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Chromatography of photosynthetic pigments
Students’ Worksheet
Introduction
Follow the instructions on the students’ sheet for the chromatography of photosynthetic
pigments and complete this worksheet to show evidence of the work you have done.
What did you do?
Which species (or material) did you investigate? Describe the material you used to
include any aspects that might influence the pigments that are present. Also include
what time of year the chromatography was conducted.
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Why did you use a relatively non-polar solvent to extract the pigments and run the
chromatogram?
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What is the sand for in the method? Did you use it?
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How could you prove that the pigments came from the plant material and not from the
sand if someone did use sand?
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Why is it important to mark positions in pencil rather then pen (particularly the starting
position of the concentrated extract)?
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Chromatography of photosynthetic pigments - Student worksheet: p. 1
Why is repeated application of the plant extract onto the same spot required?
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Why is it important to let the spot dry in between each repeated application of the plant
extract?
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Why is it important that the spot of plant extract is above the surface of the
solvent when the chromatography plate is placed in the vial?
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Why is it important that the chromatogram is stopped before the solvent front
reaches the top? What would happen to the pigment spots if the chromatogram
was left running for a long time?
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Why is it important to mark the solvent front quickly?
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Why is it useful to mark the positions of the pigment spots or take a photograph
of the chromatogram soon after it has been run?
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Chromatography of photosynthetic pigments - Student worksheet: p. 2
What safety precautions did you, and the class as a whole take when using the
solvent? Include ways in which you reduced the amount of solvent that
evaporated into the air.
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Chromatography of photosynthetic pigments - Student worksheet: p. 3
Your results
If possible stick your chromatogram in the space below. Even better, take a photograph
of your chromatogram and stick that here. If not possible then draw and annotate a
sketch of your chromatogram.
How far did the solvent front travel from the original position of the concentrated extract
spot?
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Draw a table to catalogue the information about your pigment spots (colour, distance
travelled and Rf value). Include a column for which pigment you think each spot is and
another column explaining why you have come to that conclusion.
Explain how Rf values are calculated (write the equation).
Is it possible to have an Rf value greater than 1? Explain.
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Chromatography of photosynthetic pigments - Student worksheet: p. 4
Diagrams of molecular structure are often “skeletons”, which don’t write out the presence
of the hydrogens or carbons. The 4 molecules below all have 40 carbon atoms, many
hydrogen atoms and from none to 4 oxygen atoms. The oxygen atoms increase the
polar nature of the molecule. A hydroxyl (OH) group is more polar than an epoxy group
(an oxygen in a triangle with two carbon atoms).
A
B
C
D
Figure 1: Molecular structures of some yellow photosynthetic pigments
You may have identified all or only some of these in your chromatogram but they are all
yellow molecules in the data tables of Rf values on the students’ sheet (tables 1 and 2).
Use the information above (in the text and the diagrams) and the Rf values of known
molecules on the students’ sheet to identify each of the molecules in the diagrams.
Explain your reasoning below.
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Image A author – Yikrazuul
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Chromatography of photosynthetic pigments - Student worksheet: p. 5
The error in your results
How accurately is it possible to measure the distance that the solvent front and each
pigment spot has travelled (to the nearest 1cm, 0.1cm, 0.01cm or something different)?
(Consider the limitations of the ruler you’re using and the difficulty in locating the exact
position of the solvent front or centre of the pigment spot)? How far out could an actual
distance measurement be from the recorded one?
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What is the percentage error in the measurements taken and how could you modify the
equipment/experiment only very slightly in order to get a lower percentage error?
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Based on your maximum and minimum possible values for the measurements of the
solvent front and each pigment spot work out a range for each Rf value that would
include the true Rf value for that spot in your chromatogram. Present these ranges in a
table below and comment on whether this casts any doubt on your identification of each
pigment.
What could be done to confirm the identity of each pigment if the results were unclear or
if two molecules were a similar colour and/or their Rf values were very similar?
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Chromatography of photosynthetic pigments - Student worksheet: p. 6
The biology of what you see and the science of chromatography
Where, exactly have you extracted the photosynthetic pigments from?
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How does the fact that these pigments dissolve in an organic (relatively non-polar
solvent) help explain the precise location of these pigments in plant cells?
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Assuming that the distance the pigment travelled is based on the solubility of the
molecule in the organic, relatively non-polar solvent, and its ability to bind to the polar
stationary phase (the TLC pate) suggest what might be concluded about the differences
in molecular structure between different pigments.
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What is the role of these photosynthetic pigments in photosynthesis?
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Chromatography of photosynthetic pigments - Student worksheet: p. 7
Why is it useful for a plant to have several different photosynthetic pigments?
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Chromatography of photosynthetic pigments - Student worksheet: p. 8
Scientific curiosity: further experiments into pigments and research into
plant pigments
Further experiments into pigments
The core of this investigation is just to separate (and to try to identify) photosynthetic
pigments from a plant specimen using Thin Layer Chromatography but once this
technique is mastered then more exploratory experiments could be done. It may be
possible for you to study:
a) Whether storage or freezing affects the pigment content of leaves.
b) If there are differences in pigment content of new and old leaves of a plant.
c) If there are differences in pigment content of different species of plant (and of
different parts of a plant – e.g. flower, fruit, leaves, stem).
d) If there are differences in pigment content of nutrient deprived plants or plants
that look to be in poor condition.
e) If there are differences in pigment content of plants growing in shady versus
sunny positions.
f) Whether the yellow of a hen egg yolk is the same molecule as any of the yellow
pigments found in grass. Extend the design of this experiment to be able to
investigate whether eggs from hen reared in different conditions (e.g. barn, freerange, corn fed) have different pigments in their yolks. Incorporate into the design
a way to see if the yellow pigment in this corn (sweet corn/maize) is different to
any of those found in grass.
g) The presence and variety of polar plant pigments called anthocyanins by
modifying the solvents used (Cloeus spp are good plants to investigate).
h) Is the red of strawberries and the red of tomatoes is the same pigment molecule.
For all of the above you could use different equipment to investigate concentrations of
pigments present rather than just presence or absence of different types.
Exploring plant pigments
There is a wealth of fascinating areas to research (online or in journals and other
scientific literature) in relation to plant pigments in addition to researching the topics
mentioned in the practical investigations above. You could write a short report on one or
more of the topics below (remember to reference your sources of information in an
appropriately detailed format).
1) Do animals use any of these pigments? If so what for and how do they get them?
 Look at links between these pigments and vitamin A
 Can any animals produce these pigments themselves? Look into horizontal gene
transfer from fungi to aphids and spider mites.
 Feather colour in some bird species, lobster shells
 How is “golden rice” relevant to this topic?
2) The variety of pigments
a) Map different pigments to different colours found in nature. Are all colours
pigment based? Are there pigments that we can’t see?
b) Design a tour of the variety of plant pigments using produce found in
supermarkets.
3) What do plants use these pigments for?
 How do these pigments link with fruit ripening?
 Explore the details of the molecules used in the light dependent stage of
photosynthesis.
 Explore pigments found in flowers.
 Why do some plants have leaves, or parts of leaves that are not green? Can they
still photosynthesise?
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Chromatography of photosynthetic pigments - Student worksheet: p. 9