Chromatography & Photosynthetic Pigments

Revise Chromatography & Photosynthetic Pigments for Biology 6 (IAL) WBI16 (A2 Level) — revision notes and instant AI marking.

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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

centre