Required practical 7
Use of chromatography to investigate the pigments isolated from leaves of different plants, eg, leaves from shade-tolerant and shade-intolerant plants or leaves of different colours.
Overview
Plants contain different types of photosynthetic pigments (e.g. chlorophyll).
Why would it be advantageous for a plant to have different pigments in the leaf?
Why would it be advantageous for a plant to have different pigments in the leaf?
So they can absorb more different wavelengths for photosynthesis.
Chromatography can be used to separate and then compare these photosynthetic pigments present in leaves from different plants (e.g. shade-tolerant vs shade-intolerant plants). Note there are different types of chromatography, e.g. thin layer, paper, but they all apply similar principles.
General Principles
-
Solvent (mobile phase)
- This is the liquid moving up the paper
- It carries the pigments with it
- If a pigment dissolves easily in the solvent (higher solubility) it will travel further
-
The paper (stationary phase)
- This stays still
- Pigments can stick to the paper
- If a pigment is attracted more strongly to the paper, it moves more slowly (higher affinity)
Method
- Grind leaf tissue with solvent (e.g. propanone) to extract pigments.
- Draw a pencil baseline near the bottom of chromatography paper
- Use pipette to apply pigment extract to the pencil line (known as point of origin)
- Place paper in solvent (propanone/acetone) ensuring the pigment spot is above the solvent (i.e. the solvent is below the pencil line)
- Allow solvent to rise until near the top
- When the solvent has nearly reached the top, remove the paper from the solvent and mark in pencil the point (solvent front)
Why would you grind the leaf tissue with a solvent?
Why would you grind the leaf tissue with a solvent?
- This helps to break down the cell walls of leaf cells, and the chloroplast membranes, so we can analyse the chlorophyll pigments
- It enables the pigment to dissolve in the solvent (solvent can dissolve lipids, e.g. cell and chloroplast membranes)
Why would you not draw the line in pen?
Why would you not draw the line in pen?
Ink would dissolve in the solvent and mix with the leaf pigments. A pencil line doesn’t dissolve, so it stays visible in the same place, and you can measure from it.
Tip for method
Ask ‘why’ do we perform each of these steps, it will help you understand and remember the method better
Calculating Rf value
Used to identify and compare pigments.
$$ R_f = \frac{\text{distance moved by pigment from baseline}}{\text{distance moved by solvent front from baseline}} $$
- Each pigment has a characteristic Rf value in a given solvent.
- Rf values depend on the solvent used, so comparisons must use the same solvent.
- Rf values can be compared between different plants to identify differences in pigment composition.
What would you expect to see in a shade-tolerant plant?
What would you expect to see in a shade-tolerant plant?
Shade-tolerant plants often have more chlorophyll, so they can absorb as much of the dim light as possible.
Example Rf Calculation
From the chromatogram:
-
A = distance travelled by the solvent front from the pencil line
-
B = distance travelled by the pigment spot from the pencil line
-
Distance travelled by solvent front (A) = 8.0 cm
-
Distance travelled by pigment spot (B) = 3.2 cm
$$ R_f = \frac{B}{A} $$
$$ R_f = \frac{3.2}{8.0} = 0.40 $$
- The pigment has an Rf value of 0.40 in this solvent.
- This value can be compared with:
- known pigment Rf values
- pigments from other plants (using the same solvent)
See also: Dehydrogenase activity in chloroplasts (required practical 8).
Chromatography is examined as part of photosynthesis. See how it's tested on that page →
Tips from examiner reports
What students commonly get wrong
- Don't mix up the pigment solution and the solventWatch out: the pigment (chlorophyll) solution goes on the pencil line. The solvent goes in the container, with its level below the line. Use an organic solvent, never water. 2023 P2 Q1.1 · 2019 P2 Q7.4
- Pencil, because ink would mix with the pigmentsWatch out: don't just say ink would "run" or "smudge". Say the ink would dissolve and mix with the leaf pigments, or that a pencil line stays visible in the same place. 2019 P2 Q7.3
Exam Question Practice
A student wanted to find out which pigments are in nettle leaves. She ground some leaves with a solvent to make a pigment extract. She was then given:
- a thin-layer chromatography (TLC) plate
- a pencil and a ruler
- a capillary tube
- a beaker with a lid
- a running solvent (solvent X).
Describe how she could use these to separate the pigments in the extract.
(4 marks)
Hint
Where on the plate does the extract go, and how do you mark that place? Where must the solvent level be? When do you take the plate out?
Mark Scheme
- Draw an origin line in pencil near the bottom of the plate (1 mark)
- Use the capillary tube to put a spot of extract on the origin (1 mark)
- Stand the plate in the beaker with the solvent below the origin (1 mark)
- Remove the plate before the solvent reaches the top, or mark the solvent front (1 mark)
Explanation (not marked): Pencil is used because ink would dissolve in the solvent and separate too. The solvent must start below the origin, or the pigments would dissolve into the solvent in the beaker instead of moving up the plate. Marking the solvent front lets you work out an Rf value for each pigment.
Also accepted / rejected
Tips
- Draw the origin in pencil and put the extract on it. Don’t mix up the extract with the solvent
- The solvent in the beaker must be below the origin
- Take the plate out before the solvent reaches the top, or mark the solvent front
- Use the solvent you are given, not water
A student extracted pigments from nettle leaves. Then the student used thin-layer chromatography (TLC) to separate the pigments.
Before applying the pigment solution, the student drew the origin line on the TLC plate in pencil.
Explain why the student marked the origin using a pencil rather than using ink.
(1 marks)
Hint
What would happen to an ink line when the solvent moves up the plate? Why do you still need the origin line after running the chromatogram?
Mark Scheme
Ink and (leaf) pigments would mix
OR (With ink) origin/line in different position
OR (With pencil) origin/line in same position
OR (With pencil) origin/line still visible (1 mark)
Tips
- Give the consequence: ink would dissolve in the solvent and mix with the leaf pigments, whereas a pencil line stays visible in the same place
- ‘Ink would run, smudge or affect the results’ isn’t enough, and make clear whether you mean pencil or ink
A student extracted pigments from nettle leaves. Then the student used thin-layer chromatography (TLC) to separate the pigments.
Describe the method the student used to separate the pigments after the solution of pigments had been applied to the origin.
(2 marks)
Hint
Where should the solvent be compared with the origin line? When should you take the plate out?
Mark Scheme
- Level of solvent below origin/line (1 mark)
- Remove/stop before (solvent) reaches top/end (1 mark)
Also accepted / rejected
1. Reject water or any named aqueous solution.
1. Accept named organic solvent.
Tips
- Give practical detail from required practical 7: the solvent level is below the origin, and the plate is removed before the solvent reaches the top
- Use an organic solvent, not water, glucose or acid
- Describe the method, not the principle of chromatography
A student extracted pigments from nettle leaves. Then the student used thin-layer chromatography (TLC) to separate the pigments.
Figure 1 shows the TLC plate after it was removed from the solvent, next to a ruler. The solvent front was not marked.
Calculating the Rf values of the pigments can help to identify each pigment. An Rf value compares the distance the pigment has moved from the origin with the distance the solvent front has moved from the origin.
Rf = distance pigment has moved from the origindistance solvent front has moved from the origin
The distance each pigment has moved is measured from the middle of each spot.
Pigment A has an Rf value of 0.92
Use Figure 1 to calculate the Rf value of pigment C.
Rf value of pigment C = ________
(1 marks)
Hint
The solvent front isn’t drawn. Use pigment A’s distance and its Rf value to work out how far the solvent moved.
Mark Scheme
Accept any answer in range of 0.42 to 0.46 (1 mark)
(Solvent front = 69 ÷ 0.92 = 75 mm; pigment C = 33 ÷ 75 = 0.44)
Also accepted / rejected
Accept 0.42 or 0.46.
Ignore any numbers which follow numbers in range.
Also accepted