Development of chromatofocusing techniques employing mixed-mode column packings for protein separations

• Demonstrated the chromatofocusing of proteins using mixed-mode column packings.
• Demonstrated the use of additives such as urea and neutral salts for manipulating the retention times of proteins in mixed-mode chromatofocusing.
• Demonstrated the use of numerical simulation methods for adjusting the pH gradient shape in mixed-mode chromatofocusing.
• Demonstrated the use of mixed-mode chromatofocusing for blood serum fractionation.

Recent studies reported in the literature using mixed-mode chromatography (MMC) column packings have shown that multiple modes of interactions between the column packing and proteins can be usefully exploited to yield excellent resolution as well as salt-tolerant adsorption of the target protein. In this study, a mixed-mode separation method using commercially available column packings was explored which combines the techniques of hydrophobic-interaction chromatography and chromatofocusing. Two different column packings, one based on mercapto-ethyl-pyridine (MEP) and the other based on hexylamine (HEA) were investigated with regard to their ability to separate proteins when using internally generated, retained pH gradients. The effects of added salt and urea on the behavior of the retained pH gradient and the protein separation achieved when using MMC column packings for chromatofocusing were also investigated. Numerical simulations using methods developed in previous work were shown to agree with experimental results when using reasonable physical parameters. These numerical simulations were also shown to be a useful qualitative method to select the compositions of the starting and elution buffers in order to achieve desired shapes for the pH and ionic strength gradients. The use of the method to fractionate blood serum was explored as a prototype example application.