Revise Chromatography & Photosynthetic Pigments for Biology 6 (IAL) WBI16 (A2 Level) — revision notes and instant AI marking. Free to start.
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Edexcel IAL Biology • Unit 6: Practical Skills in Biology II
Chromatography & Photosynthetic Pigments
🌈 Big idea: A leaf looks green because chlorophyll dominates, not because it contains only one pigment. Chromatography separates them by exploiting a single difference: how strongly each pigment is held by the paper compared with how readily it dissolves in the solvent.
Summary — What This Topic Covers
How chromatography separates a mixture, in terms of solubility and attraction
Carrying out the technique — and the mistakes that ruin a run
Calculating and interpreting an Rf value
The pigments you should expect to see, and their order
Why Rf is comparable between labs but distance travelled is not
1. How the Separation Works
The principle
Two competing effects: how soluble a pigment is in the moving solvent, and how strongly it is attracted to the stationary paper. A pigment that is highly soluble and weakly held travels far; one that is poorly soluble and strongly held stays near the origin.
stationary phase the chromatography paper (or the silica of a TLC plate)
mobile phase the solvent, moving up by capillary action
travels FAR → very soluble in the solvent, weakly attracted to paper
travels LITTLE → poorly soluble, strongly attracted to paper
Analogy
A crowd leaving a stadium along a corridor lined with people handing out leaflets. Those who stop for every leaflet barely move; those who ignore them reach the exit first. Same corridor, same time, different distances.
2. The Technique
1 grind leaf with a little propanone and sand to break the cells
2 draw the ORIGIN LINE IN PENCIL, above the solvent level
3 spot the extract, dry, and RESPOT many times on the same spot
4 suspend the paper so the origin sits ABOVE the solvent
5 cover the vessel; run until the solvent front nears the top
6 MARK THE SOLVENT FRONT IMMEDIATELY before it evaporates
Pencil, never pen — ink is itself a mixture of dyes and will run
Respot repeatedly, drying between, to concentrate the pigment without making the spot wide
Keep the spot small — a broad spot gives broad, overlapping bands
The origin must stay above the solvent, or the pigments dissolve off the paper into the reservoir
Cover the vessel so the atmosphere is saturated and the solvent does not evaporate from the paper as it rises
Mark the solvent front at once — it is invisible within a minute
The two that ruin a run
Origin below the solvent line, and forgetting to mark the solvent front. Neither can be fixed afterwards; the chromatogram is simply unusable.
3. Rf Values
The formula
Rf = distance travelled by the pigment ÷ distance travelled by the solvent front
Both measured from the origin line, to the centre of the spot.
solvent front travelled 8.0 cm from the origin
centre of the yellow spot 6.4 cm from the origin
Rf = 6.4 ÷ 8.0 = 0.80
Rf has NO UNITS — it is a ratio of two lengths
Rf is always between 0 and 1
Why use Rf at all
The distance a pigment travels depends on how long the run lasted and how far the solvent got. The ratio is constant for a given pigment, solvent and paper — so Rf can be compared between different runs and different laboratories, while raw distance cannot.
What mark schemes look for
Measure to the centre of the spot, and state that Rf has no units. Both are commonly dropped and both are marks.
4. The Pigments You Should See
PIGMENT COLOUR TYPICAL Rf POSITION
─────────────────────────────────────────────────────────────────
carotene orange-yellow ~ 0.95 highest
xanthophyll yellow ~ 0.70
chlorophyll a blue-green ~ 0.45
chlorophyll b yellow-green ~ 0.35 lowest
(values vary with solvent — the ORDER is what to remember)
Carotene is a hydrocarbon with no polar groups, so it is very soluble in a non-polar solvent and barely attracted to the paper — hence the highest Rf. Chlorophyll b carries more polar groups than chlorophyll a, is held more strongly by the paper, and travels least.
Easy marks
If asked to explain why carotene has the highest Rf, the answer is about polarity: least polar, most soluble in a non-polar solvent, weakest attraction to the paper.
Practice Questions
Practice Question 1
Explain why the origin line must be drawn in pencil and must sit above the level of the solvent.
Practice Question 2
A pigment spot travels 5.6 cm while the solvent front travels 8.0 cm. Calculate the Rf and state one reason Rf is preferred to the distance travelled.
Practice Question 3
Explain why carotene has a higher Rf value than chlorophyll b.
Practice Question 4
A student's chromatogram shows broad, overlapping bands. Suggest two causes and how to avoid them.
What to Memorise
Stationary = paper, mobile = solventFar = soluble + weakly heldPencil origin, above the solventRespot to concentrate, keep it smallCover the vesselMark the solvent front immediatelyRf = pigment ÷ solvent frontMeasure to the CENTRE of the spotRf has no units, 0 to 1Carotene highest, chlorophyll b lowest
Concepts Checklist
Exam Tips
What mark schemes look for
Measure to the centre of the spot; state Rf has no units; give the value to two decimal places. Three easily dropped marks.
The trap
Origin line below the solvent. The pigments dissolve into the reservoir and the run is worthless — a favourite examiner scenario.
Easy marks
Pencil rather than pen, with the reason: ink is a mixture of dyes and would separate too.
Worth remembering
Rf order is fixed even though the values shift with solvent: carotene, xanthophyll, chlorophyll a, chlorophyll b, top to bottom.