Separating Leaf Pigments on a Coffee Filter
Spinach looks green because four pigments overlap and your eye averages them. Paper chromatography pulls them apart in about twenty minutes and shows you the yellow and orange that were there all along.
Materials
| Item | Amount | If you do not have it |
|---|---|---|
| Spinach or fresh nettle leaves | 20 g | Any dark leafy green; spinach has the clearest xanthophyll band |
| Rubbing alcohol, 70% isopropyl | 30 ml | Nail varnish remover, which is faster but harsher |
| Coffee filter paper | 2, unbleached | Chromatography paper if you have it; kitchen roll is too fluffy |
| Glass jar with a lid | 1, tall enough for a 12 cm strip | A tall drinking glass with cling film over the top |
| Coin or spoon | 1 | A pestle, for crushing the leaves |
| Pencil and ruler | 1 each | A pen will run in the solvent; use pencil only |
Method
Tear the leaves into a small cup, add 20 ml of alcohol, and grind them with the back of a spoon for three minutes.
Watch for: The alcohol turns dark green within a minute and the leaf fragments go pale and papery.
Cut a strip of filter paper 12 cm long and 2 cm wide, and draw a pencil line 2 cm from the bottom.
Watch for: Do not use ink: the ink's own pigments will run and swamp the leaf bands.
Dip the strip into the green extract and let it dry, then repeat eight to ten times to build a concentrated dark spot on the line.
Watch for: Each dip leaves a thin green film; after ten dips the spot is nearly black.
Pour 5 mm of alcohol into the jar, lower the strip in so the line sits above the liquid, and seal the jar.
Watch for: Solvent climbs the paper at about 1 mm per second and the dark spot starts to streak upward within four minutes.
Take the strip out when the solvent front is 2 cm from the top, mark the front with pencil, and let it dry.
Watch for: Four bands appear, top to bottom: yellow-orange, bright yellow, blue-green and yellow-green.
Why it works
Chromatography separates molecules by how they divide their time between a mobile phase, the alcohol climbing the paper, and a stationary phase, the water held in the paper fibres. A pigment that dissolves well in alcohol travels with the solvent and moves far up the strip. A pigment that clings to the water in the paper lags behind. Chlorophyll a is the blue-green band and chlorophyll b the yellow-green; both travel moderately. The carotenes are pure hydrocarbons, dissolve best in alcohol and run furthest, giving the top yellow-orange band. Xanthophylls carry oxygen groups and hydrogen-bond to the paper's water, so they stay near the start. Two bands that look identical in a leaf are different molecules with different polarities.
Think of it like: It is a race between swimmers of different styles through water with a rope hanging in it. Some grab the rope and slow down; some are streamlined and shoot ahead. Everyone starts at the same line.
Clean-up and disposal
Isopropyl alcohol is flammable and must not be poured down the sink in quantity; let the small amount in the jar evaporate in a ventilated room or seal it and take it to a household chemical collection. The used paper strips can be taped into a notebook once dry. Wash the cup and spoon with hot soapy water before using them for food again.
Bench notes
Grinding properly
Most failed strips come from a weak extract. Crush the leaves until the alcohol is dark and the leaf fragments look bleached, then let the debris settle for two minutes and use only the clear liquid.
Observed / 实测Twenty grams of spinach in twenty millilitres of alcohol gives an extract dark enough that a single dip produces a visible spot after the strip dries.
Measuring the bands
Observed / 实测The retention factor, the distance a band travels divided by the distance the solvent travels, is about 0.95 for carotene, 0.7 for chlorophyll a, 0.55 for chlorophyll b and 0.3 for the xanthophylls. Reproducing those four numbers on a kitchen bench is a genuine quantitative result, not a party trick.
Why the strip must dry flat
Observed / 实测A wet strip curling on the bench smears every band into its neighbour within thirty seconds of leaving the jar.
Variations worth running
- Run the same strip with a green felt-tip pen and compare how many dyes the pen contains with how many pigments the leaf contains.
- Use a red or purple leaf in autumn and look for the anthocyanin band that sits between the xanthophylls and the chlorophylls.
- Change the solvent to pure water and to white spirit, and compare how far each band travels in each.
Questions that come up on the bench
- Why is the top band orange when leaves look green?
- Carotene is always there, but in a living leaf its orange colour is swamped by the much larger amount of chlorophyll around it. Removing the chlorophyll would reveal the orange; putting the pigments on paper does exactly that.
- Why must the spot be above the liquid level?
- If the line dips into the solvent, the pigments wash straight off the paper into the jar instead of being carried up the strip. The solvent has to travel past the spot, not through it.
- Can I use water instead of alcohol?
- You can, but the pigments barely dissolve in water and the strip takes hours. Alcohol is the standard solvent for leaf pigments because carotene and chlorophyll are both soluble in it.