Chromatography

Absorption chromatography (the original type of chromatography) depends on physical forces such as dipole attraction to hold the molecules onto the surface of the solid packing. In gas chromatography and HPLC, however, the solubility of the mixture's molecules in the stationary phase coating determines which ones progress through the column more slowly. Polarity can have an influence here as well. In gel filtration (also called size-exclusion or gel permeation) chromatography, the relative sizes of the molecules in the mixture determine which ones exit the column first. Large molecules flow right through; smaller ones are slowed down because they spend time trapped in the pores of the gel. Ion exchange chromatography depends on the relative strength with which ions are held to an ionic resin. Ions that are less strongly attached to the resin are displaced by more strongly attached ions. Hence the name ion exchange: one kind of ion is exchanged for another. This is the same principle upon which home water softeners operate. Affinity chromatography uses a stationary phase composed of materials that have been chemically altered. In this type of chromatography, the stationary phase is attached to a compound with a specific affinity for the desired molecules in the mobile phase. This process is similar to that of ion exchange chromatography, and is used mainly for the recovery of biological compounds. Hydrophobic Interaction Chromatography is used for amino acids that do not carry a positive or negative charge. In this type of chromatography, the hydrophobic amino acids are attracted to the solid phase, which is composed of materials containing hydrophobic groups.

Chemists choose the mobile and stationary phases carefully because it is the relative interaction of the mixture's compounds with those two phases that determines how efficient the separation can be. If the compounds have no attraction for the stationary phase at all, they will flow right through the column without separating. If the compounds are too strongly attracted to the stationary phase, they may stick permanently inside the column.