The protein separation actually occurs on a chromatography column that contains a charged stationary phase. Oppositely charged proteins bind to the stationary phase and like charged or uncharged proteins wash through the column with the void volume. The mobile phase travels through the column, carrying unbound proteins with it. The bound proteins are then eluted from the column by increasing the salt concentration in the mobile phase.

The chromatography column is the heart of the separation process. For protein separations the column usually contains porous beads of a hydrophilic polymer, such as cellulose or some other type of carbohydrate polymer. The surface of the polymer beads is chemically modified to give it properties that would make it suitable for various types of chromatography: ion exchange, molecular exclusion, hydrophobic interaction or affinity. The appropriate stationary phase is suspended in the desired mobile phase and poured into the chromatography column. Once the stationary phase has been fully equilibrated with the mobile phase, the protein sample can be introduced onto the column. The separation then occurs based on the attraction between the protein, the stationary phase and the mobile phase. For example, a positively charged protein would bind to a negatively charged stationary phase when the mobile phase has a low ionic strength (see Salts). An increase in the salt concentration may displace the protein from the stationary phase when positive ions in the mobile phase compete with the protein for binding sites on the stationary phase.

A typical chromatographic separation of a four component mixture is shown under Chromatography.

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