Separating Leaf Pigments by Chromatography

A leaf looks simply green, but it contains a mixture of coloured molecules. Some are green, some yellow, some orange — the same yellow and orange pigments that become visible in autumn when chlorophyll breaks down. In this practical you’ll extract the pigments from spinach and separate them by chromatography, producing a strip of paper or a plate with distinct coloured bands. It’s a classic experiment that combines plant biochemistry with a core technique of analytical chemistry.

Purpose

To extract photosynthetic pigments from leaves, separate them by chromatography, calculate their R_f values and explain the order of separation in terms of polarity.

Principle

The pigments

Leaves contain two main families of pigments:

• Chlorophylls (green): chlorophyll a (blue-green) and chlorophyll b (yellow-green). Each has a large, flat ring system with a magnesium ion at its centre, and a long hydrocarbon tail (see chlorophyll and magnesium). The ring contains several polar C=O groups and nitrogen atoms. Chlorophyll b has an aldehyde group (–CHO) where chlorophyll a has a methyl group (–CH₃), which makes chlorophyll b slightly more polar.
• Carotenoids (yellow–orange): β-carotene is a pure hydrocarbon, C₄₀H₅₆ — long, non-polar and orange. Xanthophylls (such as lutein) have a similar carbon skeleton but carry oxygen-containing –OH groups, so they’re more polar and yellow.

Carotenoids help plants absorb light in the blue-green range and protect chlorophyll from damage by excess light.

How chromatography separates them

Chromatography separates substances by how they distribute themselves between two phases (see chromatography):

• The stationary phase: paper (cellulose, with a thin layer of bound water) or a thin layer of silica on a TLC plate. Both are polar.
• The mobile phase: a solvent that moves up the paper or plate by capillary action.

A molecule that is strongly attracted to the polar stationary phase — through hydrogen bonds or dipole interactions — spends more time stuck and moves slowly. A molecule that dissolves well in a relatively non-polar solvent and interacts weakly with the stationary phase moves quickly.

So with a mainly non-polar solvent, the expected order from top (fastest) to bottom (slowest) is:

1. β-carotene (non-polar hydrocarbon) — near the solvent front
2. Chlorophyll a
3. Chlorophyll b
4. Xanthophylls (most polar, with –OH groups) — lowest

(The relative positions of xanthophylls and chlorophylls can vary with the exact solvent and stationary phase; on silica TLC plates, xanthophylls often run between chlorophyll a and b, or below them. Recording what you actually see is more important than matching a textbook.)

R_f values

Each spot’s position is described by its retention factor:

R_f = distance moved by the spot ÷ distance moved by the solvent front

R_f is always between 0 and 1. For a given solvent and stationary phase, it’s characteristic of a substance.

Equipment

• Fresh spinach leaves (or nettle, parsley)
• Pestle and mortar, a pinch of clean sand
• Propanone (acetone), about 5 cm³
• Chromatography paper strip (or silica TLC plate), pencil and ruler
• Fine capillary tube or glass rod
• Boiling tube or chromatography tank with a lid or bung
• Solvent: e.g. a mixture of petroleum ether (a low-boiling hydrocarbon mixture) and propanone, around 9 : 1 by volume, prepared by a technician (see safety)
• Hair dryer on a cool setting (optional)
• Eye protection, gloves

Safety

• Propanone and petroleum ether are highly flammable. No flames anywhere in the room. Use small volumes.
• Their vapours are harmful and can cause drowsiness — work in a fume cupboard or a very well-ventilated room, and keep containers closed.
• Wear eye protection; avoid skin contact.
• Dispose of solvents as instructed by your teacher — never down the sink.

Method

1. Extracting the pigments

1. Tear two or three spinach leaves into small pieces, avoiding the thick veins.
2. Grind them in the mortar with a pinch of sand and about 3–5 cm³ of propanone until you have a dark green liquid. The sand helps break cell walls; propanone dissolves the pigments, which are too non-polar to dissolve in water.
3. Tilt the mortar and let the solids settle. Use the clear green liquid.

2. Preparing the chromatogram

4. On the paper or TLC plate, draw a light pencil line about 1.5 cm from the bottom. (Pencil, not pen — ink would dissolve and run.)
5. Using a capillary tube, place a small spot of extract on the line. Let it dry (blow gently or use a cool hair dryer).
6. Repeat 10–20 times on the same spot, drying between each application, until the spot is small but dark green. A small, concentrated spot gives sharp bands.

3. Running the chromatogram

7. Pour the solvent into the tube or tank to a depth of about 1 cm — below the pencil line.
8. Carefully lower the paper in so it hangs straight and doesn’t touch the sides. Seal the container so the air inside becomes saturated with solvent vapour.
9. Leave it undisturbed. After a few minutes, as the solvent nears the top (about 1 cm from the end), remove the paper.
10. Immediately mark the solvent front with a pencil line, before it evaporates.
11. Let the chromatogram dry. Work quickly — the pigments fade in bright light.

Results

You should see several distinct bands. A typical result:

Band (top → bottom)	Colour	Pigment	Example R_f
1	Orange-yellow	β-carotene	~0.9
2	Grey or pale yellow (sometimes)	Pheophytin (chlorophyll that has lost its Mg²⁺)	~0.8
3	Blue-green	Chlorophyll a	~0.6
4	Yellow-green	Chlorophyll b	~0.5
5	Yellow	Xanthophylls	~0.3–0.4

Your R_f values will differ depending on the solvent, the paper and the temperature. Measure from the pencil line to the centre of each band.

Worked example: the solvent front moved 8.0 cm. The blue-green band’s centre is 4.6 cm from the origin. R_f = 4.6 ÷ 8.0 = 0.58.

Explaining the results

• β-carotene moves furthest because it’s a non-polar hydrocarbon: it dissolves well in the non-polar solvent and forms almost no attractions with the polar paper.
• Chlorophylls are intermediate: the long hydrocarbon tail makes them fairly soluble, but the polar groups on the ring interact with the stationary phase.
• Chlorophyll b runs below chlorophyll a because its extra aldehyde group makes it slightly more polar.
• Xanthophylls have –OH groups that hydrogen-bond strongly to the stationary phase, so they move slowly.

The appearance of pheophytin is a nice extra: chlorophyll easily loses its Mg²⁺ ion in acidic conditions or with age, forming a greyish-olive pigment. The same chemistry turns overcooked green vegetables drab olive-green — acids released during cooking replace Mg²⁺ with H⁺.

Sources of error and limitations

• Spot too large or too dilute: bands overlap or are too faint. Use many small applications.
• Solvent level above the origin line: pigments dissolve into the solvent pool instead of moving up.
• Container not sealed: solvent evaporates from the paper, giving uneven, streaky results.
• Paper touching the sides: solvent front becomes uneven.
• Fading: chlorophylls break down in light and air, so mark and measure bands quickly.
• Identification is tentative: R_f values alone don’t prove identity. Running pure standards alongside, or measuring each band’s absorption spectrum, would confirm it.
• Co-running pigments: some bands may contain more than one pigment.

Extensions

• Compare leaves of different ages or colours (young vs old, green vs red or variegated leaves, autumn leaves).
• Try different solvent ratios and see how R_f values change.
• Compare paper and TLC — TLC is usually faster and sharper (see TLC vs paper chromatography).
• Cut out each band, dissolve it in propanone and measure its absorption spectrum with a colorimeter or spectrophotometer.

Key takeaways

• Leaves contain chlorophyll a and b (green) and carotenoids (β-carotene and xanthophylls, yellow–orange).
• In chromatography, less polar pigments travel further with a non-polar solvent over a polar stationary phase.
• R_f = spot distance ÷ solvent front distance.
• Use a pencil origin line, small concentrated spots, a sealed container, and mark the solvent front immediately.

For how these pigments drive photosynthesis, see light-dependent reactions.