Comparison of the separation of proteins of wide-ranging molecular weight via trilobal polypropylene capillary-channeled polymer fiber, commercial superficiously porous, and commercial size exclusion columns.

Reversed phase and size-exclusion chromatography methods are commonly used for protein separations, although they are based on distinctly different principles. Reversed phase methods yield hydrophobicity-based (loosely-termed) separation of proteins on porous supports, but tend to be limited to proteins with modest molecular weights based on mass transfer limitations. Alternatively, size-exclusion provides complementary benefits in the separation of higher mass proteins based on entropic, not enthalpic, processes, but tend to yield limited peak capacities. In this study, microbore columns packed with a novel trilobal polypropylene capillary-channeled polymer fiber were used in a reversed phase modality for the separation of polypeptides and proteins of molecular weights ranging from 1.4 to 660 kDa. Chromatographic parameters including gradient times, flow rates, and trifluoroacetic acid concentrations in the mobile phase were optimized to maximize resolution and throughput. Following optimization, the performance of the trilobal fiber column was compared to two commercial-sourced columns, a superficially porous C4-derivatized silica and size exclusion, both of which are sold specifically for protein separations and operated according to the manufacturer-specified conditions. In comparison to the commercial columns, the fiber-based column yielded better separation performance across the entirety of the suite, at much lower cost and shorter separation times.

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.

Separation of water-soluble polymers using capillary-channeled polymer fiber stationary phases.

Chromatographic separations of synthetic and natural polymers are usually affected by a size exclusion chromatography (SEC) mechanism. Although SEC is a proven method of separation based on hydrodynamic size, a chromatographic method based solely on chemical interactions would present certain advantages. This laboratory has been investigating the use of capillary-channeled polymer (C-CP) fibers as stationary phases in HPLC for the separation of biomacromolecules. C-CP fibers allow highly efficient fluid transport and an amorphous surface structure, minimizing mass transfer effects commonly associated with porous, packed-bed technologies. Choice of the base fiber identity allows flexibility in the potential types of solute-surface interactions. Two water-soluble polymers, glycolic acid ethoxylate 4-nonylphenyl ether, and poly(4-vinylpyridine hydrochloride), were used as test solutes because of their similarities to polymers of interest in the consumer products industry. SEC separation of this pair was not possible due to the similarities in hydrodynamic size. Poly(ethylene terephthalate), polyester and nylon-6 C-CP fibers were evaluated as stationary phase materials. The former was found to offer superior chromatographic separations and recoveries when operating under what would be considered to be typical RP separation conditions: a flow rate of 1 mL/min and gradient of 0-100% H(2)O/ACN with 0.06% TFA over 5 min.