IB Biology HLPhotosynthesisPaper 1 & 2~11 min read
Separating Photosynthetic Pigments (Skills)
A leaf looks like it contains one green substance. Grind it up, run it up a strip of paper, and four or five coloured bands appear. Chromatography is how you turn “leaves are green” into actual evidence.
📚 What you need to know
Chromatography separates a mixture of pigments using a stationary phase (the paper or the thin layer) and a mobile phase (the solvent).
Pigments separate because they differ in solubility in the solvent and in how strongly they are attracted to the stationary phase.
A pigment that is more soluble and less attracted to the paper travels further.
The concentrated spot is applied to the origin, above the solvent level, and built up by repeated drying.
The solvent must not touch the origin, or the pigments dissolve into the solvent instead of running up the paper.
The solvent front is marked in pencil the moment the paper is removed, because the solvent evaporates.
Rf = distance moved by the pigment ÷ distance moved by the solvent front.
Rf is always between 0 and 1 and has no units. It is compared with database values to identify a pigment.
Typical order from the top: carotene, then xanthophyll, then chlorophyll a, then chlorophyll b.
How the separation works
Two things pull on each pigment molecule at once.
The solvent is moving up the paper by capillary action, and it drags dissolved pigment with it. The more soluble a pigment is, the more of it gets carried along.
The paper holds pigment molecules back. A pigment strongly attracted to the stationary phase spends more time stuck and travels less.
Every pigment has its own balance of those two forces, so every pigment travels a characteristic fraction of the way up. That fraction is the Rf value.
A tug of war is the right picture. The solvent pulls each molecule up; the paper pulls it back. Carotene is barely held by the paper and dissolves readily, so it almost keeps pace with the solvent front. Chlorophyll b clings hardest and is left near the bottom.
The method
🧩 Running a leaf chromatogram
Grind fresh leaf tissue with a small volume of solvent (such as propanone) using a pestle and mortar, to break the cells and release the pigments.
Draw the origin as a pencil line near the bottom of the paper. Pencil is insoluble, so it will not run.
Apply a drop of extract to the origin, let it dry, and repeat many times to build a small, concentrated spot.
Suspend the paper in a sealed vessel so the solvent is below the origin, never touching it.
Leave it until the solvent has almost reached the top, then remove the paper and immediately mark the solvent front in pencil.
Measure from the origin to the centre of each pigment band, and to the solvent front. Calculate Rf for each.
Bands are not always sharp lines. Measuring to the centre of a band, consistently for every pigment, matters more than the band being neat.
Calculating Rf
Retention factor
Rf = distance moved by the pigment ÷ distance moved by the solvent front
Because the pigment can never overtake the solvent carrying it, Rf is always between 0 and 1, and it has no units — it is a length divided by a length.
Pigment
Colour on the chromatogram
Distance moved / cm
Rf
Carotene
Orange-yellow
7.6
0.89
Xanthophyll
Pale yellow
5.9
0.69
Chlorophyll a
Blue-green
4.4
0.52
Chlorophyll b
Yellow-green
3.2
0.38
All four values above were calculated with a solvent front of 8.5 cm. Notice the order: the least soluble, most strongly held pigment sits lowest.
Rf only identifies a pigment if conditions match. Change the solvent, the paper or the temperature and the values shift. That is why you compare your results with database values obtained under the same conditions, and why the solvent used must always be recorded.
Worked examples
WE 1
Calculate an Rf value
On a chromatogram the solvent front moved 8.5 cm from the origin, and a green band moved 4.4 cm. Calculate the Rf value and suggest which pigment it is. (3 marks)
Step 1: the formula
Rf = distance moved by pigment ÷ distance moved by solvent front
Step 2: substituteRf = 4.4 ÷ 8.5 = 0.52Step 3: identify
A blue-green band with an Rf near 0.5 matches chlorophyll a, though this must be confirmed against database values for the same solvent.
Rₜ = 0.52, most likely chlorophyll ano units, and two significant figures is plenty
WE 2
Work backwards from Rf
A pigment has an Rf value of 0.89. If the solvent front travelled 9.0 cm, calculate how far the pigment travelled. (2 marks)
Step 1: rearrange
distance moved by pigment = Rf × distance moved by solvent front
Step 2: substitute0.89 × 9.0 = 8.0 cm8.0 cm from the originsense-check it: an Rₜ near 1 should give a distance close to the solvent front, and 8.0 out of 9.0 is
WE 3
Explain a failed chromatogram
A student’s chromatogram shows no separated bands, and the solvent in the vessel has turned green. Explain what went wrong. (3 marks)
Point 1: the error
The solvent level was above the origin, so the spot was submerged.
Point 2: what happened instead
The pigments dissolved directly into the solvent in the vessel rather than being carried up the paper.
Point 3: the consequence
No pigments travelled up the stationary phase, so no bands formed and no Rf values can be calculated.
The origin was below the solvent, so the pigments washed offa very faint chromatogram usually means too few drops applied to the origin instead
💡 Exam tips
Always name both phases: the paper is the stationary phase, the solvent is the mobile phase.
Explain separation using solubility and attraction to the stationary phase — both, not one.
Give a reason for pencil (insoluble) and for the sealed lid (stops the solvent evaporating).
Measure to the centre of a band and state that you did.
Rf has no units and cannot exceed 1. An answer above 1 means the distances were swapped.
Say results must be compared with values obtained under the same conditions.
⚠ Common mistakes
Dividing the wrong way round. Pigment distance goes on top.
Measuring from the bottom of the paper. All distances start at the origin.
Using pen for the origin line. The ink separates and ruins the chromatogram.
Forgetting to mark the solvent front immediately. Once it evaporates, the measurement is gone.
Giving Rf units of cm. It is a ratio.
Saying a pigment travelled far because it is lighter. It is about solubility and attraction, not mass.
Up next: Absorption Spectra. You have now proved a leaf holds several different pigments. The obvious question is why — and the answer is that each one catches a different part of the spectrum.
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