Chromatography has been used for more than a century to separate and purify proteins and other compounds. Traditionally, the compounds to be separated are dissolved in a fluid called the mobile phase, which is passed through a solid structure called the stationary phase, which typically consists of a porous bed of particles packed into a column. As the fluid flows through the column, different compounds in the mobile phase are partitioned into distinct bands based on their different rates of mobility along the column. These mobility rates vary according to differences in the compounds' affinities for the stationary phase, which are related to the physicochemical properties of the solutes.
Chromatography is a commonly used method found in the majority of laboratories performing protein isolation, purification, and production. Chromatography is typically one part of a larger laboratory workflow to separate a mixture of compounds for the collection of purified fractions. This is called preparative chromatography. Alternatively, the technique can be performed to identify unknown components in a mixture or to quantitate highly purified compounds — this is called analytical chromatography. Chromatography applications can be classified both by the type of chromatographic method used (such as ion exchange or affinity) and by the types of compounds being isolated and their characteristics such as antibodies or recombinant protein).
Biomolecule purification is a challenging and evolving technique. The range of molecular structures, properties, and quantities of both the valuable components and their contaminants make each separation distinct. In this section, we highlight several common chromatographic separations as examples of purification and quantification strategies. To learn more about different chromatography techniques, visit our Chromatography technology section.