pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations |
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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 |
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Institution: | Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. t.ahamed@tudelft.nl |
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Abstract: | 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 (pI<6) and basic (pI>8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH approximately 6 to approximately 9, neutral proteins indeed exhibit nearly zero net charge at pH approximately 9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, 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. |
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