Chromatography of photosynthetic pigments

Introduction and context

In this practical, students use Thin Layer Chromatography (TLC) to separate photosynthetic pigments from grass. They take measurements from and observations of the chromatogram to suggest the possible identity of each pigment separated.

It is interesting for students to see the range of pigments that can be separated from our ubiquitous “green grass”, highlighting the importance of separating molecules to see what is present. In this method an organic solvent is used for extraction and for running the chromatogram, so pigments that run successfully are the non-polar, membrane-bound, photosynthetic pigments not the polar, vacuolar, anthocyanin colouration pigments.

As well as comparing the photosynthetic pigments present in different plant species or varieties, extension activities could include comparing photosynthetic pigment extraction with anthocyanin extraction and the possibility of identifying photosynthetic pigments from other non-plant sources.

Safety Notes

·         The solvents used to extract the pigments from the leaves and run the chromatogram are highly flammable. Ensure that there are no naked flames or other sources of ignition in the laboratory.

·         Wear eye protection when using the solvent.

·         Avoid inhaling vapour as much as possible by minimising the quantity of solvent used and minimising the escape of the vapour.

·         Ensure laboratory is well-ventilated.

·         The vapours may cause drowsiness or dizziness.

Check CLEAPSS guidelines (Hazcards 45A and 85) for further guidance on the use, preparation, and disposal of the extraction and running solvents.

Apparatus – per chromatogram

• 1 x Thin Layer Chromatography strip ~ 13mm x 67mm*
• Glass specimen vial (~2cm x 7.5cm) / universal vial
• Cork / bung for glass vial, cut in half lengthways
• Pestle and Mortar
• 0.5g fresh grass leaves (from a succulent grass lawn ~5 pinches, or other green plant material)
• Dissecting scissors
• Silver sand – a pinch
• Propanone – approximately 2cm³
• 2x Plastic pipettes (3cm³) one for propanone and one for the running solvent
• 1cm³ (approx.) running solvent (5 cyclohexane: 3 propanone: 2 petroleum spirit 80-100°C by volume) – per chromatogram depending on glassware used
• Plastic micro-pipette tip/ fine glass pipette
• White tile
• Pencil & ruler

*TLC plates are commonly available as 40mm x 80mm plates (50 per box), each plate can be economically cut into 3 strips lengthways to provide a plate for each student - adjust in length to fit your vials (see supplier details below).

Other apparatus for extension activities

• It may be possible to extract carotenoids from other plant material using this method (e.g. carrots, maize, dandelions, buttercups, bananas, sweet potatoes, cantaloupe melons, tomatoes)
• It may be possible to extract anthocyanins from red or blue fruits, flowers and leaves but a different extraction and running solvent would be required as these anthocyanins are much more polar molecules (CLEAPSS has suggestions for anthocyanin extraction solvents). Strawberries or the leaves of Coleus sp. could be used.
• It may be possible to extract carotenoids from egg yolks or butter using this method and a comparison of the types of carotenoids found in eggs from hens reared in different conditions (e.g. barn, free range, corn fed) may be an interesting investigation.

Suppliers

TLC plates

Thin layer Chromatograph plates are available from standard school science suppliers; we recommend the Macherey Nagel POLYGRAM brand, product code 805021. Costs for these vary between suppliers, but average around £40 for a pack of 50 plates (cutting the plates into 3 gives an end cost of around 25p per run). See for example Timstar 

Chromatography vessels:

Glass specimen vials or wide necked McCartney vials are ideal, but any glass vessel or approx. 2x7.5cm will suffice. Drosophila culture vials are also ideal for this purpose. Cork stoppers/bungs (if available) are easiest to split in half lengthways to hold the TLC plate.

Teaching Notes

General practical tips

• Selecting the plant material – grass is suggested, partly as this is ubiquitous so students will see it regularly and may relate this back to their chromatogram. Also because it produces a good range of different pigments when developing the method. Any plant material can be tried and it may be useful to trial different species to see what works well at the time you are carrying out the practical. Spinach can work well, we suggest using bagged ‘organic’ spinach (‘organic’ leaves tend to be freshest, and not pre-treated with bleaches).
• Time frame to collect data – The extraction of the photosynthetic pigments should only take 2-3minutes, the spotting on the TLC plate will take a further 3 minutes or so depending on the rigour of drying between each spot and the number of spots re-applied. The chromatogram will take a couple of minutes to set up and then about 8 minutes to run. Removal of the chromatography plate, marking the location of the solvent front and each pigment spot should be done quickly ideally within a minute.
• Drying and concentrating spots – the results are clearer if the spot is small and concentrated. The chromatogram will still show the spread of pigments if there is a bit of spreading but students should try their best to make the spot as small and as concentrated as possible (~3 mm dia. max).
• Solvents can be dispensed using plastic pipettes, students should be encouraged to use the minimum volumes required – just enough to produce a liquid in the bottom of the mortar and just enough running solvent to so it will touch the chromatography plate in the glass vial.
• Measuring the solvent front – the front starts evaporating immediately on removal of the plate from the glass vial. Within a couple of seconds the solvent front shrinks lower and will disappears within a few more seconds. Students need to be made aware that recording the location of the solvent front is a critical point in the practical.
• When to stop the chromatogram – running the chromatogram for longer gives better separation of the pigments but it is critical to stop the chromatogram before solvent front reaches the cork/bung – further than this will prevent measurement of the solvent front, so Rf values cannot be calculated. Time spent running the chromatogram is not critical as the calculation of Rf values standardises this, however students should keep an eye on their chromatogram and remove it when the location of the solvent front is approaching the cork bung.
• Measuring distances – measuring the distance each pigment spot and the solvent front has travelled to the nearest mm is an appropriate level of accuracy. If spread widely the centre of each pigment spot should be used to determine the distance each pigment has travelled. As some pigments fade each pigment band should be recorded lightly by pencil on one side of the plate.
• Rf Values – The Rf values obtained by students may be dependent upon the specific TLC plates used, the temperature of the laboratory and the age of the running solvent. Accompanying Student Notes give a suggested range of Rf values for each photosynthetic pigment using this method, these ranges may vary under different conditions.

Students may need to be taught or reminded about:

• Calculating Rf values
• Polar and non-polar molecules
• Calculating percentage error

Notes on student tasks

• As a minimum students should extract the photosynthetic pigments from grass, measure the distances the pigment spots and the solvent front have travelled, calculate Rf values and use these values, as well as colour observations, to make suggestions for the identity of each pigment spot.
• This activity should take about 20mins in total.
• Extension activities:
• Students could run several chromatograms simultaneously with minimal increase in the total practical time - different students could carry out extraction of different plant materials with all extracts then available to all students.
• Students could consider the relationship between Rf value and molecular structure for each of the photosynthetic pigments. This protocol uses a non-polar extraction and running solvent. The solvent extracts relatively non-polar molecules such as the photosynthetic pigments embedded within the thylakoid membrane of chloroplasts. It is straightforward to explain the relative Rf values of the different carotenoids based on their molecular structure as well as the relative Rf values of the chlorophylls and pheophytins based on their molecular structure. It is more difficult to develop suggestions to explain the relative Rf values of the different carotenoids in comparison to the different pheophytins and chlorophylls. (see the “Background information” section for further discussion of the categories of photosynthetic pigments and their molecular nature)
• One potential extension is to compare extraction of both the relatively non-polar photosynthetic pigments and the polar anthocyanins (used by plants for colouration). A different extraction and running solvent would be needed to extract anthocyanins. Extending the practical in this way would help encourage students to think carefully about the nature of chromatography, how it separates molecules. It also helps students to make the link between the chemical nature of the pigments and how this determines how they can be extracted (and their Rf value with a particular solvent). Additionally links can also be made between the function and location of the pigments in plant – photosynthetic pigments being non-polar due to the functional requirement that they are membrane-bound, with anthocyanins being polar as they are accumulated in the watery contents of the vacuole.
• Further potential investigations could be to study the carotenoids found in egg yolks. Using grass as a standard it could be possible to make suggestions regarding which carotenoids may be present or absent in egg yolks. It may also be possible to compare barn reared, free range and corn fed hens eggs to see if there are differences in carotenoids present. Maize may be used as a comparison. It may also be possible to incorporate work with a colorimeter to investigate quantities of pigments present.

Possible questions for students

1.     Follow the protocol and answer the following questions

Name the method of chemical separation you have used.

What species did you investigate?

Why is it important to use an organic (non-polar) solvent and not a polar one like water?

What is the sand for? How could you prove that the pigments came from the plant material and not the sand?

Why is it important to mark positions in pencil rather than pen (particularly that starting position of the concentrated extract)?

Why is repeated application of the plant extract onto the same spot required?

Why is it important to let the spot dry in between each repeated application of the plant extract?

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?

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?

Why is it important to mark the solvent front quickly?

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?

How accurate is your measurement of the distance the solvent front has travelled and how far 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 do you calculate an Rf value?

How could you modify the equipment/experiment, only very slightly in order to get more accurate measurements of the Rf values for each pigment?

What is the percentage error in the Rf values you get for each pigment spot?

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.

2.     The findings from your investigation

How many different pigment spots did you obtain?

Have you managed to identify any pigments? If so which ones and how confident are you about their identity? What factors affect your confidence (both positively and negatively)?

Is it possible to have an Rf value greater than 1? Explain.

Does it matter if different chromatograms are run for different lengths of time? Explain.

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?

If you were interested in the concentration of each pigment in the plant material what practical technique could you use?

3.     The Biology of photosynthetic pigments and exploring the photosynthetic molecules

Where, exactly have you extracted the photosynthetic pigments from?

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?

Assuming that the distance the pigment travelled is based on the solubility of the molecule in the organic solvent suggest what might be concluded about the differences in structure between different pigments.

What is the role of these photosynthetic pigments in photosynthesis?

Why is it useful for a plant to have several different photosynthetic pigments?

How do the molecular structures of chlorophyll a and b differ? Explain why chlorophyll a has a larger Rf value than chlorophyll b? (show molecular structures) (clue: relatively non-polar solvent = mobile phase and a relatively polar stationary phase = the TLC plate).

How do the molecular structures of the chlorophylls vary in comparison to the pheophytins explain why the pheophytins have a larger Rf value than the chlorophylls? (show molecular structures) (clue: relatively non-polar solvent = mobile phase and a relatively polar stationary phase = the TLC plate).

How does the molecular structure of beta-carotene explain why it has such a large Rf value? (show structure) (clue: relatively non-polar solvent = mobile phase and a relatively polar stationary phase = the TLC plate).

How do the molecular structures of the 3 different xanthophylls explain why they all have relatively small Rf values and how do the 3 different structures explain the order in which these pigments appear in the chromatogram? (show structures and give clue as above. Also extra information is likely to be required: Hydroxyl (OH) and epoxy (C-O-C in a triangle) groups are polar groups and the hydroxyl group is more polar than the epoxy group.

4.     Exploring pigments in other biological material

Other plant pigments called anthocyanins are not membrane bound, relatively non-polar molecules, but are pigments that are polar and dissolved in the watery contents of plant vacuoles. What would you need to do to modify this experimental method in order to investigate anthocyanins in leaves of flowers?

Design an experiment to determine whether the red of strawberries and the red of tomatoes is the same pigment molecule.

If you got a pigment spot from one of the strawberry or the tomato but not the other it might be possible that the pigment from the one without the spot is still the same molecule but is held more tightly within the cells or organelles and so just didn’t move up the chromatography plate. What other reason could there be for a pigment not moving from the original location and what could you do to further investigate if this was the case?

Do some research into colour pigments in the natural world. Investigate keywords such as carotenoids, carotenes and xanthophylls, flavonoids, anthocyanins, and anthoxanthins. Pick 3 interesting facts to share.

Design an experiment to determine whether the yellow of 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 hens reared in different conditions (e.g. barn, free-range, 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. You could also research this online.

Would it be possible to use this experiment to see if artificial “yolk-yellowers” are added to the feed of some hens? Would it also be possible to use this experiment to see if supplements of the natural “yolk-yellowing” pigments are added to the feed of some hens?

How could quantities of pigments in egg yolk be investigated rather than just presence or absence of a particular pigment?

Getting learning value from the practical

It is important to consider the purpose of practical work in Biology lessons and the following information provides suggested areas where this practical can be used to help students develop their practical, mathematical and subject-knowledge-based skills and understanding. The latter part of this section shows how this practical (or an extension of it) can also be used to meet some of the A-level specification requirements for the Use of apparatus and techniques as well as the Common practical assessment criteria (CPAC).

PRACTICAL SKILL DEVELOPMENT

You should be able to:

·       use a pestle and mortar to extract photosynthetic pigments from plant material

·       follow instructions carefully to successfully produce a chromatogram of photosynthetic pigments from the plant extract

·       accurately measure the distance between the solvent front and the original concentrated extract spot as well as the distance between each pigment spot and the original concentrated extract spot

·       calculate Rf values for each of the pigment spots

·       Compare calculated Rf values with those of known pigments to suggest the identity of each molecule

·       record Rf values, observations and suggested identity of pigments in a suitable table

·       use information about the nature of each molecule to explain their Rf values relative to other similar molecules

EXTENSION TO PRACTICAL SKILL DEVELOPMENT

You should be able to:

·       suggest ways to improve the accuracy of the Rf values calculated

·       suggest factors that would need to be controlled, and/or modifications to the method if comparisons of pigments from different sources were to be made

·       describe a suitable control experiment to ensure that the pigment come from the plant extract and not from other equipment or chemicals used in the process

·       design an experiment to investigate the possible presence of both polar and non-polar pigments in different plant extracts (e.g. comparing tomatoes and strawberries)

·       design an experiment to explore the pigments in hen egg yolks and how this may relate to how the hens are reared and/or what they are fed

·       design an extension to the main experiment, or any of the others mentioned above to investigate the concentration of particular pigments rather than just their presence or absence

MATHS SKILLS DEVELOPMENT

You should be able to:

·       calculate Rf values for each of the pigment spots

·       calculate the percentage error in the measurements taken

EXTENSION TO MATHS SKILL DEVELOPMENT          

You should be able to:

·       work out a range for each Rf value that would include the true Rf value for that chromatogram

·       calculate the percentage error in the Rf values calculated

ASSOCIATED SUBJECT KNOWLEDGE DEVELOPMENT

You should be able to:

1.     State the precise location of the pigments involved in photosynthesis within the leaf

2.     Name 5 photosynthetic pigments (or types of pigment) found in leaves

3.     Outline the role of the leaf pigments in photosynthesis

4.     Explain how the terms “light harvesting systems”, “photosystems” and “reaction centres” are related

5.     Explain why many plants have a variety of photosynthetic pigments

6.     Describe how to conduct chromatography to separate pigments from a leaf, explain importance of each step

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

8.     Describe how to calculate the Rf value for a particular substance

9.     Explain how a chromatogram can be used to identify an unknown substance

RECORDING EVIDENCE OF STUDENTS’ WORK

Students can use the worksheet provided to produce evidence of their work in this practical that can contribute to their practical skills accreditation.

MEETING ASPECTS OF THE USE OF APPARATUS AND TECHNIQUES

The full practical can allow students to meet various components of Appendix 5c of the GCE AS and A level subject content for biology, chemistry, physics and psychology (DfE, April, 2014) in bold (with clarification where needed):

1.     Use appropriate apparatus to record a range of quantitative measurements (to include mass, time, volume, temperature, length and pH).

2.     Use appropriate instrumentation to record quantitative measurements, such as a colorimeter or potometer.

3.     Use laboratory glassware apparatus for a variety of experimental techniques to include serial dilutions.

4.     Use of light microscope at high power and low power, including use of a graticule.

5.     Produce scientific drawing from observation with annotations.

6.     Use qualitative reagents to identify biological molecules.

7.     Separate biological compounds using thin layer/paper chromatography or electrophoresis.

8.     Safely and ethically use organisms to measure:

a.     Plant or animal responses

b.     Physiological functions

9.     Use microbiological aseptic techniques, including the use of agar plates and broth

10.  Safely use instruments for dissection of an animal organ, or plant organ.

11.  Use sampling techniques in fieldwork.

12.  Use ICT such as computer modelling, or data logger to collect data, or use software to process data.

MEETING ASPECTS OF THE COMMON PRACTICAL ASSESSMENT CRITERIA (CPAC)

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Competency	Practical Mastery	Evidence from this practical / or opportunity to train students in this skill using this practical
Follows written procedures	Correctly follows instructions to carry out the experimental techniques or procedures.	Observational evidence from the teacher. Positive outcome for the student’s chromatogram. Being able to answer questions about the procedure.
Applies investigative approaches and methods when using instruments and equipment	Correctly uses appropriate instrumentation, apparatus and materials (including ICT) to carry out investigative activities, experimental techniques and procedures with minimal assistance or prompting.	Use of pestle and mortar, running the chromatogram and measuring Rf values.
	Caries out techniques or procedures methodically, in sequence and in combination, identifying practical issues and making adjustments when necessary.	Follow procedure – set up the chromatography plate in the glassware appropriately, adjusting during trial if necessary.
	Identifies and controls significant quantitative variables where applicable, and plans approaches to take account of variables that cannot readily be controlled.	If comparing between plant species/varieties then running on the same plate or ensuring the same protocol is followed. Standardising extraction method.
	Selects appropriate equipment and measurement strategies in order to ensure suitably accurate results.	Can answer questions about modifications to increase the accuracy of the Rf value measurement and to clarify the identification of the different pigments.
Safely uses a range of practical equipment and materials	Identifies hazards and assesses risks associated with these hazards when carrying out experimental techniques and procedures in the lab or field.	Can answer questions about the risks from this practical and the safety precautions taken.
	Uses appropriate safety equipment and approaches to minimise risks with minimal prompting. Identifies safety issues and makes adjustments when necessary.	Evidence from actions that minimise use and evaporation of the extraction and running solvent.
Makes and records observations	Makes accurate observations relevant to the experimental or investigative procedure.	Accurate descriptions of colour and measurements of distances the solvent front and the pigments travel.
	Obtains accurate, precise and sufficient data for experimental and investigative procedures and record this methodically using appropriate units and conventions.	Measurements and observations from the chromatogram taken and recorded and used appropriately to make suggestions about the identity of the pigments.
Researches, references and reports	Uses appropriate software and/or tools to process data, carry out research and report findings.	Report writing packages (e.g. Word). Internet search engines.
	Sources of information are cited demonstrating that research has taken place, supporting planning and conclusions.	Referencing in final report or presentation.

 

 

Background information

The learning outcomes document for this practical summarises the key background information relating to the chromatography of photosynthetic pigments.

Rf value terminology – The definition of an Rf value, based on how it is calculated, is consistently applied throughout the literature however what the “R” and the “f” stand for varies from source to source. You may find it referred to as “relative front”, “rate of flow”, “retention value” or “retardation factor”.

Below is information about the different pigments that may be useful when discussing the reasons behind the relative distances the different pigments travel in the chromatogram.

The chlorophylls (a and b) and pheophytins (a and b) are very similar molecules (figure 1A-C). They all have a porphyrin-like ring structure (a complex ring of carbon atoms with 4 Nitrogen atoms near the centre) with a long hydrophobic hydrocarbon tail. The chlorophylls have a magnesium atom at the centre of the porphyrin-like ring whereas the pheophytins are missing the magnesium atom and have two hydrogen atoms instead. As well as the magnesium and nitrogen atoms there are some oxygen atoms around the ring that contribute to the polar nature of this part of the molecule. The absence of the magnesium atom in the pheophytins makes them less polar than the chlorophylls and so they have a larger Rf value in the solvent used here. Chlorophyll b and pheophytin b have one more oxygen atom around the ring than their a equivalents, making them more polar than their a equivalents, so they have smaller Rf values than their a equivalents.

Pheophytins used to be considered solely as breakdown products of chlorophyll, however, they are now considered to be early electron acceptors of photosystem II (they may still be breakdown products as well).

Carotenoids are a group of molecules with 40 carbon atoms arranged, mostly, linearly, often terminating at each end in a 6 carbon ring. The linear component has alternating carbon-carbon single and double bonds. These molecules act as both antenna pigments and photoprotective agents as well as giving colour to plant structures. The carotenoids are split into two groups: the carotenes (hydrocarbons) and the xanthophylls (molecules that also contain some oxygen atoms). The carotene in grass is mostly beta-carotene (figure 1D) and, being a hydrocarbon, is non-polar and so has a very large Rf value. The xanthophylls most abundant in grass, are lutein, violaxanthin and neoxanthin (figure 1E-G). Being xanthophylls they all contain oxygen atoms, making them polar so they have relatively small Rf values. The least polar of the 3 common xanthophylls is lutein with one hydroxyl group at each end of the molecule only, therefore lutein has the largest Rf value of these 3 xanthophylls. As well as the two hydroxyl groups that lutein has, violaxanthin has two epoxy groups (carbon-oxygen-carbon in a triangle). Neoxanthin has 3 hydroxyl groups and one epoxy group. Epoxy groups are polar, but not as polar as hydroxyl groups so neoxanthin is the most polar of the 3 xanthophylls and therefore has the smallest Rf value of these 3 molecules.

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Sample data

The Chromatogram to the left (figure 2) and the table below shows data collected from grass picked from an amenity lawn outside the laboratory in July, using propanone as the extraction solvent, no sand, and the suggested running solvent (5 cyclohexane: 3 propanone: 2 petroleum ether 80-100°C). It was run for about 8mins and photographed very soon after removal from the running solvent. The solvent front travelled 44mm.

Pigment colour	Distance pigment has travelled / mm	Rf value	Suggested identity of the pigment
Bright yellow	41	0.93	Carotene
Grey	20	0.45	Pheophytin a
Pale Brown	16	0.36	Pheophytin b
Blue green	12	0.27	Chlorophyll a
Yellow	11	0.25	Xanthophyll – Lutein
Green	10	0.23	Chlorophyll b
Pale yellow	7	0.16	Xanthophyll – Violaxanthin
Pale yellow	3	0.07	Xanthophyll – Neoxanthin

Further Investigations

As mentioned within this document there is scope to develop this practical in a range of directions including:

·         Investigating the presence of carotenoids in other plant material (e.g. carrots, maize, dandelions, buttercups, bananas, sweet potatoes, cantaloupe melons, tomatoes).

·         Investigating the presence of carotenoids in animal products (e.g. egg yolk and butter) and possibly comparing eggs produced by hen reared in different conditions (e.g. barn, free range, corn fed).

·         Comparing the pigments extracted using polar and non-polar solvents to investigate and compare anthocyanins and the photosynthetic pigments (e.g. comparing the red of tomatoes (lycopene, a non-polar carotenoid) and the red for strawberries (an anthocyanin), or investigating both photosynthetic pigments and anthocyanins in Cloeus spp.)

References

Davies, K. (ed) (2004) Plant pigments and their manipulation. Annual Plant Review, Vol. 14.

Tomkins, S. P. and Miller, M. B. (1994), A rapid extraction and fast separation of leaf pigments using thin layer chromatography. School Science Review 75 (273), 69 - 72. (Available from the SAPS website)

Acknowledgements

Protocol and support materials re-developed by Dr Chris Graham, Long Road Sixth Form College, Cambridge and Gail Webdell, Faculty of Education, University of Cambridge. Based on an original protocol developed by Stephen Tomkins, Homerton College, Cambridge, and Barry Miller, Douglas Ewart High School, Newton Stewart.