We have performed experiments and derived mathematical models for packed bed columns used for liquid phase chromatographic separations of proteins with impulse input of feed solutions. These models can now be used to describe the relationships between the elution characteristics (peak height, peak position, and shapes) and the operating conditions (flow rate and buffer conditions) of ion exchange and gel permeation column chromatography for protein separations.
The surface adsorption model was discussed relative to the nature of the mobile and stationary phases in ion exchange column chromatography for two distinct cases: with and without pore diffusion. For large solute molecules, such as proteins and enzymes, the surface adsorption model without pore diffusion is adequate for prediction of elution profiles from ion exchange columns. This model is shown to be sufficient, since the solute molecules cannot readily diffuse into the solid matrix of column packings. For smaller solute molecules, such as amino acids and peptides, one must consider both the pore diffusion in the solid matrix and the axial dispersion in the mobile phase. A separate gel permeation model for chromatography was developed to focus on the diffusion of solute molecules involving no adsorption on solid phase.
The retention times of the large solute molecules are less than that of smaller molecules because of the lower probability for diffusion into the solid matrix of column packings.Thus, the application of a specific model depends on the origin of packing materials in the chromatography column, the size of the solute molecules, and the interactions between the solid and mobile phases. Effects of model parameters (column length, cross sectional area, flow rate, effective contact area, void fraction, particle size, axial dispersion, mass transfer coefficient, equilibrium constant, and pore diffusivity) on the calculated elution profiles are discussed based on the "series mass transfer mechanism". These effects are incorporated to describe the transport behaviors of solute molecules between the solid and liquid phases.
The model protein system of hemoglobin and an albumin mixture was experimentally separated by cycling the change of pH in ion exchange column chromatography experiments, in order to study the transport relationship between the protein elution profile and transient pH wave. A pH phase lag within the column is needed to define for the pH cyclic zone operation in order to verify the elution characteristics between the experimental and predicted elution profiles. The success of our cycling techniques and models is further shown on the real protein system where we purified alkaline phosphatase from human placenta on an ion exchange packed bed with cycling of the buffer concentration. The optimal protein separation technique resulted in a high recovery and high purity product for this real protein enzyme system. The concentration phase lag and iso-ionic points are defined and combined with the relationships between the buffer concentrations and model parameters in order to predict the elution characteristics. The calculated and the experimental profiles are shown to be in good agreement when using the surface adsorption model without pore diffusion.
The derived models can also be applied to determine the Number of Theoretical Plates (N) and Height Equivalent to Theoretical Plates (HETP) from the calculated profiles (peak height, peak width, retention time, and retention volume). The model parameters can be obtained from the limited experimental data for the desired operating conditions (mobile phase composition, flow rate, and column dimensions) in order to evaluate the column efficiency and optimization of column operation.
|
![[NJIT logo]](http://archives.njit.edu/vhlib/images/njit_logo.gif){kind=logo}
![[Van Houten Library logo]](images/vhl-top-image.jpg)
