Chromatofocusing is a form of gradient elution chromatography performed using an ion‐exchange column packing and an internally generated pH gradient that travels through the column as a retained front. The interaction between the pH gradient and amphoteric substances such as proteins causes these substances to exit the chromatographic column at characteristic locations in the effluent as focused bands. The method was first introduced in the 1970s. Over the last several decades, the performance and the range of applications of the method have been significantly increased through the efforts of numerous investigators.
Key Concepts
Proteins can be focused into narrow bands similar to those produced in isoelectric focusing using an ion‐exchange chromatography column and a pH gradient that moves through the column packing more slowly than the interparticle fluid.
The focusing effects produced in chromatofocusing yield unique dynamic behaviour for the species being separated which, if properly optimised, can produce high‐resolution separations.
Numerical simulation methods and analytical local‐equilibrium theories can be used to optimise the compositions of the elution and presaturation buffers used in chromatofocusing.
Chromatofocusing can be applied not only to proteins, but also to related types of amphoteric species including small peptides and macromolecular complexes such as viruses.
Chromatofocusing can usefully employ mixed‐mode column packings where both electrostatic and hydrophobic interactions determine the adsorption affinity of the species being separated.
