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Required practical 7: chromatography of leaf pigments

A green leaf holds several pigments, and chromatography pulls them apart. Spot an extract on a pencil line, stand the paper in a non-polar solvent, and each pigment climbs a different distance. The ratio of that distance to the solvent's, the Rf value, identifies the pigment. Run two leaves on one paper and you can compare which pigments each has, and roughly how much.

The capability this develops

This guide is one investigation under capability P10: Chromatography and the separation of pigments, which sets out the techniques the investigation belongs to, the calculations it carries and the errors that cost marks in all of them. The notes behind the biology are in Photosynthesis and primary productivity.

Where this sits on the specifications

This library maps a qualification to its own specification one numbered heading at a time, against a document whose sha256 is recorded, and publishes the result as a route map. These are the headings whose practical work this investigation serves, on the five qualifications mapped so far. Each links to the route map the row belongs to.

Some boards number their practical activities, and those lists were read here from the same specifications as the headings above. This investigation is numbered on them as:

• AQA 7401/7402 required practical 7
• OCR Biology A H020/H420 PAG 6
• Pearson Edexcel Biology B 8BI0/9BI0 core practical 11

The other qualifications above are matched by specification heading only, because none of them numbers an activity that is this investigation. Use your awarding organisation’s current specification and its practical handbook as the authority on which activities your course requires and how they are assessed.

The principle

Pigments separate because they differ in how strongly they are attracted to the paper and how soluble they are in the solvent. A pigment that is very soluble and holds weakly to the paper travels a long way; one that clings to the paper barely moves. Carotene, a hydrocarbon with no polar groups, runs almost to the solvent front. Chlorophyll b, the most polar of the group, lags at the back.

The Rf value is the distance moved by the spot divided by the distance moved by the solvent, both measured from the pencil line. It has no unit and cannot be more than 1.

Because the ratio cancels the units, an R_f value is the same whether the solvent ran three centimetres or thirteen, so it is worth quoting. It is only reproducible for a given solvent, though: change the solvent and every value changes, so a table of R_f values always comes with the solvent named.

Running two leaves on the same paper means they share the solvent, the run and the solvent front. A pigment in both leaves then gives spots at the same height, and any difference between the leaves is in which spots are present and how strong they are.

R_f value

The distance moved by a spot divided by the distance moved by the solvent front, both from the origin line.

Origin

The pencil line on which the extract is spotted, drawn above the level of the solvent.

Solvent front

The furthest point the solvent reached up the paper, marked as soon as the paper is taken out.

Apparatus

• Two kinds of leaf: one from a plant in full sun, and one from a shade-tolerant plant on the floor of a wood, such as ivy
• A mortar and pestle, a little clean sand, and propanone
• Chromatography paper, or a thin-layer chromatography plate, and a pencil and ruler
• Fine glass capillary tubes for spotting
• A tall tank or boiling tube with a lid or bung
• Running solvent: petroleum ether and propanone, about 9 : 1, or a commercially prepared chromatography solvent
• A hair dryer on a cool setting, used away from the solvents, or time to let each spot dry
• Eye protection

Safety

Work to your school’s risk assessment. Propanone and the running solvent are highly flammable and their vapour is harmful, so use them in a well-ventilated room or a fume cupboard, with no naked flame anywhere in the room, and keep the tank lidded. Check the CLEAPSS Hazcard for each solvent before starting. Grinding leaves with sand can flick grit and solvent, so wear eye protection while grinding. Glass capillaries break easily and go in the broken-glass container.

Variables

Independent variable

The leaf: one from a plant growing in full sun and one from a shade-tolerant plant growing in shade.

Dependent variable

The pigments present, identified by their Rf values, and the size and depth of colour of each spot as a rough measure of how much of it there is.

Control variables

• The solvent and the run. Both extracts on one paper, run in one tank, so the solvent and the front are the same for both.
• The extract. The same mass of leaf ground with the same volume of propanone, so spot strength reflects the leaf and not the extraction.
• The spotting. The same number of applications for each extract, each dried before the next.
• The origin. One pencil line, above the solvent surface, for both spots.

Method

Extracting the pigments

1. Cut 2 g of each leaf into small pieces, avoiding the main veins, and grind each separately with a little sand and 5 cm³ of propanone until the liquid is dark green.
2. Let the debris settle, or filter the liquid, and keep each extract in a stoppered tube out of bright light.

Spotting and running

1. Draw a pencil line 2 cm from the bottom of the paper, and mark two points on it 3 cm apart, one for each leaf.
2. Spot the extract onto a pencil origin line, let it dry and spot it again, five or six times, so the spot is concentrated but small.
3. Then stand the paper in a shallow layer of solvent with the origin line above the solvent surface.
4. Keep a lid on the tank so the air inside stays saturated with solvent vapour; otherwise solvent evaporates from the paper as it rises and the front runs unevenly.

Describe the drawing

A rectangle of chromatography paper with a pencil origin line near the bottom and the solvent front marked near the top. Five pigment spots sit at different heights up the paper, each joined by a dotted line to its name and its Rf value, from carotene highest at nought point nine five down to chlorophyll b lowest at nought point four five. Measuring arrows show the eight point five centimetres the solvent ran and the five centimetres chlorophyll a moved, and the division is worked out beneath.

Marking and measuring

1. Take the paper out when the solvent is about 1 cm from the top, and mark the solvent front in pencil at once.
2. Draw round each spot in pencil before the colours fade, and mark the centre of each.
3. Measure from the origin line to the centre of each spot, and from the origin line to the solvent front, and calculate each Rf.

Sample data

These results are idealised. The Rf values are the ones the lesson's chromatogram shows, run here over a 10.0 cm solvent front, and the spot descriptions were made up to show how two leaves are compared. Nothing here is a record of an experiment.

The solvent front was 10.0 cm from the origin. For chlorophyll a, Rf = 5.9 ÷ 10.0 = 0.59.

Both leaves gave all five spots at the same heights, because they shared the paper and the solvent. So the two leaves contain the same pigments, and the difference between them is in the amounts.

The shade leaf's chlorophyll b spot is the clearest difference. Shade leaves often carry more chlorophyll b relative to chlorophyll a than sun leaves do. Chlorophyll b absorbs some wavelengths that chlorophyll a absorbs poorly, which helps a leaf in the dim light under a canopy.

Reading the chromatogram

Identify by Rf, not by colour. Two yellow spots, carotene and xanthophyll, are told apart by how far they ran, and a spot is matched to a pigment by comparing its Rf with values found in the same solvent.

The order of the spots is the order of polarity. Carotene, with no polar groups, is most soluble in the non-polar solvent and least held by the paper, so it is at the top. Chlorophyll b, the most polar, is at the bottom.

Phaeophytin is not a pigment the leaf was using. It is chlorophyll that lost its magnesium ion while it was being extracted, which is why the spot is grey and faint, and why an extract made quickly and kept out of the light gives less of it.

Comparing amounts. Spot size and depth of colour are a judgement by eye, enough to say that the shade leaf's chlorophyll b spot is larger and not enough to say by how much. To put a number on it, cut out each spot, dissolve the pigment out in a fixed volume of propanone and read its absorbance in a colorimeter.

Leaves of different colours. The same method compares a green leaf with a red or purple one. A red leaf still shows the chlorophyll spots; its red pigment dissolves in water rather than in the non-polar solvent, so it stays at or near the origin.

Evaluation

Each of these is a real source of error in this investigation, and the middle column is the part an answer earns marks for: what it does to the result, and in which direction.

Questions

Each answer opens where you tap it.

Coverage maps are available for AQA 7401/7402, OCR Biology A H020/H420, Cambridge International 9700, Pearson Edexcel Biology A (Salters-Nuffield) 8BN0/9BN0 and Pearson Edexcel Biology B 8BI0/9BI0. Use the relevant route map to check what InkBiology covers, and your awarding organisation’s current specification as the final authority on examinable content.