Separating Photosynthetic Pigments by Chromatography
Chromatography separates a leaf's pigments because each one dissolves differently in the solvent and sticks differently to the paper, so they travel different distances and form separate bands.
A green leaf looks like it has one color, but it actually holds a mix of pigments: chlorophyll a, chlorophyll b, carotenoids, and xanthophylls. Crush the leaf and spot the extract near the bottom of a strip of chromatography paper, then stand the paper in a shallow pool of solvent. The solvent climbs the paper by capillary action, and as it passes over the pigment spot, it carries each pigment along with it. Pigments that are more soluble in the solvent and cling less to the paper fibers get carried further; pigments that are less soluble or bind more tightly to the paper lag behind. After a few minutes you see four bands stretched out: yellow-orange carotenes furthest up, then xanthophylls, then blue-green chlorophyll a, then yellow-green chlorophyll b closer to the start.
To compare runs done on different papers or for different times, biologists use the Rf value: distance traveled by the pigment divided by distance traveled by the solvent front, measured from the same starting spot. Rf is always less than 1, since no pigment can outrun the solvent that is dragging it. A pigment that moved 4.2 cm while the solvent front moved 7.0 cm has an Rf of 4.2/7.0 = 0.60, a number you could reproduce in any lab with the same paper and solvent.
This investigation sits among the required labs because it is a clean example of separating mixtures by a physical property and turning a measurement into a ratio, the same science-practice skill tested with gel electrophoresis distances or enzyme-rate graphs elsewhere in the unit.
Key terms
- Rf value
- The ratio of the distance a pigment travels to the distance the solvent front travels, always less than 1.
- Solvent front
- The leading edge of the solvent as it moves up the chromatography paper.
- Capillary action
- The tendency of a liquid to move through narrow spaces, like paper fibers, without outside force.
- Pigment
- A colored compound, such as chlorophyll a or a carotenoid, that absorbs specific wavelengths of light.
Watch it explained
- How it worksHow does solubility in the solvent decide how far each pigment travels?
- How it worksWalk me through the solvent front climbing the paper and dragging pigments unevenly.
- Worked exampleFind the Rf value of a band that moved 4.2 cm when the solvent front moved 7.0 cm.
- Common mistakeWhy isn't the pigment that travels furthest the one that absorbs the most light?
Check yourself
End of topic.