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, enabling the separation of the different compounds
When the purpose of this separation is the collection of purified fractions for downstream applications, this is referred to as preparative chromatography. Preparative chromatography is typically one part of a more extensive laboratory workflow designed to separate a mixture of compounds, for example, preparative protein purification.
The goals of preparative protein purification are as diverse as isolating enzymes for biochemical characterization or structure determination, process-level purifications of enzymes for industrial applications, and purification of therapeutic molecules for clinical applications.
Alternatively, chromatography can be performed to identify unknown components in a mixture or to quantitate highly purified compounds — this is called analytical chromatography. Like preparative chromatography, analytical chromatography has a wide range of applications, from metabolic profiling to environmental testing.
Chromatography for protein purification can be classified by the type of chromatographic method used (such as ion exchange or affinity) and by the types of compounds being isolated and their characteristics.
Protein 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.
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