Comparative analysis of trilobal capillary‐channeled polymer fiber columns with superficially porous and monolithic phases toward reversed‐phase protein separations

Abstract

A trilobal capillary‐channeled polymer fiber stationary phase is evaluated for its performance for intact protein separations under reversed‐phase high‐performance liquid chromatography conditions. The separation quality, operational characteristics, and protein dynamic loading capacity on the fiber phases are compared to commercially‐available superficially porous and monolithic columns. The trilobal or “y‐shaped” polypropylene fiber phase was employed to separate a synthetic mixture of five proteins (having diverse chemistries and molecular weights). The separation quality was evaluated based on the resolution, peak heights/recoveries, peak widths, and peak areas. The present work illustrates the unique ability to operate at higher linear velocities (47.5 mm/s) while maintaining lower back pressures (∼4 MPa), faster separation times (<8 min), and faster gradient rates using the fiber columns while yielding comparable chromatographic performance to the commercial columns. The separations employing the commercial stationary phases operate at lower linear velocities (∼3.0 mm/s), higher back pressures (∼9 MPa), require longer separation times (10 min), and require slightly higher compositions of organic mobile phase to effect protein elution. Likewise, based on breakthrough loading analysis of lysozyme and bovine serum albumin, the trilobal, polypropylene C‐CP fiber column stationary phases demonstrate 3–9X greater binding capacities on a bed volume basis versus the commercial columns.

Rapid metal speciation of cell culture media using reversed‐phase separations and inductively coupled plasma optical emission spectrometry

“…PPY fibers sufficient to meet target interstitial fraction values (equating to ~630 fibers) were pulled through 30 cm of polyether ether ketone (PEEK) tubing (0.78 mm diameter) (Cole‐Palmer, Vernon Hills, IL, USA) and washed with DI‐H 2 O, ACN, and DI‐H 2 O to remove any anti‐static coatings on the fiber stationary phase. The column packing and separation conditions were optimized and described previously 35–37 …”

“…To differentiate the speciation states (free vs. ligated) of the metals in the various media, trilobal polypropylene (PPY) capillary-channeled polymer (C-CP) fibers were employed as the stationary phase during the reversed phase solid phase extraction process, with packing and separation conditions previously optimized. [35][36][37] C-CP fiber columns allow for rapid processing in a format the costs <$5 per column, which can be used over tens of cycles. This effort builds substantially on the initial demonstration of using the fiber phase to isolate free and ligate metals for a singular medium.…”

“…In addition to quantification, the speciation of the metals in CCM is an important parameter to access the bioavailability of the metals in the media. To differentiate the speciation states (free vs. ligated) of the metals in the various media, trilobal polypropylene (PPY) capillary‐channeled polymer (C‐CP) fibers were employed as the stationary phase during the reversed phase solid phase extraction process, with packing and separation conditions previously optimized 35–37 . C‐CP fiber columns allow for rapid processing in a format the costs <$5 per column, which can be used over tens of cycles.…”