Any separation technique that distributes the components of a mixture between two phases, a fixed stationary phase and a free-moving mobile phase, is known as chromatography. There are many different chromatography formats but all depend on the same underlying principle. A mixture of molecules is dissolved in a solvent and fed into the chromatography process. As the mobile phase moves over the stationary phase, the components of the mixture can interact with the molecules of both the solvent and the stationary matrix. Different components in the mixture move at different rates because of their differing affinities for each phase. Molecules with the lowest affinity for the stationary phase will move the most quickly because they tend to remain in the solvent, while molecules with the highest affinity move the most slowly because they tend to stay associated with stationary phase and are left behind. This results in the mixture being partitioned into a series of fractions, which can be eluted and collected individually.

LC is used more often than other chromatography formats because of its versatility and compatibility with MS. Unlike 2DGE, LC is suitable for the separation of both proteins and peptides, and can therefore be applied either upstream of 2DGE to prefractionate the sample, downstream to separate the peptide mixtures from single excised spots, or instead of 2DGE itself.

In LC methods, the stationary phase is a porous matrix while the mobile phase is a solvent containing dissolved proteins or peptides. The rate of flow depends of the affinity with the stationary phase.

Affinity Chromatography

Based on the ability of proteins to bind to certain molecules

Affinity chromatography partitions proteins or peptides on the basis of their specific, ligand-binding affinity. The matrix on an affinity column contains ligands that are highly selective for particular proteins or classes of proteins. Thus there is a two-step elution process. [4]

Ion exchange chromatography

Based on the charge differences (due to surface residues)

It separates proteins or peptides according to their charge. It is based on the reversible adsorption of solute molecules to a solid phase that contains charged chemical groups. Cationic (+ve) or anionic (-ve) resins may be used and these attract molecules of opposite charge in the solvent. Instead of a 2-step elution procedure, gradient elution is achieved by washing the column with buffers of gradually increasing ionic strength or pH. [4]

Reversed-phase chromatography

Based on differences in hydrophobicity (due to nonpolar surface residues)

RP involves the reversible adsorption of proteins or peptides to the stationary phase matrix, and multiple fractions are produced by gradient elution. The proteins and peptides are separated according to their hydrophobicity, and the reversed-phase resin consists of hydrophobic ligands such as C14 to C18 alkyl groups. Gradient elution is achieved by gradually increasing the amount of an organic modifier in the elution buffer, which disrupts the weakest hydrophobic interactions first. Among the different chromatography techniques, RP-HPLC is the most powerful method and has the highest resolution. [4]

Size exclusion chromatography - Gel filtration chromatography

Based on size differences

Multidimensional LC

The flexibility of LC methods in terms of combining different separating principles makes multidimensional chromatography an attractive technology. Not all methods are compatible so care must be taken before coupling two methods.