Fast fabrication of a hybrid monolithic column containing cyclic and aliphatic hydrophobic ligands via photo‐initiated thiol‐ene polymerization

Three monomers, octakis (3‐mercaptopropyl) octasilsesquioxane, 1,2,4‐trivinylcyclohexane and isophytol were employed to synthesize a novel monolithic stationary phase via photo‐initiated thiol‐ene click polymerization for reversed‐phase liquid chromatography. Several factors such as porogenic system, reaction time and the molar ratio of functional groups were investigated in detail. The resulting poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column exhibited suitable permeability for fast separation and outstanding thermal stability. Five alkylbenzenes were employed to evaluate the ability of chromatographic separation of the resulting monolithic columns at different flow rates, and showed the highest column efficiencies of 90,200–93,100 N/m (corresponding to 10.4–10.6 μm of plate height) at a velocity of 0.41 mm/s. The baseline separations of five anilines and eight phenols further proved the applicability of poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column in the separation of small molecules.     

Rapid determination of molecular parameters of synthetic polymers by precipitation/redissolution high‐performance liquid chromatography using “molded” monolithic column

Rapid high‐performance liquid chromatography (HPLC) of polystyrenes, poly(methyl methacrylates), poly(vinyl acetates), and polybutadienes using a monolithic 50 × 4.6 mm i.d. poly(styrene‐co‐divinylbenzene) column have been carried out. The separation process involves precipitation of the macromolecules on the macroporous monolithic column followed by progressive elution utilizing a gradient of the mobile phase. Depending on the character of the separated polymer, solvent gradients were composed of a poor solvent such as water, methanol, or hexane and increasing amounts of a good solvent such as THF or dichloromethane. Monolithic columns are ideally suited for this technique because convection through the large pores of the monolith enhances the mass transport of large polymer molecules and accelerates the separation process. Separation conditions including the selection of a specific pair of solvent and precipitant, flow rate, and gradient steepness were optimized for the rapid HPLC separations of various polymers that differed broadly in their molecular weights. Excellent separations were obtained demonstrating that the precipitation‐redissolution technique is a suitable alternative to size‐exclusion chromatography (SEC). The molecular weight parameters calculated from the HPLC data match well those obtained by SEC. However, compared to SEC, the determination of molecular parameters using gradient elution could be achieved at comparable flow rates in a much shorter period of time, typically in about 1 min. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2767–2778, 2000     

Monolithic Stationary Phases for Capillary Electrochromatography Based on Synthetic Polymers: Designs and Applications

Monolithic materials have quickly become a well‐established stationary phase format in the field of capillary electrochromatography (CEC). Both the simplicity of their in situ preparation method and the large variety of readily available chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials. This review summarizes the contributions of numerous groups working in this rapidly growing area, with a focus on monolithic capillary columns prepared from synthetic polymers. Various approaches employed for the preparation of the monoliths are detailed, and where available, the material properties of the resulting monolithic capillary columns are shown. Their chromatographic performance is demonstrated by numerous separations of different analyte mixtures in variety of modes. Although detailed studies of the effect of polymer properties on the analytical performance of monolithic capillaries remain scarce at this early stage of their development, this review also discusses some important relationships such as the effect of pore size on the separation performance in more detail.     

Preparation and evaluation of poly(alkyl methacrylate-co-methacrylic acid-co-ethylene dimethacrylate) monolithic columns for separating polar small molecules by capillary liquid chromatography

In this study, methacrylic acid (MAA) was incorporated with alkyl methacrylates to increase the hydrophilicity of the synthesized ethylene dimethacrylate-based (EDMA-based) monoliths for separating polar small molecules by capillary LC analysis. Different alkyl methacrylate–MAA ratios were investigated to prepare a series of 30% alkyl methacrylate–MAA–EDMA monoliths in fused-silica capillaries (250-μm i.d.). The porosity, permeability, and column efficiency of the synthesized MAA-incorporated monolithic columns were characterized. A mixture of phenol derivatives is employed to evaluate the applicability of using the prepared monolithic columns for separating small molecules. Fast separation of six phenol derivatives was achieved in 5 min with gradient elution using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. In addition, the effect of acetonitrile content in mobile phase on retention factor and plate height as well as the plate height-flow velocity curves were also investigated to further examine the performance of the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. Moreover, the applicability of prepared polymer-based monolithic column for potential food safety applications was also demonstrated by analyzing five aflatoxins and three phenicol antibiotics using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column.     

Rapid reversed-phase separation of proteins and peptides using optimized 'moulded' monolithic poly(styrene-co-divinylbenzene) columns

Monolithic macroporous poly(styrene-co-divinylbenzene) stationary phases have been prepared by free radical polymerization within the confines of 4.6-mm I.D. chromatographic columns. The optimized porous properties allow the mobile phase to flow through these columns at flow-rates of up to 10 ml/min. As opposed to the simultaneously tested columns packed with either silica or synthetic polymer beads, the monoliths exhibit only modest back pressure. The monolithic columns were able to separate mixtures of peptides and proteins in a very short time. Under the optimized conditions, the separation of five proteins can be easily achieved in less than 20 s.     

Effect of ion adsorption on CEC separation of small molecules using hypercrosslinked porous polymer monolithic capillary columns

Both poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) and poly(4-methylstyrene-co-vinylbenzyl chloride-co-divinylbenzene) monolithic columns have been hypercrosslinked and for the first time used to achieve capillary electrochromatographic separations. Although these columns do not contain ionizable functionalities, electroosmotic flow was observed due to adsorption of ions from a buffer solution contained in the mobile phase on the surface of the hydrophobic polymer. An increase of more than one order of magnitude was observed with the use of both monolithic polymers. The hypercrosslinking reaction creates a large surface area thus enabling adsorption of a much larger number of ions. Alkylbenzenes were successfully separated using the hypercrosslinked monolithic columns.     

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.     

A New Monolithic‐Type HPLC Column For Fast Separations

The application of a new silica‐based, monolithic‐type HPLC‐column for fast separations is presented. The column is prepared according to a new sol‐gel process, which is based on the hydrolysis and polycondensation of alkoxysilanes in the presence of water soluble polymers. The method leads to “rods” made of a single piece of porous silica with a defined pore structure, i. e. macro‐ and mesopores. The main feature of silica rod columns is a higher total porosity, about 15% higher than of conventional particulate HPLC columns. The resulting column pressure drop is therefore much lower, allowing operation at higher flow rates including flow gradients. Consequently, HPLC analysis can be performed much faster, as it is demonstrated by various applications.     

Monolithic systems: from separation science to heterogeneous catalysis

Recent results that have been obtained in the ring-opening metathesis polymerization (ROMP)-based synthesis of monolithic supports are summarized. We have elaborated a synthetic concept that allows modifying monolithic supports in a way that they can be used both for applications in separation science, for SEC and as supports for catalytically active systems. In all cases, a tailor-made microstructure was accessible due to the controlled character of the transition-metal catalyzed polymerization. Taking advantage of the “living” catalytic sites, an “in situ” functionalization was accomplished by subsequently grafting a variety of functional monomers and catalyst precursors onto the rod. Their design and use as supports for high-performance separation devices (e.g. for ds-DNA) and catalytic supports (e.g. supported Grubbs-type catalysts) is summarized.     

Photopolymerized monolithic capillary columns for rapid micro high-performance liquid chromatographic separation of proteins

The preparation of monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns using photoinitiated in situ polymerization within 200 microm i.d. capillaries and their application for microHPLC separations of proteins have been studied. The low resistance to flow characteristic of monolithic columns, enabled the use of very high flow rates of up to 100 microL/min representing a flow velocity of 87 mm/s. Very good separations of a model protein mixture consisting of ribonuclease A, cytochrome c, myoglobin, and ovalbumin was achieved in less than 40 s using a very simple single step gradient of the mobile phase. Interestingly, no effect of the pore size on the separations of proteins was observed for these monolithic columns within the size range of 0.66-2.2 microm. The monolithic microHPLC columns are found very robust and no changes in the long term separation performance and back pressure were observed.     

Design of the monolithic polymers used in capillary electrochromatography columns

Monolithic columns for capillary electrochromatography (CEC) are receiving quite remarkable attention. Both the simplicity of the in situ preparation and the large number of readily available chemistries make the monolithic separation media a vital alternative to capillary columns packed with particulate materials. This review summarizes the current state-of-the-art in this rapidly growing area of CEC with a focus on monolithic capillary columns prepared from synthetic polymers. Recent achievements in column technologies for both high-performance liquid chromatography and capillary electrophoresis are used as the starting point to highlight the influence of these well established analytical methods on the development of monolithic capillary columns for CEC. The effects of individual variables on the separation properties of monolithic capillaries are discussed in detail. The analytical potential of these columns is demonstrated with separations involving various families of compounds in different chromatographic modes.     

A Novel Route to Prepare Highly Reactive and Versatile Chromatographic Monoliths

The copolymer poly(isocyanatoethyl methacrylate‐co‐methyl methacrylate‐co‐ethylene glycol dimethacrylate) (poly(IEM‐co‐MMA‐co‐EGDMA)) was developed as a novel, facile, highly reactive and versatile monolithic matrix, which was amenable to surface functionalization with a variety of nucleophilic modifiers based on the reactive isocyanate group, producing monoliths of various chemistries suitable for chromatography. The specific surface area, pore size distribution, porosity and morphology of the monolithic matrix were characterized using a mercury intrusion porosimeter and scanning electron microscopy (SEM), respectively. Thermal analysis results revealed that the monolith was thermally stable up to 307 °C. The success of the chemical modification of the monolithic matrix was confirmed by FT‐IR, solid state ¹³C NMR and XPS elemental analysis, showing the high ligand density of the modified monoliths. A ligand density of up to 2.33 mmol·mL⁻¹ was obtained for the 1‐octanol modified monolith (M1) with an isocyanate group conversion of 96.9%, indicating a high efficiency of the modification reaction. The potential application of the monoliths was demonstrated by the separation of a series of compounds. The novel monolithic columns exhibited high mechanical stability, column efficiency and good repeatability and reproducibility.

     

HPLC analysis of synthetic polymers on short monolithic columns

Ultrashort monolithic columns (disks) were thoroughly studied as efficient stationary phases for precipitation–dissolution chromatography of synthetic polymers. Gradient elution mode was applied in all chromatographic runs. The mixtures of different flexible chain homopolymers, such as polystyrenes, poly(methyl methacrylates), and poly(tert‐butylmethacrylates) were separated according to their molecular weights on both commercial poly(styrene‐co‐divinylbenzene) disks (12 id × 3 mm and 5 × 5 mm) and lab‐made monolithic columns (4.6 id × 50 mm) filled with supports of different hydrophobicity. The experimental conditions were optimized to reach fast and highly efficient separation. It was observed that, similar to the separation of monoliths of other classes of (macro)molecules (proteins, DNA, oligonucleotides), the length of column did not affect the peak resolution. A comparison of the retention properties of the poly(styrene‐co‐divinylbenzene) disk‐shaped monoliths with those based on poly(lauryl methacrylate‐co‐ethylene dimethacrylate), poly(butyl methacrylate‐co‐ethylene dimethacrylate), and poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) supports demonstrated the obvious effect of surface chemistry on the resolution factor. Additionally, the results of the discussed chromatographic mode on the fast determination of the molecular weights of homopolymers used in this study were compared to those established by SEC on columns packed with sorbent beads of a similar nature to the monoliths.     

Fast reversed‐phase liquid chromatographic separation of proteins by flow‐through poly(styrene‐co‐divinylbenzene) microspheres

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High‐performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow‐through poly(styrene‐co‐divinylbenzene) microspheres characteristic of the binary pores, i.e. flow‐through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene‐co‐divinylbenzene) microspheres were suitable as the reversed‐phase stationary phase for separation of proteins. For the high permeability of the poly(styrene‐co‐divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ～2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0–99.4%. The proposed column had good pH stability of 1–13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio‐sample separation. The flow‐through poly(styrene‐co‐divinylbenzene) microspheres were promising for fast separation of large molecules.     

Polymer monolithic capillary column fabricated by using monodisperse iron oxide nanocrystal template to enhance the electrochromatographic separation of small molecules

Monodisperse iron oxide nanocrystals and organic solvents were utilized as coporogens in monolithic poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) capillary columns to afford stationary phases with enhanced electrochromatographic performance of small molecules. While the conventional monoliths using organic solvents only as a porogen exhibited poor resolution (Rs) <1.0 and low efficiency of 40 000–60 000 plates/m, addition of a small amount of nanocrystals to the polymerization mixture provided increased resolution (Rs > 3.0) and high efficiency ranged from 60 000 to 100 000 plates/m at the same linear velocity of 0.856 mm/s. It was considered that the mesopores introduced by the nanocrystals played an important role in the improvement of the monolith performance. This new strategy expanded the application range of the hydrophobic monoliths in the separation of polar alkaloids and narcotics. The successful applications demonstrated that the glycidyl methacrylate based monoliths prepared by using nanocrystal template are a good alternative for enhanced separation efficiency of small molecules.     

Development and application of polymeric monolithic stationary phases for capillary electrochromatography

Monolithic columns for capillary electrochromatography are receiving quite remarkable attention. This review summarizes results excerpted from numerous papers concerning this rapidly growing area with a focus on monoliths prepared from synthetic polymers. Both the simplicity of the in situ preparation and the large number of readily available chemistries make the monolithic separation media a vital alternative to capillary columns packed with particulate materials. Therefore, they are now a well-established stationary phase format in the field of capillary electrochromatography. A wide variety of synthetic approaches as well as materials used for the preparation of the monolithic stationary phases are presented in detail. The analytical potential of these columns is demonstrated with separations involving various families of compounds and different chromatographic modes.     

Polymethacrylate monolithic columns for capillary liquid chromatography

In recent years, continuous separation media have attracted considerable attention because of the advantages they offer over packed columns. This research resulted in two useful monolithic material types, the first based on modified silica gel and the second on organic polymers. This work attempts to review advances in the development, characterization, and applications of monolithic columns based on synthetic polymers in capillary chromatography, with the main focus on monolithic beds prepared from methacrylate-ester based monomers. The polymerization conditions used in the production of polymethacrylate monolithic capillary columns are surveyed, with attention being paid to the concentrations of monomers, porogen solvents, and polymerization initiators as the system variables used to control the porous and hydrodynamic properties of the monolithic media. The simplicity of their preparation as well as the possibilities of controlling of their porous properties and surface chemistries are the main benefits of the polymer monolithic capillary columns in comparison to capillary columns packed with particulate materials. The application areas considered in this review concern mainly separations in reversed-phase chromatography, ion-exchange chromatography, hydrophobic and hydrophilic interaction modes; enzyme immobilization and sample preparation in the capillary chromatography format are also addressed.     

New hierarchically porous titania monoliths for chromatographic separation media

Separation media based on hierarchically porous titania (TiO₂) monoliths for high‐performance liquid chromatography (HPLC) have been successfully fabricated by the sol–gel process of titanium alkoxide in a mild condition utilizing a chelating agent and mineral salt. The as‐gelled TiO₂ monoliths were subjected to a simple solvent exchange process from ethanol (EtOH) to H₂O followed by drying and calcination. The resultant monolithic TiO₂ columns consist of anatase crystallites with the typical specific surface area of more than 200 m²/g. The resultant monolithic TiO₂ column calcined at 200 and 400°C exhibited a good separation performance for organophosphates as well as for polar benzene derivatives in the normal‐phase mode.     

New synthetic ways for the preparation of high-performance liquid chromatography supports.

The latest developments and in particular important synthetic aspects for the preparation of modern HPLC supports are reviewed. In this context, the chemistry of inorganic supports based on silica, zirconia, titania or aluminum oxide as well as of organic supports based on poly(styrene-divinylbenzene), acrylates, methacrylates and other, more specialized polymers is covered. Special consideration is given to modern approaches such as sol-gel technology, molecular imprinting, perfusion chromatography, the preparation of monolithic separation media as well as to organic HPLC supports prepared by new polymer technologies such as ring-opening metathesis polymerization. Synthetic particularities relevant for the corresponding applications are outlined.     

蛋白A连续床亲和毛细管色谱柱的制备及性能考察

人免疫球蛋白 G(HIg G)是一种重要的生物大分子 ,是人血浆中的主要成分之一 ,通常采用免疫学的方法测定 .蛋白 A(Protein A)与免疫球蛋白 (HIg G)的 Fc区之间具有很强的特异性亲和作用 ,因而固载蛋白 A的亲和介质可用于免疫球蛋白及单克隆抗体的分离、纯化和分析测定[1～ 3 ] .根据固定相存在形式的不同 ,毛细管色谱柱主要有开管、填充和连续床柱 3种方式 .连续床具有相比高、易制备 (一步合成 )、孔径易控制、不需烧塞子和易改性等优点 .连续床与其它常用的亲和介质 (如球型凝胶颗粒、灌流色谱基质 [4、 5] 、膜介质 [6,7] 等 )相比具…