International A Level Biology / Photosynthesis

Separating leaf pigments by chromatography revision notes

Edexcel International A Level Biology (YBI11), chapter 14: Photosynthesis, specification point 5.7. Free lesson.

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Chromatography separates the pigments in a leaf so they can be identified. You identify them by calculating their Rf values and comparing these with known values measured under the same conditions.

How to run a leaf chromatogram

First, extract the pigments from the leaf. A solvent carries them up the paper, but different pigments travel different distances, so they form separate spots. Grind chopped leaf with sand and a little propanone to dissolve the pigments. Draw the origin line in pencil, 2 cm from the bottom. Ink would dissolve and run with the pigments. Spot extract onto the origin. Let it dry and repeat to build a small, dark spot. Stand the paper in solvent with the origin above the liquid, or the pigments will wash into the solvent. Put a lid on the container. Remove the paper before the solvent…

What the separated spots show

Each pigment travels a different distance, depending on how it moves through the paper and solvent. A typical order is: Carotene is orange-yellow and travels furthest, near the solvent front. Chlorophyll a is blue-green and sits lower. Chlorophyll b is yellow-green and sits lower still.

How to calculate an Rf value

An Rf value compares how far a pigment moves with how far the solvent front moves. It has no unit because it is one distance divided by another. It is between 0 and 1, since a pigment cannot move further than the solvent front. Measure both distances from the origin line, not from the bottom edge of the paper. Measure the pigment distance to the centre of its spot, because each spot has some width. Use the same unit for both distances.

How to identify pigments using Rf

Calculate an Rf value for each spot, then compare it with known Rf values. The comparison is valid only when the known pigments were run in the same solvent and on the same kind of paper or plate. A pigment's Rf changes if the solvent changes, because its solubility and how strongly it binds to the paper affect how far it travels. For a three-mark answer, state what distances you measure, how you calculate Rf, and what known values you compare it with.

How TLC compares with paper chromatography

Thin-layer chromatography (TLC) uses a thin layer of silica gel on glass, plastic or foil. Paper chromatography uses cellulose as its stationary phase, the material the pigments move through. TLC is usually faster and separates pigments better, so more pigments can be distinguished. Paper spots can spread out and overlap. TLC produces smaller, sharper spots that are further apart. Better-resolved spots are clearly separate, so each can be measured on its own. Calculate Rf in the same way for paper chromatography and TLC.