Ion exchange chromatography of amino acids: Cation exchange equilibria and dynamics of stepwise elution with flow reversal

Seung Un Kim, Purdue University

Abstract

Fundamental understanding of ion exchange of biochemicals is essential for developing better bioseparation techniques. Ion exchange of biochemicals differs from that of inorganic small ions because biochemicals can bind to the chromatographic materials simultaneously in several different ways. Amino acids are simple biomolecules which allow one to study the effect of heterogeneous binding behavior on the separation. In the first part of this work, equilibrium ion exchange behavior of amino acids has been examined. Batch equilibrium data were obtained and analyzed with a model based on mass action law. Fourier Transform Infrared Spectroscopy(FTIR) was also used to probe the interaction between basic amino acids and resin. The batch equilibrium study shows that the average valence of basic amino acids can be a fractional number due to their heterogeneous charge structures and multiple equilibria. The average valence and the average separation factor of basic amino acids can change with pH, salt concentration, and composition. These results have been confirmed by direct FTIR data, and the number of interaction sites of basic amino acid with resin functional group was directly measured by FTIR for the first time. In the second part of this work, peak compression (increase of peak concentration and reduction of peak tailing) due to a step pH change or flow reversal has been studied. Linear and nonlinear local equilibrium models and a detailed rate model have been used to analyze the mechanisms of peak compression. The models have been tested with L-lysine data in a cation exchange system. Peak compression, which is significant when a large difference in affinity is induced for a large dilute pulse, can achieve high resolution using relatively large sorbent particles. It is shown experimentally that a dilute lysine pulse can be compressed to about 35 times its feed concentration. For multicomponent separations, compression prior to separation can give a shorter cycle time and higher product purity than linear gradient elution. Flow reversal coupled with selectivity reversal in stepwise elution allows separation to occur during both forward and reversed elution, resulting in increased column utilization. In general, optimized stepwise elution with flow reversal can give better separation than linear gradient elution. The results of these studies can give insight to ion exchange of biochemicals. The results are also useful in optimizing column design and increasing column efficiency in downstream bioprocessing.

Degree

Ph.D.

Advisors

Wang, Purdue University.

Subject Area

Chemical engineering

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