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

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.

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

“…Herein, a systematic comparison is made between the PP C‐CP fibers having a y‐shaped perimeter (termed PPY fibers) and commercially‐available superficially porous and monolithic columns, sold explicitly for RP protein separations. The studies presented here complement those of a recent report from this laboratory, wherein the PPY format was benchmarked versus a different RP superficially porous column and an SEC platform in terms of performance across biomolecules ranging up to a molecular weight of 660 kDa [66]. Efficient fiber column separations spanned the entirety of the protein suites, whereas the other two formats were limited to the low and high ends of the mass scale, respectively.…”

Identification of peptides of cinobufacini by gel filter chromatography and peptidomics

“…SEC is a method of separation and purification based on the difference in molecular weight of the substances to be separated accord-ing to the nature of the packing material. It is commonly used for the purification and enrichment of substances with large molecular weight differences [11].…”

UV‐based solvent system screening for high‐speed counter‐current chromatography fractionation of compounds with similar UV absorption from complex samples followed by preparative HPLC purification: Flavonoids from barley seedlings as sample

“…It takes advantage of high‐performance separation, online detection, and automatic control to realize the efficient separation of target compounds [1]. The various modes available to date, for example, normal phase [2], reverse phase [3], size exclusion [4], ion exchange [5], two‐dimensional [6], and even three‐dimensional [7] can be used to purify most classes of natural products. However, it has high requirements for samples, and the stationary phase filler is expensive.…”