The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column.

FPLC is generally applied only to proteins; however, because of the wide choice of resins and buffers it has broad applications.

[5] A typical laboratory FPLC consist of one or two high-precision pumps, a control unit, a column, a detection system and a fraction collector.

The majority of systems utilize two two-cylinder piston pumps, one for each buffer, combining the output of both in a mixing chamber.

Some simpler systems use a single peristaltic pump which draws both buffers from separate reservoirs through a proportioning valve and mixing chamber.

This permits the experimenter to identify when peaks in protein concentration occur, indicating that specific components of the mixture are being eluted.

Distribution of the eluate into separate containers are determined by fixed volumes or specific fractions detected at peaks of protein concentration.

The columns used in FPLC are large (inner diameters on the order of millimeters) tubes that contain small (micrometer-scale) particles or gel beads as the stationary phase.

[7] For easy use, a wide range of pre-packed columns for techniques such as ion exchange, gel filtration (size exclusion), hydrophobic interaction, and affinity chromatography are available.

The purpose of purifying proteins with FPLC is to deliver quantities of the target at sufficient purity in a biologically active state to suit its further use.

The quality of the end product varies depending the type and amount of starting material, efficiency of separation, and selectivity of the purification resin.

The ultimate goal of a given purification protocol is to deliver the required yield and purity of the target molecule in the quickest, cheapest, and safest way for acceptable results.

On the other hand, if the starting material is enough to develop more complete protocol, the amount of work to reach the separation goal depends on the available sample information and target molecule properties.

A typical multistep purification protocol starts with a preliminary capture step which often utilizes ion exchange chromatography (IEC).

Polishing is used to achieve the final level of purification required and is commonly performed on a gel filtration column.

This flexibility is imperative for a functional purification system as all proteins behave differently and often deviate from predictions.