Microbore polypropylene capillary channeled polymer (C-CP) fiber columns for rapid reversed-phase HPLC of proteins

Abstract

The performance of microbore columns with polypropylene (PP) capillary-channeled polymer (C-CP) fibers as the support/stationary phase for separation of macromolecules has been investigated. Polypropylene C-CP fibers (40 μm diameter) were packed in fluorinated ethylene propylene (FEP) tubing of inner diameter 0.8 mm and lengths of 40, 60, 80, and 110 cm. The performance of PP fiber packed microbore columns (peak width, peak capacity, and resolution) was evaluated for separation of a three-protein mixture of ribonuclease A, cytochrome c, and transferrin under reversed-phase gradient conditions. The low backpressure characteristics of C-CP fiber columns enable operation at high linear velocities (up to 75 mm s(-1) at 1.5 mL min(-1)). In contrast with the performance of other phases, such velocities enable enhanced resolution of the three-protein mixture, because peak widths decrease with velocity. Increased column length resulted in increased resolution, because the peak widths remained essentially constant, although retention times increased. In addition, it was found that the peak capacity increased with column length and linear velocity. Radial compression of the microbore tubing enhanced the homogeneity of the packing and, thereby, separation efficiency and resolution. Radial compression of columns resulted in a decrease in the interstitial fraction (~5%), but increased resolution of ~14% between ribonuclease A and cytochrome c. Even so, a linear velocity of 75 mm s(-1) required a backpressure of 9.5 MPa only. It is clear that the fluid and solute-transport properties of the C-CP fiber microbore columns afford far better performance than is obtainable by use of standard format columns. The ability to achieve high separation efficiencies, rapidly and with low volume flow rates, holds promise for high-capacity protein separations in proteomics applications.

Roles of interstitial fraction and load conditions on the dynamic binding capacity of proteins on capillary‐channeled polymer fiber columns

“…In the case of the C-CP fiber separation of proteins, evidence for the first phenomenon has been seen through the need for lesser elution solvent strengths as the separation linear velocities increase. 36,74 The increase in the calculated DBC over the EBC for the C-CP fiber columns is thus interpreted as improved mass transport and higher degrees of surface ordering vs. the static case. For this reason as well, a much broader "linear" range of the adsorption isotherm is observed.…”

“…As a cumulative result, C-CP fiber columns can be used for analytical scale protein separations at extremely high linear velocities (U o 100 mm s 21 ), and low backpressures (<2,000 psi). 35,36 Conversely, small molecule separations are more favorable performed a low linear velocities. Interestingly, for small molecules, there is a trade-off between achieving longer retention times at high fiber packing densities (low interstitial fractions) and more narrow peaks at higher interstitial fractions.…”

Polyethylenimine modified poly(ethylene terephthalate) capillary channeled-polymer fibers for anion exchange chromatography of proteins

“…This high permeability, coupled with very efficient solute mass transport from the mobile-to-stationary phases permits high velocity analytical protein separations and high throughput downstream processing [7,9,10]. These advantages could certainly be compromised in the course of any on-column surface modification.…”

“…The C-CP fiber microbore columns were prepared following previously reported methods [10,36]. PET C-CP fibers were obtained from the Clemson University School of Materials Science and Engineering.…”

“…This allows for the direct loading of complex and viscous feedstock, such as cell fermentation broths, for target molecule capture. It is believed that the high column permeability, low mass transfer resistance and the low cost of the C-CP fiber chromatography columns make them promising choices for fast protein separations at various scales [7,10].…”