Chromatofocusing

Douglas D Frey, University of Maryland Baltimore County, Maryland, USA
Chittoor R Narahari, University of Maryland Baltimore County, Maryland, USA
Ronald C Bates, University of Maryland Baltimore County, Maryland, USA

Published online: April 2001

DOI: 10.1038/npg.els.0002681

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 pH front.

Keywords: chromatofocusing; chromatography; preparative chromatography; pH gradient; protein purification; polyampholyte buffer

Figure 1. (a) pH profile and concentration profiles of proteins focused into bands in the effluent from a chromatofocusing column. (b) Corresponding relation between the apparent isoelectric points, the curves giving the velocities of protein bands as functions of the liquid-phase pH, and the curves giving the velocities of pH values as a function of those values inside the chromatofocusing column.
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 References
    Bates R and Frey DD (1998) Quasi-linear pH gradients for chromatofocusing using simple buffer mixtures: local equilibrium theory and experimental verification. Journal of Chromatography A 814: 43–54.
    Frey DD (1996) Local-equilibrium behavior of retained pH and ionic strength gradients in preparative chromatography. Biotechnology Progress 12: 65–72.
    Hearn MTW and Lyttle D (1981) Buffer-focusing chromatography using multicomponent electrolyte elution systems. Journal of Chromatography 218: 483–495.
    Hutchens TW, Li CM and Besch PK (1986) Performance evaluation of a focusing buffer developed for chromatofocusing on high-performance anion exchange columns. Journal of Chromatography 359: 169–179
    Liu Y and Anderson DJ (1997) Chromatofocusing high-performance liquid chromatography: 1. Practical aspects. Journal of Chromatography A 762: 207–217.
    Logan KA, Lagerlund I and Chamow SM (1999) A simple, two-component buffer enhances use of chromatofocusing for processing of therapeutic proteins. Biotechnology and Bioengineering 62(2): 208–215.
    Narahari CR, Strong JC and Frey DD (1998) Displacement chromatography of proteins using a self-sharpening pH front formed by adsorbed buffering species as the displacer. Journal of Chromatography A 825(2): 115–126.
    Sluyterman LA AE and Elgersma O (1978) Chromatofocusing: isoelectric focusing on ion-exchange columns: 1. General principles. Journal of Chromatography 150: 17–30.
    Sluyterman LA AE and Wijdeness J (1978) Chromatofocusing: isoelectric focusing on ion-exchange columns: 2. Experimental verification. Journal of Chromatography 150: 31–44.
    Strong JC and Frey DD (1997) Experimental and numerical studies of the chromatofocusing of dilute proteins using retained pH gradients formed on a strong-base anion-exchange column. Journal of Chromatography A 769: 129–143.
 Further Reading
    book Amersham Pharmacia Biotechnology (1987) Chromatofocusing with Polybuffer and PBE. Uppsala: Amersham.
    Giri L (1990) Chromatofocusing. Methods in Enzymology 182: 380–392.
    book Hutchens TW (1989) "Chromatofocusing". In: Janson J and Ryden L (eds) Protein Purification, chap. 5, pp. 149–174. New York: VCH.
    Li CM and Hutchens TW (1992) Chromatofocusing. Methods in Molecular Biology, 11: 237–248.

Related Sub-Topics:

Biochemical and Biophysical Techniques | Proteins: Structure, Function, Metabolism
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Article Title: Chromatofocusing
 
How to Cite close
Frey, Douglas D, Narahari, Chittoor R, and Bates, Ronald C(Apr 2001) Chromatofocusing. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002681]