Table 2–

Peptide purification methods.

Method	Principle	Uses
Reversed‐phase chromatography	Based on hydrophobicity. Consisting of a stationary phase of lower polarity and a mobile phase of higher polarity	Enables rapid detection and purification of a peptide sequence from a mixture
Ion exchange chromatography	The distribution and surface charge of the peptide determines the interaction of charged groups with the surface of the stationary phase	Used for purification of peptides and proteins
Exclusion liquid chromatography	Based on separation process according to the size of the peptide relative to pore sizes in the stationary phase. Used primarily in the early stages of purification of the peptide, when performed in multiple steps	Used to separate low‐molecular‐weight impurities from a mixture of peptides. However, the separation of the peptide of interest with other closely related peptides is virtually impossible
Affinity chromatography	Based on the biological specificity of the peptide. Consists of a ligand (small specific biomolecule such as an antibody) that is immobilized in the column. The separation occurs because of highly specific biochemical interactions between the peptide and the ligand	Used when a high degree of specificity is required, for example, isolation of a target protein present in low concentration in a biological fluid or a cell extract
Capillary electrophoresis	Based on the migration of the peptide according to its charge in solution, depending on the application of an electric field. Complementary technique to reversed‐phase chromatography	Used for peptides and proteins