This thesis studies the ionic strength dependence of protein retention in ion-exchange chromatography (IEC). It discusses some aspects of the use and applicability of retention models in describing the interaction between a protein and the highly charged stationary phase. The two models that are used in the four papers of this thesis are based on the Poisson-Boltzmann equation that describes the distance dependent interaction energy due to the double layer outside a charged surface. The used models have different degrees of simplification in the assumptions regarding the physical properties of the system.
Two of the papers use a linear model based on the assumption of two planar surfaces and a linearised potential (the slab model). The non-linear model used in the two remaining papers studies the interaction between a spherical protein of defined size and a planar stationary phase (the sphere model). The latter model does not require a linearised electrostatic potential. In the models, it is assumed that the proteins have an evenly distributed surface charge. Thus, the net charge of the protein can be obtained from an evaluation of the retention data. Experiments performed with proteins in IEC often require gradient elution.
Consequently, the two models have been extended to gradient elution conditions. The results are discussed in terms of how well the models describe the ionic strength dependence of the interaction. Further, an evaluation of the parameters obtained by fitting the model to the experimental data is made.
Conclusively, this thesis demonstrates the impact of the protein charge distribution on protein retention in IEC through evaluating a set of very similar, well-defined proteins. It shows that the slab model can be applied in gradient elution mode which enables accurate retention prediction in gradient elution mode basedon gradient experiments. By using this procedure determination of isocratic parameters from gradient experiment is also possible.
The determination of the isocratic parameters is difficult and requires a model with closer fit to the experimental data, the sphere model. Thereby, it was possible to accurately predict the retention time for human albumin at isocratic elution form gradient data. By using the sphere model, it was shown that the elution behaviour for late eluting proteins are the same in both elution modes whereas it differs for early eluting proteins.
Stockholm: Stockholm University , 1999. , 38 p.