pH-gradient ion-exchange chromatography: An analytical tool for design and optimization of protein separations

• Published in: Journal of Chromatography A Vol. 1164; no. 1; pp. 181 - 188
• Main Authors: Ahamed, Tangir, Nfor, Beckley K., Verhaert, Peter D.E.M., van Dedem, Gijs W.K., van der Wielen, Luuk A.M., Eppink, Michel H.M., van de Sandt, Emile J.A.X., Ottens, Marcel
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 14.09.2007; Elsevier
• Subjects: Analytical chemistry; Anion Exchange Resins - chemistry; Anion-exchange chromatography; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, Ion Exchange - methods; E. coli lysate; Elution-pH; Exact sciences and technology; Other chromatographic methods; p I; pH-gradient; Proteins - chemistry; Proteins - isolation & purification; Proton-Motive Force; Reproducibility of Results; Titration curve; pH-gradient; E. coli lysate; Anion-exchange chromatography; p I; Elution-pH; Titration curve; Elution; pH gradient; Lysate; Escherichia coli; Optimization; Protein; Ion exchange chromatography; Separation method; Anion exchanger; pH; Bacteria; pI; Enterobacteriaceae
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2007.07.010

This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (p I < 6) and basic (p I > 8) proteins elute roughly at their p I, whereas neutral proteins (p I 6–8) elute at pH 8–9 regardless their p I values. Because of the flat nature of protein titration curves from pH ∼6 to ∼9, neutral proteins indeed exhibit nearly zero net charge at pH ∼9. The elution-pH in pH-gradient IEC or the titration curve, but not the p I, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.