Gradient and isocratic high-performance liquid chromatography of proteins on a new agarose-based anion exchanger

We describe a new, simple, and mild method for the preparation of anion-exchangers, based on the coupling of alkylamines to epoxy-activated agarose (prepared by the reaction of agarose with butanediol diglycidyl ether). Since a polar OH-group is formed when an epoxide reacts with an OH or NH2 group, the ion-exchanger did not show any hydrophobic interaction. This is important, since it may be impossible to desorb a protein from an ion exchanger having a hydrophobic character, because increasing the salt concentration of the eluent to decrease the electrostatic binding inevitably strengthens the hydrophobic interaction. By the method described, 3-diethylamino-2-hydroxy-propyl agarose (DEAHP-agarose) was prepared. High resolution of proteins was obtained by gradient elution at both high and low degrees of substitution. However, isocratic separations required a low degree of substitution, in accordance with a hypothesis previously put forward in connection with a theoretical and experimental study of the conditions for isocratic elution of macromolecules on amphiphilic gels. A study of the retention times of several proteins at different pH levels and buffer compositions indicated that different pH levels should be tested for maximal resolution and that, in many cases, the best resolution can be obtained if the DEAHP-agarose is operated in a buffer containing sodium acetate instead of sodium chloride. A quaternary amine agarose, 3-methyldiethylamino-2-hydroxy-propyl agarose (QAE-agarose), can be synthesized easily from DEAHP-agarose by alkylation with methyl iodide. The titration curves of DEAHP-agarose and QAE-agarose showed pK values around 9.5 and 11.3, respectively.