Chromatofocusing

Abstract

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.

Keywords: bioseparations; bioprocessing; chromatofocusing; ion‐exchange chromatography; protein chromatography

Figure 1. (a) Method for forming an externally generated pH gradient where the starting buffer A and elution buffer B are combined in a certain ratio that varies with time in a proportionating valve PV. This mixture then passes through pump P and then into column C. As shown by the uniform purple colour in the column, the spatial composition variation in the column tends to be small. (b) Method for forming an internally generated pH gradient where the column is first uniformly equilibrated with starting buffer A. Elution buffer B is then introduced into column C as a step change at the column entrance using pump P. A sequence of compositions spanning the range between buffers A and B and travelling in a retained manner with respect to the fluid velocity are then formed inside the column, as illustrated by the sequence of purple shades travelling in the column. For the two cases a similar effluent pH time history may be observed at the column outlet despite the different spatial composition gradients inside the column.
Figure 2. (a) pH and concentration profiles of proteins focused into bands in the column effluent during chromatofocusing. (b) Corresponding relation between the apparent isoelectric points (i.e. the pH values at which proteins exit the column), the protein band velocity curves (i.e. the functional relation between pH and the velocity of the protein band in the column) and the pH gradient (i.e. functional relation between pH and the velocity of that pH on the gradient).
Figure 3. Chromatofocusing of purified monoclonal antibody illustrating the separation of different antibody charge variants using a cation‐exchange column and simple buffer mixtures. The pH gradient used was generated inside the column and had a composite shape consisting of a shallow upstream portion for achieving a high‐resolution separation in a narrow pH range, and a steep downstream portion for desorbing late eluting variants. The separated variants exhibited apparent isoelectric points of 6.45, 6.50, 6.55 (major peak), 6.62 and 6.7. Data from Kang et al. (2013) © Elsevier.
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Further Reading

GE Healthcare Bio‐Sciences (2004) Ion Exchange Chromatography and Chromatofocusing: Principles and Methods. Piscataway, NJ: GE Healthcare Bio‐Sciences.

Giri L (1990) Chromatofocusing. Methods in Enzymology 182: 380–392.

Li CM and Hutchens TW (1992) Chromatofocusing. Methods in Molecular Biology 11: 237–248.

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How to Cite close
El‐Sayed, Mayyada MH, and Frey, Douglas D(May 2015) Chromatofocusing. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002681.pub2]