Evaluation of protein separations based on hydrophobic interaction chromatography using polyethylene terephthalate...

Poly(ethylene terephthalate) (polyester, PET) capillary-channeled polymer fibers are used as the stationary phase for the separation of a synthetic protein mixtures composed of cytochrome c, myoglobin, ribonuclease A, lysozyme, α-chymotrypsinogen A, α-chymotrypsin and holo-transferrin. Polyester is a useful phase for HIC, having a mixed alkyl and aromatic backbone to yield mildly hydrophobic properties, an aromatic functionality allowing for π-π interactions, and carboxylate end groups adding hydrophilicity. In addition, the lack of extraneous hydrophobic ligands or charged surface species alleviates secondary interactions between proteins and common support phases. Breakthrough experiments with lysozyme solution were operated at various loading flow rates (0.1-0.5 mL min^-1) and protein concentrations (0.025-1.0 mg mL^-1). Dynamic binding capacity (DBC) was plotted and results fit to two isotherm models: Langmuir and Freundlich. Column stability and reproducibility were tested via 10 continuous lysozyme load/elution cycles. Organic solvents, acetonitrile and isopropanol, were tested with proportions ranging from 0 to 40% in the elution buffer, aiding protein elution, with the best separation efficiency employing ∼15-20% organic modifier. Resolution was evaluated at solvent pH values between 5 and 7.5, flow rates ranging from 0.2 to 0.6 mL min^-18^-1) and at gradient slopes between 5 and 16.7% B min^-1. Optimum resolution and fast analysis times were achieved employing a steep gradient using high linear velocities. Finally, a comparison of separation efficiency between a commercial HIC phase (TSKgel Phenyl-5 P W) and the PET fiber column is presented for a standard, six protein mixture, using optimal operation conditions for each column type.