Dynamic evaluation of a trilobal capillary-channeled polymer fiber shape for reversed phase protein separations and comparison to the eight-channeled form.

A new, trilobal-shaped capillary-channeled polymer fiber is under development to address the issues of poor A-term performance of the previous eight-channeled form. The trilobal geometry should provide better packing homogeneity due to the fewer potential orientations of the symmetric fiber geometry. Comparisons of separation efficiency and peak shape were made between the two fiber shapes through several dynamic parameters. Column hydrodynamics were investigated with two marker compounds, uracil and bovine serum albumin, with van Deemter plots of those two compounds revealing differences in the packing qualities between the different fiber shapes. Parametric fitting to the van Deemter, Knox, and Giddings equations provides insights into the column physical structures. Separation quality for both shapes was evaluated across differences in fiber packing density, gradient rate, and mobile phase linear velocity for the reversed phase separation of a four protein mixture, containing ribonuclease A, cytochrome c, lysozyme, and myoglobin. The results of this study lay the ground work for future efforts in the use of trilobal fibers for the separation of biomacromolecules.

Preliminary assessment of the modification of polystyrene-divinylbenzene resin with lipid-tethered ligands for selective separations.

Functionalized lipid tethered ligands use physical adsorption to anchor reactive head groups to hydrophobic supports. We previously demonstrated the use of these species adsorbed onto polypropylene capillary-channeled polymer fibers. The general use of lipid tethered ligands on other hydrophobic chromatographic supports is demonstrated here for polystyrene-divinylbenzene. Evaluation of ligand adsorption conditions was performed using a fluorescein isocyanate head group to quantify the extent of loading by UV-Vis absorbance and by fluorescence microscopy. Selective protein capture was demonstrated by the detection of Texas Red labeled streptavidin (using fluorescence microscopy imaging, with quantification assessed through the depletion of solution-phase protein using UV-Vis absorbance. A second demonstration of the coupling involved an iminodiacetic acid head group lipid tethered ligand to capture the cationic dye, methylene blue. Two common means of alleviating nonspecific binding, adsorption in detergent media and use of a bovine serum albumin block, were evaluated. The first was found to cause release of the ligands, while the second was nominally effective. Indeed, the lipid tethered ligands itself may be most effective at impeding nonspecific binding. While further optimization and chromatographic evaluation is required, the general viability of this ligand immobilization method onto this common polymer support is demonstrated.