IBDP Biology Required Practical

Required
Practical 4
Chromatography

Skill:
Separation of photosynthetic pigments by chromatograph.
 Chromatography
IBDP Biology Required Practical

Required Practical 4
Skill:
Separation of photosynthetic pigments by chromatograph.

Key Terms
Chromatography Chlorophyll Xanthophyll
Chromatograph Resolution Retardation Factor (Rf)
Photosynthesis Accessory Pigments Solvent front
Pigment Carotene

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Skill: Separating Photosynthetic Pigments
Notes:
1. Chromatography separates soluble pigments based on density. Pigments with low
density travel faster and further than pigments with high density.
2. Most pigments in a leaf are soluble in organic solvents.
3. If possible, use thin layer chromatography paper. However, if thin layer
chromatography paper is not available, then cut 1cm wide strips of filter paper.
4. Spinach leaves are a good source of photosynthetic pigments.
5. Use a pencil to draw the origin line on the chromatography paper (not a pen, which
contains pigments that will be separated).
6. Pigment can be transferred with capillary tubes, a narrow pipette, or by repeated
use of a toothpick or straightened paper clip.
7. Suggested running solution mixtures include: a mixture of propanone : petroleum
ether (1:9) or a mixture of diethylether : petroleum ether (3:2)
Materials:
• Mortar and pestle (+ pinch of • Running solution
sand) • Hair dryer
• Spinach leaves • Dropping pipette
• Scissors • Watch glass
• Chromatography paper (or strips of • Measuring cylinder
filter paper) • Boiling tube and stopper with
• Capillary tubes (very fine) boiling tube rack
• Ethanol

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 Chromatography
IBDP Biology Required Practical

Procedure
(Modified from SAPS)
Preparation of chromatography paper
1. Cut the chromatography paper 2 cm longer than the boiling tube
2. Draw a pencil line 2 cm (Origin) from the bottom of the chromatography paper

Preparation of spinach pigment solution

1. Place two spinach leaves in hot water (about 60oC) for 2 minutes – to kill cells and
make them more soluble to the solvent.
2. Carefully remove the spinach leaves, and allow to cool.
3. Cut the spinach leaves and place in a mortar with some sand.
4. Add 5ml of ethanol, and crush the spinach leaves with the pestle until a dark green
extract is present.
5. Use a capillary tube to place 20 drops of the extract onto a watch glass, and heat
with a hairdryer (to evaporate the water and ethanol).
6. Add 3 drops of ethanol to the dry pigments
Preparation of the chromatograph
1. Add running solution to a height of 1cm into a boiling tube.
2. Use a capillary tube to transfer a drop of pigment from the watch glass
to the center of the origin line on the chromatography paper – allow to
dry/dry with a hair dryer.
3. Continue adding drops of pigment and drying until a dark green spot is
present.
4. Carefully place the chromatography paper into the boiling tube – the
chromatography paper should be in the running solution, but the origin
line with the dark green spot should be above the solution.
5. Wait until the running solution has almost reached the top of the
chromatography paper, then remove the paper from the boiling tube
and mark, with a pencil, the highest level the solvent has moved up the
chromatography paper (This is called the Solvent Front).
6. Outline each spot of separated pigments on the chromatograph. Do this quickly as
some pigments (especially the beta-carotene) may fade in a short time.

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 Chromatography
IBDP Biology Required Practical

Calculating retardation factor (Rf)

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑝𝑖𝑔𝑚𝑒𝑛𝑡

𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑟𝑢𝑛𝑛𝑖𝑛𝑔 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

1. Measure the distance from origin to the solvent front -This is the distance travelled
by the running solvent.
2. Measure the distance from the origin to the center of each outlined pigment – This is
the distance travelled by the pigment.
3. Calculate the retardation factor (Rf) for each pigment, and complete the following
table.

Pigment Color Pigment Rf Value

orange yellow Carotene
blue green Chlorophyll a
green Chlorophyll b
deep yellow Xanthophyll

IB Biology Exam Practice:

See:

• November 2017 Standard Level Paper 3, Question 2

• November 2017: Higher Level Paper 3, Question 1 [Accessible for SL students]
• May 2018 Time Zone 2: Standard Level Paper 3, Question 1
• May 2019 Time Zone 2: Standard Level Paper 3, Question 1
• November 2019: Standard Level Paper 3, Question 3
• November 2019: Standard Level Paper 3, Question 3 (d)
All examination papers are available on Follett’s IB store.

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 Thank You!
Chromatography
IBDP Biology Required Practical

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