Dynamic evaluation of polypropylene capillary‐channeled fibers as a stationary phase in high‐performance liquid chromatography

2014

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Capillary-channeled polymer (C-CP) fibers are used as a stationary phase for ion-exchange chromatography of proteins. Collinear packing of the fibers permits operation at high linear velocities (Uo > 100 mm s(-1)) and low backpressure (<2,000 psi) on analytical-scale columns. Rapid solvent transport is matched with very efficient solute mass transfer as fibers are virtually non-porous with respect to the size of the target protein molecules. Lack of porosity of course limits the equilibrium binding capacity of stationary phases. Breakthrough curves and frontal analysis are used to better understand trade-offs between the kinetic and thermodynamic properties as C-CP fibers are applied in preparative situations. Fiber columns packed to different interstitial fraction values affect both the total fiber surface area (e.g., equilibrium binding capacity [EBC]) and the permittivity to flow and mass transport characteristics (e.g., dynamic binding capacity [DBC]). The EBC of the nylon 6 C-CP fibers was found to be 1.30 mg g(-1), with isotherms that were best matched by a Moreau model, showing linearity up to solute concentrations of ∼0.4 mg mL(-1). Isotherms generated under flow conditions were equally well approximated using Langmuir, Freundlich, and Moreau isotherm models. Fairly linear responses were seen up to the maximum load concentration of 1.2 mg mL(-1). Counterintuitively, dynamic studies revealed that conditions of high column porosity yielded a DBC that is ∼70% higher than the EBC. These findings point to potential advantages in terms downstream processing applications, where protein throughput and yield are critical metrics.

Section: Dynamic Binding Capacitymentioning

confidence: 92%

Section: Role Of Fiber Packing Density At Fixed Volume Flowmentioning

confidence: 97%

Section: Fiber Loading Studiesmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 98%

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

Wang

2014

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Initial evaluation of protein throughput and yield characteristics on nylon 6 capillary‐channeled polymer (C‐CP) fiber stationary phases by frontal analysis

Randunu

2013

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Nylon 6 capillary-channeled polymer (C-CP) fibers are investigated as an alternative support/stationary phase for downstream processing of macromolecules. Ionizable amine and carboxylic acid end groups on the native fiber surface allow for ion exchange chromatography (IEC). The low cost and ability to operate at high linear velocities and low back pressures are practical advantages of C-CP fibers for preparative-scale macromolecule separations. The lack of fiber porosity ensures facile adsorption/desorption that is conducive to high throughput and recoveries/yields. Described here is a preliminary investigation of the processing characteristics of lysozyme on nylon 6 fibers with an eye toward downstream processing applications. Fibers were packed into microbore (0.8 mm i.d.) and analytical-size (2.1 mm i.d.) columns for the evaluation of the role of linear velocity on pressure drop, frontal throughput, and yield. Protein isolation by frontal development involved three steps: loading of the column to breakthrough, an aqueous wash, and a salt wash to recover the protein. Frontal throughput was evaluated with different salt concentrations (0-1000 mM NaCl) and different linear velocities (6-24 mm s(-1)). The observed throughput values are in the range of 0.12-0.20 mg min(-1) when 0.25 mg mL(-1) lysozyme (in 20 mM Tris-HCl) is loaded onto 78 mg of C-CP fiber in 0.52 mL volume analytical columns. Increased throughput and yield were found when protein was loaded and eluted at high linear velocity. Results of this study lend credence to the further development of C-CP fibers for biomacromolecule processing on larger scales.

Overload Effects in Reversed Phase Protein Separations using Capillary‐Channeled Polymer Fiber Columns

Wang

2018

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In preparative chromatography, overloading effects are a fact of life. However, peak distortion is problematic in analytical scale chromatography, especially in the case of bio-macromolecule separations. Capillary-channeled polymer fibers have been employed for fast protein separations in reversed phase, ion exchange, and hydrophobic interaction liquid chromatography. Although the primary advantage of the phase is operation at high linear velocities (~100 mm s ) without van Deemter C-term limitations, the limited specific surface area (<5 m g ) suggests that a thorough understanding of overloading effects is needed. We evaluate important factors (injected mass and volume) affecting overload in terms of peak height, width and shape (asymmetry). Overload conditions readily apparent as the peak shape changes from a Gaussian distribution to a left-triangle with slight tailing; more-or-less classical overload behavior. Three methods were used to compute column efficiency in terms of plate counts (N), including the area height, half-height, and the Dorsey-Foley approaches. The half-height method is best-suited for describing column overload, accounting for peak distortion and achieving high levels of consistency. The limiting plate count (N ) and the column sample loading capacity (ω ) were key metrics to characterize overall column performance. Peak capacity (P) was used to assess gradient elution separation performance. Increased flow rates and column length result in enhanced loading capacity. pH mismatch between the sample matrix and the mobile phase, a common cause of protein overload with other phases, was not responsible for distorted peak shapes unless the solvent pH is close to the pI of protein samples.