R. Dario Arrua

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.

Monolithic cryopolymers with embedded nanoparticles. II. Capillary liquid chromatography of proteins using charged embedded nanoparticles

The preparation of composite monolithic cryopolymers is presented. These novel porous materials were prepared in capillary format at -70°C using poly(ethyleneglycol) diacrylate (PEGDA) Mw 258 as the single monomer and a mixture of dioxane and water as the porogen. Positively (NR4(+)) or negatively (SO3(-)) charged nanoparticles were incorporated within the polymeric structure by direct addition of their suspensions to the polymerisation mixture. In contrast to our previous report using neutral nanoparticles, the trapping of charged nanoparticles is mostly observed at the polymer surface. The incorporation of these nanostructures improved the chromatographic separations of standard proteins under a hydrophobic interaction chromatography (HIC) separation mode. Moreover, the presence of ionic groups on the polymer surface allowed the application of these columns under ion-exchange (IEX) conditions. The results obtained in this work show that the functionalisation of monolithic columns by direct addition of nanoparticles is a good alternative towards the modification of monolithic polymers without altering the polymeric scaffold.

Highly ordered monolithic structures by directional freezing and UV-initiated cryopolymerisation. Evaluation as stationary phases in high performance liquid chromatography

Rigid aligned polymers were prepared by directional freezing and photo-initiated cryopolymerisation. Poly[poly(ethyleneglycol) diacrylate] (polyPEGDA) and poly(butyl methacrylate-co-ethylene glycol dimethacrylate) [poly(BuMA-co-EDMA)] macroporous polymers were prepared by directional freezing of the polymerisation mixture in liquid nitrogen and photo-initiated polymerisation in the frozen state. The polymerisation mixtures consisted of monomer/s (total monomer concentrations > 25 wt%), dioxane as solvent and 2,2-dimethoxy-2-phenylacetophenone as photo-initiator. The porous properties of the macroporous polymers were tuned by modifying the monomer concentration in the polymerisation mixture and the immersion rate. The results obtained suggest that the freezing of the solvent crystals occurs in the direction of the temperature gradient from the surface of the reactor to its centre rather than in the freezing direction. The aligned polymers were prepared in situ within pre-treated cycloolefin copolymer (COC) tubing and the resulting materials were tested as stationary phases for the separation of biomolecules under reversed-phase and hydrophobic interaction chromatography.

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.

Preparation of inverse polymerized high internal phase emulsions using an amphiphilic macro-RAFT agent as sole stabilizer

Oil-in-water (‘inverse’) High Internal Phase Emulsions (HIPEs) have been prepared using an amphiphilic macro-RAFT agent with toluene as the internal dispersed phase (∼80 vol%) and an aqueous monomer solution as the continuous phase. The water phase consisted of the monomers acrylamide (AM) and N,N′-methylenebisacrylamide (MBAM), an initiator as well as the amphiphilic macro-RAFT agent, that is 2-(butylthiocarbonothioylthio)-2-poly(n-butyl acrylate)-b-poly(acrylic acid), which was used as an anionic polymeric surfactant. The presence of these amphiphilic species allowed the successful preparation of a polyHIPE upon polymerization. The effect of concentration of macro-RAFT agent, pH, initiator, hexadecane as an organic modifier and the polymerization temperature on the morphology of the resulting porous materials was investigated. Varying the lengths of the hydrophilic and hydrophobic blocks of the macro-RAFT agent resulted in polyHIPEs with different porous structures. The presence of RAFT functionality in the polyHIPE was confirmed by elemental analysis, EDX-SEM, Raman and FT-IR spectroscopies. Raman mapping revealed full coverage of the void walls with dithiocarbamate groups.

Characterization of Polymer Monoliths Containing Embedded Nanoparticles by Scanning Transmission X-ray Microscopy (STXM)

The structural and chemical homogeneity of monolithic columns is a key parameter for high efficiency stationary phases in liquid chromatography. Improved characterization techniques are needed to better understand the polymer morphology and its optimization. Here the analysis of polymer monoliths by scanning transmission X-ray microscopy (STXM) is presented for the first time. Poly(butyl methacrylate-co-ethyleneglycoldimethacrylate) [poly(BuMA-co-EDMA)] monoliths containing encapsulated divinylbenzene (DVB) nanoparticles were characterized by STXM, which gives a comprehensive, quantitative chemical analysis of the monolith at a spatial resolution of 30 nm. The results are compared with other methods commonly used for the characterization of polymer monoliths [scanning electron microscopy (SEM), transmission electron microscopy (TEM), mercury porosimetry, and nitrogen adsorption]. The technique permitted chemical identification and mapping of the nanoparticles within the polymeric scaffold. Residual surfactant, which was used during the manufacture of the nanoparticles, was also detected. We show that STXM can give more in-depth chemical information for these types of materials and therefore lead to a better understanding of the link between polymer morphology and chromatographic performance.

PEO-based brush-type amphiphilic macro-RAFT agents and their assembled polyHIPE monolithic structures for applications in separation science

Polymerized High Internal Phase Emulsions (PolyHIPEs) were prepared using emulsion-templating, stabilized by an amphiphilic diblock copolymer prepared by reversible addition fragmentation chain transfer (RAFT) polymerization. The diblock copolymer consisted of a hydrophilic poly(ethylene glycol) methyl ether acrylate (PEO MA, average Mn 480) segment and a hydrophobic styrene segment, with a trithiocarbonate end-group. These diblock copolymers were the sole emulsifiers used in stabilizing “inverse” (oil-in-water) high internal phase emulsion templates, which upon polymerization resulted in a polyHIPE exhibiting a highly interconnected monolithic structure. The polyHIPEs were characterized by FTIR spectroscopy, BET surface area measurements, SEM, SEM-EDX, and TGA. These materials were subsequently investigated as stationary phase for high-performance liquid chromatography (HPLC) via in situ polymerization in a capillary format as a ‘column housing’. Initial separation assessments in reversed-phase (RP) and hydrophilic interaction liquid chromatographic (HILIC) modes have shown that these polyHIPEs are decorated with different microenvironments amongst the voids or domains of the monolithic structure. Chromatographic results suggested the existence of RP/HILIC mixed mode with promising performance for the separation of small molecules.

Robust open cellular porous polymer monoliths made from cured colloidal gels of latex particles

The coagulation of oppositely charged latexes, prepared from the soap-free emulsion polymerisation of styrene using water as the reaction medium, resulted in the obtainment of colloidal gels that were porous in nature and held together by electrostatic interactions. Chemical crosslinking, involving the introduction of a water-soluble crosslinker, resulted in the obtainment of stronger chemical bonds between particles affording a rigid porous material known as a monolith. It was found that, in a simpler approach, these materials could be prepared using a single latex where the addition of ammonium persulfate both resulted in the formation of the colloidal gel and initiated the crosslinking process. The pore size of the resulting monoliths was predictable as this was observed to directly correlate to the particle diameter, with larger pores achieved using particles of increased size. All gels obtained in this work were highly mouldable and retained their shape, which allowed for a range of formats to be easily prepared without the requirement of a mould.

Characterization of oligo(acrylic acid)s and their block co-oligomers

Oligo(acrylic acid), oligoAA are important species currently used industrially in the stabilization of paints and also for the production of self-assembled polymer structures which have been shown to have useful applications in analytical separation methods and potentially in drug delivery systems. To properly tailor the synthesis of oligoAA, and its block co-oligomers synthesized by Reversible-Addition Fragmentation chain Transfer (RAFT) polymerization to applications, detailed knowledge about the chemical structure is needed. Commonly used techniques such as Size Exclusion Chromatography (SEC) and Electrospray Ionization-Mass Spectrometry (ESI-MS) suffer from poor resolution and non-quantitative distributions, respectively. In this work free solution Capillary Electrophoresis (CE) has been thoroughly investigated as an alternative, allowing for the separation of oligoAA by molar mass and the RAFT agent end group. The method was then extended to block co-oligomers of acrylic acid and styrene. Peak capacities up to 426 were observed for these 1D CE separations, 10 times greater than what has been achieved for Liquid Chromatography (LC) of oligostyrenes. To provide a comprehensive insight into the chemical structure of these materials 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used to provide an accurate average chain length and reveal the presence of branching. The chain length at which branching is detected was investigated with the results showing a degree of branching of 1% of the monomer units in oligoAA with an average chain length of 9 monomer units, which was the shortest chain length at which branching could be detected. This branching is suspected to be a result of both intermolecular and intramolecular transfer reactions. The combination of free solution CE and NMR spectroscopy is shown to provide a near complete elucidation of the chemical structure of oligoAA including the average chain length and branching as well as the chain length and RAFT agent end group distribution. Furthermore, the purity in terms of the dead chains and unreacted RAFT agent was quantified. The use of free solution CE and 1H NMR spectroscopy demonstrated in this work can be routinely applied to oligoelectrolytes and their block co-oligomers to provide an accurate characterization which allows for better design of the materials produced from these oligomers.