Surfactant-bound monolithic columns for separation of proteins in capillary high performance liquid chromatography

Abstract

A surfactant bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA-EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties (i.e. morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase.

A strategy to decorate porous polymer monoliths with graphene oxide and graphene nanosheets

“…The inner wall of the capillary was vinylized to enable covalent attachment of the monolith. 26 Poly(GMA-EDMA) monoliths were prepared using modied conditions developed previously. 27 We varied the weight percents of GMA, EDMA, cyclohexanol and 1-dodecanol, but xed that of AIBN (1% of w/w with respect to the total monomer content) in polymerization mixture solution to prepare four monolithic columns with different porosities (see Table 1).…”

Fast separations of intact proteins by liquid chromatography

“…Using the optimized column, the fast separation of three proteins was obtained in 2.5 min, and a tryptic digest of myoglobin was also successfully separated on the high‐resolution column. These experimental results showed that the D‐optimal method, which optimizes the five factors involved in the preparation of monolithic columns via preliminary experiments and Design‐Expert software is a very promising approach for obtaining truly optimum polymerization conditions, allowing the successful development of new monolithic stationary phases . Some typical examples are listed in Table .…”

“…These mixed‐mode packings have special characteristics, not only for protein separations and simultaneous renaturation processes, which can substantially shorten the purification process of recombinant proteins , but also for protein separations with two modes and operations, either by off‐line or on‐line 2D‐LC, enabling fast protein purification . The exploration of new column technologies, such as columns for high‐temperature separations , monolithic columns , and chromatographic cakes , also provides many choices for separating proteins at very high flow rates. When target proteins are obtained from E. coli , a protein renaturing step is typically required before subjecting the target proteins to several LC steps for further purification.…”

Preparation and Evaluation of Long Chain Alkyl Methacrylate Monoliths for Capillary Chromatography

“…Capillary HPLC offer several advantages over conventional normal scale HPLC. The advantages include increased chromatographic resolution, higher efficiency, lower sample and solvent consumption, the ability to analyze and isolate rare compounds of interest, greater mass sensitivity and ease of on-line connection to mass spectrometer [1][2][3].…”