Column chromatography separates a mixture by repeatedly partitioning each compound between a moving mobile phase (the solvent, or "eluent") and a stationary solid packed into a glass column. Compounds that prefer the stationary phase lag behind; compounds that prefer the mobile phase race ahead — so a single mixed sample loaded at the top emerges as separate bands at the bottom, at different times.
This scene uses four classic plant pigments — β-carotene, chlorophyll a, chlorophyll b and a xanthophyll — the compounds separated in the textbook "leaf pigment" column chromatography experiment.
Mikhail Tswett invented column chromatography around 1900 while separating plant pigments — and even coined the name "chromatography" (Greek for "colour writing") because the technique literally painted the separated compounds as coloured bands down the column.
A mixed sample of plant pigments loaded onto a packed glass column separates into distinct coloured bands that travel down the column at different speeds, based on each compound's partition coefficient between the mobile solvent and the stationary packing.
Each pigment band's speed depends on how strongly it favours the stationary phase versus the moving solvent. Compounds that resemble the stationary packing lag behind; compounds that resemble the solvent race ahead and elute first.
Adjust solvent polarity and flow rate, and switch between polar silica and nonpolar C18 packing. Watch the four bands separate and drip into collection tubes at the bottom in a new order.
Mikhail Tswett invented column chromatography around 1900 while separating plant pigments — and coined the very word "chromatography," Greek for "colour writing."