Effect of ethoxylated sorbitan ester surfactants on the chromatographic efficiency of poly(ethylene glycol)-based monoliths.

The effect of adding ethoxylated sorbitan ester surfactants (Tweens®) to poly(ethylene glycol) diacrylate-based monolithic recipes was investigated. Five different Tweens® have been evaluated to investigate the exact role of non-ionic surfactants in poly(ethylene glycol) diacrylate-based monolith preparations. These monoliths were characterized by scanning electron microscopy, infrared spectroscopy, and nitrogen physisorption analysis. Different morphological features, and surface areas were observed when different types of Tween® were included in the recipe; Tween® 20 and 85 showed small globules, while Tween® 40, 60 and 80 exhibited larger globular structures with different sizes and degrees of coalescence. The different Tween®-based monoliths were investigated for the chromatographic separation of mixtures consisting of hydroxybenzoic acids and alkylbenzenes. These columns were mechanically stable, except for Tween® 80. The highest methylene selectivity and the best overall performance were achieved by Tween® 60. The efficiency was increased by increasing the concentration of the Tween® 60 and the amount of poly(ethylene glycol) diacrylate Mn 700 in the recipes up to 30 wt%, each. Further increases in either Tween® 60 or poly(ethylene glycol) diacrylate Mn 700 led to formation of non-permeable columns. The optimized column was successfully used for separation of mixtures of nonsteroidal anti-inflammatory and sulfa drugs, with a maximum efficiency of 60,000 plates/m.

Utilizing RAFT Polymerization for the Preparation of Well-Defined Bicontinuous Porous Polymeric Supports: Application to Liquid Chromatography Separation of Biomolecules.

Polymer-based monolithic high-performance liquid chromatography (HPLC) columns are normally obtained by conventional free-radical polymerization. Despite being straightforward, this approach has serious limitations with respect to controlling the structural homogeneity of the monolith. Herein, we explore a reversible addition-fragmentation chain transfer (RAFT) polymerization method for the fabrication of porous polymers with well-defined porous morphology and surface chemistry in a confined 200 µm internal diameter (ID) capillary format. This is achieved via the controlled polymerization-induced phase separation (controlled PIPS) synthesis of poly(styrene-co-divinylbenzene) in the presence of a RAFT agent dissolved in an organic solvent. The effects of the radical initiator/RAFT molar ratio as well as the nature and amount of the organic solvent were studied to target cross-linked porous polymers that were chemically bonded to the inner wall of a modified silica-fused capillary. The morphological and surface properties of the obtained polymers were thoroughly characterized by in situ nuclear magnetic resonance (NMR) experiments, nitrogen adsorption-desorption experiments, elemental analyses, field-emission scanning electron microscopy (FESEM), scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) spectroscopy, and X-ray photoelectron spectroscopy (XPS) as well as time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealing the physicochemical properties of these styrene-based materials. When compared with conventional synthetic methods, the controlled-PIPS approach affects the kinetics of polymerization by delaying the onset of phase separation, enabling the construction of materials with a smaller pore size. The results demonstrated the potential of the controlled-PIPS approach for the design of porous monolithic columns suitable for liquid separation of biomolecules such as peptides and proteins.

Poly(ethylene glycol)-based monolithic capillary columns for hydrophobic interaction chromatography of immunoglobulin G subclasses and variants.

Polymer monoliths were prepared in 150 µm id capillaries by thermally initiated polymerization of PEG diacrylate for rapid hydrophobic interaction chromatography of immunoglobulin G (IgG) subclasses and related variants. Using only one monomer in the polymerization mixture allowed ease of optimization and synthesis of the monolith. The performance of the monolith was demonstrated by baseline resolution of IgG subclasses and variants, including mixtures of the κ variants of IgG1, IgG2, and IgG3 as well as the κ and λ variants associated with IgG1 and IgG2. The effect of eluent concentration and pH on the separation efficiency of studied proteins was also explored, allowing almost baseline resolution to be achieved for mixtures of the κ variants of IgG1, IgG2, IgG3, and IgG4 but also for the κ and λ variants of IgG1 and IgG2. The results showed significant improvement in the separations in terms of the tradeoff between analysis time and resolution, while maintaining a simple methodology, in comparison to previous reports. The synthesized monolith was also used for the separation of isoforms of a therapeutic monoclonal antibody.

Review of recent advances in the preparation of organic polymer monoliths for liquid chromatography of large molecules.

In recent years the use of monolithic polymers in separation science has greatly increased due to the advantages these materials present over particle-based stationary phases, such as their relative ease of preparation and good permeability. For these reasons, these materials present high potential as stationary phases for the separation and purification of large molecules such as proteins, peptides, nucleic acids and cells. An example of this is the wide range of commercial available polymer-based monolithic columns now present in the market. This review summarizes recent developments in the synthesis of monolithic polymers for separation science, such as the incorporation of nanostructures in the polymeric scaffold as well as the preparation of hybrid structures. The different methods used in the surface functionalization of monolithic columns are also reviewed. Finally, we critically discuss the recent applications of this column technology in the separation of large molecules under different chromatographic mode.