
phase)
further it will travel
How it interacts with the plate used
Molecules that interact more strongly with the plate
(stationary/solid phase)
used will not travel as far
Size of the pigment molecule
The smaller the molecule the further it will travel
(Note: the size of the molecule is much less of a factor than the other two)
Science & Plants for Schools: www.saps.org.uk
Chromatography of leaf pigments – Learning outcomes: p. 2
8. Describe how to calculate the Rf value for a particular substance
Rf value
=
distance moved by the substance from its original location
distance moved by the solvent from the same location
Note: usually the distance moved by
the substance is measured using the
centre of the pigment spot’s final
location
Note: Each chemical (in this case,
pigment) has its own Rf value. So in
this diagram there are 8 different
pigments each with their own Rf value.
Distance moved by
the solvent from the
same location
Distance moved by
the substance from
its original location
9. Explain how a chromatogram can be used to identify an unknown substance
Calculate the Rf value for the substance and compare this to the Rf values for known substances (specific
to that solvent and that type of chromatography plate). The unknown substance should be one of the
substances with the same Rf value.
However more than one substance can have the same Rf value for a particular solvent and
chromatography paper and so it’s possible that multiple chromatograms will need to be run with different
solvents (and/or chromatography paper) in order to find out the exact identity of the unknown substance.
(other characteristics of the chemical may also help identification – e.g. the colour of photosynthetic
pigments).
Science & Plants for Schools: www.saps.org.uk
Chromatography of leaf pigments – Learning outcomes: p. 3