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Abstract 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.
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This content will become publicly available on August 1, 2026
Preparation, characterization, and simulation of continuous stationary phase gradients on butyl functionalized liquid chromatography columns for protein and peptide separations
This work seeks to explore the capabilities of stationary phase gradients (SPGs) for the large-molecule chromatographic separations of proteins and peptides through a combination of experimental studies and simulations. Continuous SPGs are fabricated on commercial Phenomenex Jupiter C4 columns using the time-based infusion of trifluoroacetic acid through the column. The resultant gradients are characterized using smallmolecule chromatography and physical gradient profiling via thermogravimetric analysis. The gradient columns are then tested using two different protein analyte mixtures and a peptide standard analyte mixture to examine the efficacy of the separations. Collected experimental data from these tests are used to establish fitted parameters for the Linear Solvent Strength (LSS) model to computationally predict the retention behavior of the analytes on the gradient columns. Successful chromatographic separation of all three analyte mixtures was achieved. The re-parameterized LSS model successfully simulates the analyte retention on the gradient columns with only minor deviations from observed experimental retention data. Collectively, this work demonstrates the ability to create a C4 continuous stationary phase gradient starting with a uniform commercial column that can be effectively used to separate mixtures of biomolecules, and the ability to accurately simulate and predict retention times. The computational model described herein could be used to effectively predict retention behavior in silico, significantly reducing the time needed for experimental design.
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- Award ID(s):
- 2305102
- PAR ID:
- 10615067
- Publisher / Repository:
- Journal of Chromatography A
- Date Published:
- Journal Name:
- Journal of Chromatography A
- Volume:
- 1756
- Issue:
- C
- ISSN:
- 0021-9673
- Page Range / eLocation ID:
- 466048
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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