Grind up a leaf and you get one murky green liquid. Run it up a strip of paper and it splits into four distinct colours — proof that “chlorophyll” was never one substance at all.
📚 What you need to know
Plants contain several pigments in two groups: chlorophylls and carotenoids.
Each pigment absorbs a different range of wavelengths, so together they capture more of the spectrum.
Chromatography separates a mixture because its components travel through the material at different speeds.
The solvent is the mobile phase; the paper or silica gel is the stationary phase.
Each pigment has a characteristic Rf value = distance moved by pigment ÷ distance moved by solvent.
More soluble, smaller pigments travel further and have a higher Rf.
Always draw the start line in pencil, keep it above the solvent, and measure to the centre of each spot.
Why one pigment is not enough
Chlorophyll absorbs light well in some parts of the spectrum and badly in others. A plant relying on it alone would waste a lot of the light landing on its leaves.
So chloroplasts also contain carotenoids, which sit around the chlorophyll and absorb wavelengths that chlorophyll misses. They pass the energy on. The effect is to widen the range of wavelengths the plant can actually use.
Pigment group
Pigment
Colour of the pigment
Chlorophylls
Chlorophyll a
Blue-green
Chlorophylls
Chlorophyll b
Yellow-green
Carotenoids
β carotene
Orange
Carotenoids
Xanthophyll
Yellow
Careful with the word “colour”. The table gives the colour the pigment appears. That is the light it reflects, which is the opposite of the light it absorbs. Chlorophyll looks green precisely because green is the light it does not use.
How chromatography separates a mixture
Two things are competing for each pigment molecule. The solvent (mobile phase) is trying to carry it up the paper; the paper or gel (stationary phase) is trying to hold it back.
A pigment that dissolves readily in the solvent and sticks to the paper weakly is carried a long way.
A pigment that clings to the stationary phase — often a larger molecule — is carried a short way.
Because each pigment strikes a different balance, they end up at different heights and separate into bands.
Paper or thin-layer?
Paper chromatography uses filter paper (cellulose). Simple, cheap, good enough to tell the pigments apart.
Thin-layer chromatography (TLC) uses a thin layer of an adsorbent such as silica gel. The mixture travels faster and separates more sharply, so TLC gives better results.
Notice the orange carotene has travelled almost to the solvent front while chlorophyll b has barely moved. That gap is what the Rf value measures.
🧩 The method
Draw a pencil line about 1 cm above the bottom of the paper. Pencil, because ink would separate into its own colours and ruin the result.
Choose a healthy leaf that has been in direct sunlight, so it is full of active photosynthetic cells.
Grind the leaf in a mortar with a few drops of propanone. The solvent dissolves the lipid membranes and the grinding breaks open the chloroplasts, releasing the pigments.
Spot the extract onto the pencil line with a capillary tube. Let it dry and spot again several times to build up a concentrated, small spot.
Stand the paper in the solvent so the level is below the pencil line, and leave it until the solvent nears the top.
Mark the solvent front in pencil as soon as you remove the paper, before it evaporates.
Measure and calculate the Rf value of each spot.
Calculating Rf
Retardation factor
Rf = distance moved by pigment ÷ distance moved by solvent
Both distances are measured from the pencil line, and the pigment distance is measured to the centre of the spot. Because the pigment can never overtake the solvent carrying it, Rf is always between 0 and 1 and has no units.
Marking the solvent front the moment you lift the paper out matters — once the solvent evaporates there is nothing left to measure against.
What the Rf value tells you
A high Rf means the pigment is very soluble in the mobile phase, or a small molecule, so it travelled far.
A low Rf means it has a strong affinity for the stationary phase, or is a large molecule, so it lagged behind.
In a given solvent each pigment has a characteristic value, so Rf is used to identify spots.
Pigment
Typical Rf
Why
β carotene
Highest, close to 1
Very soluble in the solvent, travels almost to the front
Xanthophyll
High
Still travels a long way, but less than carotene
Chlorophyll a
Middle
Between the carotenoids and chlorophyll b
Chlorophyll b
Lowest
Held most strongly by the stationary phase
You do not need to memorise specific Rf numbers. What you do need is the order, and the reason for it — solubility in the mobile phase versus attraction to the stationary phase.
Worked examples
WORKED EXAMPLE
On a chromatogram the solvent travelled 10.0 cm from the pencil line. The centre of the orange spot is 9.5 cm from the pencil line and the centre of the yellow-green spot is 4.5 cm. Calculate both Rf values and identify the pigments.
Step 1: orange spot9.5 ÷ 10.0 = 0.95Step 2: yellow-green spot4.5 ÷ 10.0 = 0.45Step 3: match to the colours
Orange with the highest value is a carotenoid; yellow-green with the lowest is chlorophyll b.
0.95 for β carotene, 0.45 for chlorophyll bno units — the centimetres cancel out
WORKED EXAMPLE
A pigment has an Rf value of 0.65. On a chromatogram where the solvent travels 12.0 cm, calculate how far from the pencil line the spot will be.
Step 1: rearrange the formulapigment distance = Rf × solvent distanceStep 2: substitute0.65 × 12.0 = 7.87.8 cm from the pencil linesense check: 0.65 is a bit over half, and 7.8 cm is a bit over half of 12.0 cm
WORKED EXAMPLE
A student sets up a chromatogram but the solvent level is above the pencil line. Predict what they will see and explain why. [2]
Step 1: what happens to the spot
The spot is submerged, so the pigments dissolve straight into the solvent in the beaker.
Step 2: what appears on the paper
Very little is carried up the paper, so there is no clear separation into bands.
The pigments wash off into the solvent and no useful chromatogram formsthis is why “solvent below the line” is worth stating in any method answer
Limitations
Paper chromatography is less precise than TLC, though it is enough to separate and identify the main pigments.
It tells you which pigments are present, not how much of each there is.
It says nothing about the wavelengths each pigment absorbs. For that you need a colorimeter or a spectrometer.
Rf values depend on the solvent used, so values from different experiments are not directly comparable.
💡 Exam tip
Say pencil, and say why: ink would separate into its own components.
Say the solvent must be below the start line. It is an easy mark and often asked.
Give Rf to two decimal places and remember it has no units.
Measure to the centre of the spot — state this if a question asks how to improve accuracy.
Explain a high Rf in terms of solubility in the mobile phase and weak attraction to the stationary phase.
Distinguish the colour a pigment appears from the colours it absorbs. Examiners test this deliberately.
⚠ Common mix-up
Getting the Rf fraction upside down. Pigment on top, solvent underneath. An answer above 1 means you inverted it.
Adding units to Rf. It is a ratio of two lengths, so the units cancel.
Measuring from the bottom of the paper instead of from the pencil line.
Measuring to the top edge of a spot. Use the centre.
Assuming the pigment colour is the colour it absorbs. It is the colour it reflects.
Thinking chlorophyll is a single substance. There are at least two forms, plus the carotenoids alongside them.
Forgetting to mark the solvent front before the paper dries.
Up next: Absorption Spectra — now that the pigments are separated, we can ask which colours of light each one actually absorbs, and why leaves are green.
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