Preparation of a novel polymer monolith with functional polymer brushes by two‐step atom‐transfer radical polymerization for trypsin immobilization

Novel porous polymer monoliths grafted with poly{oligo[(ethylene glycol) methacrylate]‐co‐glycidyl methacrylate} brushes were fabricated via two‐step atom‐transfer radical polymerization and used as a trypsin‐based reactor in a continuous flow system. This is the first time that atom‐transfer radical polymerization technique was utilized to design and construct polymer monolith bioreactor. The prepared monoliths possessed excellent permeability, providing fast mass transfer for enzymatic reaction. More importantly, surface properties, which were modulated via surface‐initiated atom‐transfer radical polymerization, were found to have a great effect on bioreactor activities based on Michaelis–Menten studies. Furthermore, three model proteins were digested by the monolith bioreactor to a larger degree within dramatically reduced time (50 s), about 900 times faster than that by free trypsin (12 h). The proposed method provided a platform to prepare porous monoliths with desired surface properties for immobilizing various enzymes.     

Silica monolithic columns: synthesis, characterisation and applications to the analysis of biological molecules

In recent years, proteomics has been a subject of intense research. The complexity of proteomics samples has fostered technological developments. One of these addresses the need for more efficient and faster separations. Monolithic columns prepared from organic and silica monomers offer very efficient separations at low back-pressure. Silica-based monoliths have small-sized skeletons and a bimodal pore size distribution with microm-sized throughpores and nm-sized mesopores. This gives silica-based monoliths favourable properties for high-efficiency, fast separations, like a low-pressure drop across the column, fast mass transfer kinetics and a high binding capacity.     

有机聚合物整体柱的制备与应用的研究进展

整体柱具有通透性能良好和传质速度快等特点,可实现快速、高效、高通量的分离,近年来已引起人们的热切关注。聚合物整体柱是其中应用最为广泛的一种,它是由单体、交联剂、致孔剂和引发剂等通过原位聚合得到的连续均一的棒状聚合物,具有取材广泛,使用pH范围比较宽,生物兼容性好等特点,通过化学修饰,可以用作多种色谱模式的固定相。该文主要综述了2003年至2006年期间有关聚合物整体柱制备和应用的研究进展。     

Monoliths for fast bioseparation and bioconversion and their applications in biotechnology

Monoliths have consolidated their position in bioseparation. More than 200 different applications have been reported in the past two decades and their advantages compared to conventional chromatography demonstrated. These include the high mass transfer efficiency due to the convective flow enabled by the macroporous character of the matrix. Recently plasmid DNA and viruses were separated with high efficiency and cryogels and monolithic superporous agarose were developed for capture of proteins from crude homogenates and separation of microorganisms or lymphocytes. Currently four companies manufacture monoliths mainly for analytical applications although monoliths with a volume of 0.8 liter are commercially available and 8 L are available as prototypes. A book entitled "Monolithic materials: preparation, properties and applications" was published in 2003 and became standard reference of the status of this area. This review focuses on the progress in monoliths that goes beyond the scope of this reference book. Less progress has been made in the field of bioconversions in spite of the fact that monolithic supports exhibit better performance than beads in enzymatic processing of macromolecules. It appears that the scientific community has not yet realized that supports for these applications are readily available. In addition, monoliths will further substantially advance bioseparations of both small and large molecules in the future.     

Comparison of monolithic approaches for enantioselective capillary electrochromatography involving cyclodextrins

The access to CD-modified monoliths for enantiomeric separation by CEC can be divided into two main approaches. (i) Silica-based monoliths, prepared by either a sol-gel process or by sintering of silica particles, are modified after fabrication by coating with a CD selector. Alternatively the fusion of CD functionalized silica particle via gluing is feasible. (ii) Rigid or homogeneous organic polymer-based monoliths, prepared by polymerization of organic monomers in the presence of a porogen, are modified with the CD selector either by copolymerization or by physical incorporation into the continuous bed.     

Textural characterization of native and n-alky-bonded silica monoliths by mercury intrusion/extrusion, inverse size exclusion chromatography and nitrogen adsorption

Native and n-alkyl-bonded (n-octadecyl) monolithic silica rods with mesopores in the range between 10 and 25 nm and macropores in the range between 1.8 and 6.0 microm were examined by mercury intrusion/extrusion, inverse size exclusion chromatography (ISEC) and nitrogen sorption. Our results reveal very good agreement for the mesopore size distribution obtained from nitrogen adsorption (in combination with an advanced NLDFT analysis) and ISEC. Our studies highlight the importance of mercury porosimetry for the assessment of the macropore size distribution and show that mercury porosimetry is the only method which allows obtaining a combined and comprehensive structural characterization of macroporous/mesoporous silica monoliths. Our data clearly confirm that mercury porosimetry hysteresis and entrapment have different origin, and indicate the intrinsic nature of mercury porosimetry hysteresis in these silica monoliths. Within this context some silica monoliths show the remarkable result of no entrapment of mercury after extrusion from the mesopore system (i.e. for the first intrusion/extrusion cycle). The results of a systematic study of the mercury intrusion/extrusion behavior into native silica monoliths and monoliths with bonded n-alkyl groups reveals that the macro (through) pore structure, which controls the mass transfer to and from the mesopores, here mainly controls the entrapment behavior. Our data suggest that mercury intrusion/extrusion porosimetry does not only allow to obtain a comprehensive pore structure analysis, but can also serve as a tool to estimate the mass transport properties of silica monoliths to be employed in liquid-phase separation processes.     

Mutual consistency between simulated and measured pressure drops in silica monoliths based on geometrical parameters obtained by three-dimensional laser scanning confocal microscope observations

The geometrical properties of co-continuous macroporous silica monoliths have been studied by laser scanning confocal microscopy (LSCM) and a comparison has been made with those obtained by conventional mercury intrusion method. Tetrahedral skeleton model (TMS), which mimics the gel skeleton shape of monoliths, was compared with real monoliths in terms of macropore and porosity using the geometrical parameters extracted from the LSCM observations. Liquid flow behavior through the macroporous silica monoliths was examined in comparison with those simulated using TSM, based on the geometrical properties obtained from LSCM observations. Heterogeneity in macropore topology and connectivity in pores and skeletons are suggested to contribute to the improvement of the model structure for macroporous monoliths.     

Porous polymer monoliths for small molecule separations: advancements and limitations

Porous polymer monoliths are considered to be one of the major breakthroughs in separation science. These materials are well known to be best suited for the separation of large molecules, specifically proteins, an observation most often explained by convective mass transfer and the absence of small pores in the polymer scaffold. However, this conception is not sufficient to explain the performance of small molecules. This review focuses in particular on the preparation of (macro)porous polymer monoliths by simple free-radical processes and the key events in their formation. There is special focus on the fluid transport properties in the heterogeneous macropore space (flow dispersion) and on the transport of small molecules in the swollen, and sometimes permanently porous, globule-scale polymer matrix. For small molecule applications in liquid chromatography, it is consistently found in the literature that the major limit for the application of macroporous polymer monoliths lies not in the optimization of surface area and/or modification of the material and microscopic morphological properties only, but in the improvement of mass transfer properties. In this review we discuss the effect of resistance to mass transfer arising from the nanoscale gel porosity. Gel porosity induces stagnant mass transfer zones in chromatographic processes, which hamper mass transfer efficiency and have a detrimental effect on macroscopic chromatographic dispersion under equilibrium (isocratic) elution conditions. The inherent inhomogeneity of polymer networks derived from free-radical cross-linking polymerization, and hence the absence of a rigid (meso)porous pore space, represents a major challenge for the preparation of efficient polymeric materials for the separation of small molecules.     

Applications of monolithic columns in gas chromatography and supercritical fluid chromatography

Porous monoliths are well‐known stationary phases in high‐performance liquid chromatography and capillary electrochromatography. Contrastingly, their use in other types of separation methods such as gas or supercritical fluid chromatography is limited and scarce. In particular, very few studies address the use of monolithic columns in supercritical fluid chromatography. These are limited to silica‐based monoliths and will be covered in this review together with an underlying reason for this trend. The application of monoliths in gas chromatography has received much more attention and is well documented in two reviews by Svec and Kurganov published in 2008 and 2013, respectively. The most recent studies, covered in this review, build on the previous findings and on further understanding of the influence of preparation conditions on porous properties and chromatographic performance of poly(styrene‐co‐divinylbenzene), polymethacrylate, and silica‐based monolithic columns while expanding to polymer‐based monoliths with incorporated metal organic frameworks and to vinylized hybrid silica monoliths. In addition, the potential application of porous layer open tubular monolithic columns in low‐pressure gas chromatography will be addressed.     

Synthesis of propyl-functionalized hybrid monolithic silica capillaries and evaluation of their performances in nano-LC and CEC

During the last decade, silica monolithic capillaries have focused more and more attention on miniaturized separation techniques like CEC, nano-LC, and chip electrochromatography owing to their unique chromatographic properties and to their possible in situ synthesis. Nevertheless, the preparation of conventional silica-based individual monolithic columns is time consuming, owing to the individual steps involved, including the synthesis of the silica matrix and its subsequent on-column chemical grafting. The hybrid organic-inorganic monoliths, whose synthesis is based on the polycondensation of siloxane with organosiloxane precursors, seems to be an attractive alternative since their direct synthesis leads to silica monoliths with organic moieties covalently linked to the inorganic silica matrix through hydrolytically stable Si-C bonds. This study describes the synthesis of hybrid monoliths using propyltrimethoxysilane (C3-TriMOS) as a new kind of silica coprecursor to subsequently increase the hydrophobicity of the stationary phase. The influence of several experimental parameters (pH, gelation temperature, relative proportion of the precursors) on the textural (skeleton and macropore size) and chromatographic properties (efficiency, retention, and electroosmotic mobility) of the obtained monoliths are discussed. The results show that the optimal coprecursor incorporation is obtained after a postgelation step during which the condensation of the C3-TriMOS coprecursor is favored by an increase in the pH medium. Thermal hydrolysis of urea previously added to the polymerization mixture allows this in situ pH increase. These hybrid monoliths present hydrophobic properties and allow the separation of test mixtures in the RP mode without any further modification. Moreover, they present excellent efficiencies since reduced plate height as low as 5 and 15 microm are obtained in the electrodriven mode (CEC) and in the hydrodynamic one (nano-LC), respectively.     

Porous polymer monolith for surface-enhanced laser desorption/ionization time-of-flight mass spectrometry of small molecules

Porous poly(butyl methacrylate-co-ethylene dimethacrylate), poly(benzyl methacrylate-co-ethylene dimethacrylate), and poly(styrene-co-divinylbenzene) monoliths have been prepared on the top of standard sample plates used for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the modified plates were used for laser desorption/ionization mass spectrometry (LDI-MS). The hydrophobic porous surface of these monoliths enables the transfer of sufficient energy to the analyte to induce desorption and ionization prior to TOFMS analysis. Both UV and thermally initiated polymerization using a mask or circular openings in a thin gasket have been used to define spot locations matching those of the MALDI plates. The desorption/ionization ability of the monolithic materials depends on the applied laser power, the solvent used for sample preparation, and the pore size of the monoliths. The monolithic matrices are very stable and can be used even after long storage times in a typical laboratory environment without observing any deterioration of their properties. The performance of the monolithic material is demonstrated with the mass analysis of several small molecules including drugs, explosives, and acid labile compounds. The macroporous spots also enable the archiving of samples.     

Ring-opening metathesis polymerization-derived monolithic capillary columns for high-performance liquid chromatography. Downscaling and application in medical research

Monolithic capillary columns were prepared via ring-opening metathesis polymerization (ROMP) using norborn-2-ene (NBE) and 1, 4, 4a, 5, 8, 8a-hexahydro-1, 4, 5, 8-exo,endo-dimethanonaphthalene (DMN-H6) as monomers. The monolithic polymer was copolymerized with Grubbs-type initiator RuCl(2)(PCy(3))(2)(CHPh) and a suitable porogenic system within the confines of fused silica capillaries of different inner diameter (I.D.). The first part of the study focused on batch-to-batch reproducibility of ROMP-derived capillary monoliths. Capillary monoliths of 200 microm I.D. showed good reproducibility in terms of retention times, with relative standard deviations (RSD) of 1.9% for proteins and 2.2% for peptides. However, the separately synthesized capillary monoliths revealed pronounced variation in back pressure with RSD values of up to 31%. These variations were considerably reduced by cooling of the capillaries during polymerization. Using this optimized preparation procedure capillary monoliths of 100 and 50 microm I.D. were synthesized and the effects of scaling down the column I.D. on the morphology and on the reproducibility of the polymerization process were investigated. In the second part, the applicability of ROMP-derived capillary monoliths to a separation problem common in medical research was assessed. A 200 microm I.D. monolithic column demonstrated excellent separation behavior for insulin and various insulin analogs, showing equivalent separation performance to Vydac C4 and Zorbax C3-based stationary phases. Moreover, the high permeability of monoliths enabled chromatographic separations at higher flow rates, which shortened analysis time to about one third. For the analysis of insulin in human biofluid samples, enhanced sensitivity was achieved by using a 50 microm I.D. ROMP-derived monolith.     

Hierarchically macro/mesoporous silica monoliths constructed with interconnecting micrometer-sized unit rods

Under typical dilute reactant compositions (3 ~ 5 wt% of surfactant template concentration) and conventional hydrothermal conditions for mesoporous materials synthesis, successful preparation of hierarchically macro/mesoporous silica monoliths was reported in this paper. The resultant materials were characterized by a series of techniques including powder X-ray diffraction, N₂ adsorption–desorption, SEM, TEM/EDS, and Hg porosimetry. A new kind of stable and hierarchically porous pure silica monoliths was confirmed, which are featured with highly ordered mesoporous structures, rod-shaped unit particles, large specific surface area of 492 m²/g, continuous macropores of about 4.0 μm in size and high macropore volume of about 13.1 cm³/g. Moreover, using the resultant silica monoliths as hard templates, carbon monoliths have been successfully replicated, which inherit the structural characters of parent silica materials. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Facile approach to glycidyl methacrylate-based polyHIPE monoliths with high epoxy-group content

This paper reports a facile approach to glycidyl methacrylate (GMA)-based polyHIPE monoliths with high epoxy-group content, which are fabricated using a high internal phase emulsion (HIPE) template via radiation-induced polymerization at room temperature. The effects of the polymerization temperature and the pore sizes of polyHIPE monoliths on the content of epoxy groups are investigated. Results show that the polymerization temperature is the most important factor in influencing the content of epoxy groups in GMA-based polyHIPE monoliths. To prove their superiority over monoliths with low epoxy-group contents, the as-prepared polyHIPE monoliths are applied in phenol removal from cigarette smoke through a reaction between the epoxy group and phenol. The results show that the higher the content of epoxy groups in the polyHIPE monoliths, the higher the rate of phenol removal, indicating their high performance in these specific applications for the polyHIPE monoliths with high epoxy-group contents.     

Monolithic bed structure for capillary liquid chromatography

Monolithic stationary phases show promise for LC as a result of their good permeability, ease of preparation and broad selectivity. Inorganic silica monoliths have been extensively studied and applied for separation of small molecules. The presence of a large number of through pores and small skeletal structure allows the chromatographic efficiencies of silica monoliths to be comparable to columns packed with 5 μm silica particles, at much lower back pressure. In comparison, organic polymeric monoliths have been mostly used for separation of bio-molecules; however, recently, applications are expanding to small molecules as well. Organic monoliths with high surface areas and fused morphology rather than conventional globular morphology have shown good performance for small molecule separations. Factors such as domain size, through-pore size and mesopore size of the monolithic structures have been found to govern the efficiency of monolithic columns. The structure and performance of monolithic columns are reviewed in comparison to particle packed columns. Studying and characterizing the bed structures of organic monolithic columns can provide great insights into their performance, and aid in structure-directed synthesis of new and improved monoliths.     

Imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase for hydrophilic interaction/reversed‐phase mixed‐mode chromatography

A novel imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase has been prepared by surface radical chain‐transfer polymerization. The stationary phase was characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, and element analysis. Fast and efficient separations of polar analytes, such as nucleosides and nucleic acid bases, water‐soluble vitamins and saponins, were well achieved in hydrophilic interaction chromatography mode. Additionally, a mixed mode of hydrophilic interaction and reversed‐phase could be also obtained in the analysis of polar and nonpolar compounds, including weak acidic phenols, basic anilines and positional isomers, with high resolution and molecular‐planarity selectivity, outperforming the commercially available amino column. Moreover, simultaneous separation of polar and nonpolar compounds was also achieved. In conclusion, the multimodal retention capabilities of the imidazolium‐embedded iodoacetamide‐functionalized silica‐based column could offer a wide range of retention behavior and flexible selectivity toward hydrophilic and hydrophobic compounds.     

Silica-based monoliths for rapid peptide screening by capillary liquid chromatography hyphenated with electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Capillary liquid chromatography based on particulate and monolithic stationary phases was used to screen complex peptide libraries by fast gradient elution coupled on-line to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS). A slightly modified commercial electrospray interface consisting of a fused-silica transfer capillary and low dead volume stainless steel union at which the electrospray voltage was grounded enabled the effluent of all the capillary columns to be directly sprayed into the mass spectrometer. Stable electrospray conditions were generated over a wide range of mobile phase compositions, alleviating the need for a tapered end of the spray capillary, pneumatic assistance or preheated nebulizer gas. Since the identification of complex samples containing numerous isobaric substances is facilitated by chromatographic separation prior to mass spectrometry, stationary phase materials have been employed which offer a fast, efficient elution and, due to the complexity of samples, a high loading capacity. Silica-based monolithic capillary columns combine these three characteristics in a unique manner due to a tailored adjustment of both macro- and mesopore sizes in the highly porous silica structure. As we demonstrate by a comparative study of the silica-based monolithic and packed capillaries for LC/MS analysis of complex peptide libraries, silica monoliths show superior performance over packed beds of small-diameter particles with respect to analysis time and separation efficiency. Libraries with more than 1000 different peptides could be screened in less than 20 min.     

Sponge-nested polymer monoliths: Versatile materials for the solid-phase extraction of bisphenols

Polymer monoliths are promising materials for sample preparation due to their high porosity, pH stability, and simple preparation. The use of melamine formaldehyde foams has been reported as an effective support to prepare highly robust silica and polymer monoliths. Herein, divinylbenzene monoliths based on a 50:50 (%, w/w) crosslinker/porogen ratio have been nested within a melamine-formaldehyde sponge, resulting in monoliths with a surface area higher than 400 m²/g. The extraction performance of these monoliths was evaluated for the extraction of endocrine-disrupting bisphenols from aqueous solutions. We evaluated for the first time the versatility of sponge-nested polymer monoliths by comparing three different extraction modes (vortex mixing, magnetic stirring, and orbital shaking). Vortex mixing showed a comparable recovery of bisphenols (39%–81%) in a shorter extraction time (30 min, instead of 2 h). In addition, the robustness of the sponge-nested polymer monoliths was demonstrated for the first time by reshaping a larger monolithic cube (0.125 cm³) into four smaller pieces (4 × 0.03125 cm³) leading to a 16%–21% increase in extraction efficiency. This effect was attributed to an increase in the effective contact area with the sample, obtaining a higher analyte extraction capacity.