Chromatography

Chromatography separates the substances in a mixture. A mobile phase (a liquid or gas) carries the mixture through a stationary phase (a solid or a liquid held still). Each substance moves at its own speed, depending on how strongly it is attracted to the stationary phase, so they come out at different times. The record of when each comes out, as a series of peaks, is a chromatogram. (You may have seen ink separate into colours on wet paper: the same idea.)

Types of chromatography:

• Adsorption: substances stick to the surface of a polar solid (silica gel, alumina, charcoal).
• Partition: substances divide themselves between two liquids that do not mix.
• Ion-exchange: charged substances bind to oppositely charged resins (with carboxymethyl or DEAE groups), and are released by changing the pH or the salt concentration.
• Gel filtration (size exclusion): porous beads separate molecules by size. Small molecules wander into the pores and take longer; large ones pass around the beads and come out first.
• Affinity: a molecule (ligand) fixed on the column holds on only to the molecules that bind it specifically, such as antibodies, enzymes or receptors. This gives very selective purification.

Two powerful modern forms:

• HPLC (high-performance liquid chromatography) packs the column with very fine particles for sharp separation. Liquid cannot flow through such fine packing on its own, so it is pumped at high pressure.
• Gas chromatography (GC) uses a gas as the mobile phase, for volatile compounds (those that turn easily into vapour), such as alcohols and ketones.