Organic polymer monoliths as stationary phases for capillary HPLC

Modern rigid porous polymer monoliths were conceived as a new class of stationary phases in classical columns in the early 1990s and later extended to the capillary format. These monolithic materials are typically prepared using a simple molding process carried out within the confines of the capillary. Polymerization of a mixture comprising monomers, initiator, and porogenic solvent affords macroporous materials with large through-pores that enable applications in a rapid flow-through mode. Since all the mobile phase must flow through the monolith, convection considerably accelerates mass transport within the monolithic separation medium and improves the separations. As a result, monolithic columns perform well even at very high flow rates. Various mechanisms including thermally and UV initiated free radical polymerization as well as ring opening metathesis copolymerizations were demonstrated for the preparation of monolithic capillary columns. The versatility of these preparation techniques was demonstrated by their use with hydrophobic (styrene, divinylbenzene, butyl methacrylate, ethylene dimethacrylate), hydrophilic (2-hydroxyethyl methacrylate, methacrylamide, methylenebisacrylamide), ionizable (vinylsulfonic acid, 2-acrylamido-2-methyl-propanesulfonic acid), and tailor-made (norborn-2-ene, 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene) monomers. Variation of polymerization conditions enables control of the porous properties of the monolith over a broad range and mediates the hydrodynamic properties of the monolithic columns. The applications of polymer-based monolithic capillary columns are demonstrated for numerous separations in the microHPLC mode.     

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.     

Advances in the preparation of porous polymer monoliths in capillaries and microfluidic chips with focus on morphological aspects

Porous polymer monoliths have emerged as unique materials for many applications, including liquid-chromatographic analyses at an unrivaled speed, solid-phase extraction, and enzyme immobilization in capillary and microfluidic chip format. This article reviews the state of the art in the preparation of monoliths in narrow-bore capillaries and microfluidic chips and their miniaturization under conditions of spatial confinement. New developments in their preparation mainly using free radical polymerization techniques with a focus on morphological aspects in view of homogeneous porous materials are described. The suitability of monoliths for analysis of both large and small molecules is also discussed.     

Porogens and porogen selection in the preparation of porous polymer monoliths

Porogens are key components required for the preparation of porous polymer monoliths for application in separation science. Porogens determine the stability, selectivity, and permeability of polymer monoliths. This review summarizes the role of porogens in the preparation of porous polymer monoliths with a focus on clear understanding of effect of porogens on morphological properties, porosity, surface area, mechanical stability, and permeability of monoliths, particularly targeting the field of separation science. This review also includes the use of different types of porogens with the focus on various approaches used to set criteria for their systematic selection, including porogen‐free techniques recently used for synthesis of porous monoliths. It discusses the current state‐of‐the‐art applications of porogens in column preparation as well as where the future developments in this field may be directed.     

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。     

Liquid-phase synthesis and application of monolithic porous materials based on organic–inorganic hybrid methylsiloxanes, crosslinked polymers and carbons

Our recent progress in porous materials based on organic–inorganic hybrids, organic crosslinked polymers, and carbons is summarized. Flexible aerogels and aerogel-like xerogels with the polymethylsilsesquioxane (PMSQ) composition are obtained using methyltrimethoxysilane (MTMS) as the sole precursor. Preparation process and the flexible mechanical properties of these aerogels/xerogels are overviewed. As the derivative materials, hierarchically macro- and mesoporous PMSQ monoliths and marshmallow-like soft and bendable porous monoliths prepared from dimethyldimethoxysilane /MTMS co-precursors have been obtained. Organic crosslinked polymer monoliths with well-defined macropores are also tailored using gelling systems of vinyl monomers under controlled/living radical polymerization. The obtained polymer monoliths are carbonized and activated into activated carbon monoliths with well-defined pore properties. The activated carbon monoliths exhibit good electrochemical properties as the monolithic electrode. Some possibilities of applications for these porous materials are also discussed.     

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.     

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.     

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.     

Towards stationary phases for chromatography on a microchip: molded porous polymer monoliths prepared in capillaries by photoinitiated in situ polymerization as separation media for electrochromatography

Photoinitiated free radical polymerization has been used for the preparation of porous polymer monoliths within UV transparent fused silica capillaries and quartz tubes. These formats were used as models for the preparation of the separation media within channels of microfabricated devices. A mixture of ethylene dimethacrylate, butyl methacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid was polymerized in the presence of a porogenic solvent consisting of 1-propanol, 1,4-butanediol, and water at room temperature under UV irradiation. Modification of the porogen composition enables the tailoring of pore size within the broad range from ca. 100 to 4000 nm. Scanning electron micrographs confirmed the homogeneity of the porous structure of the materials prepared, even in a quartz tube with a diameter as large as 4 mm. Separation properties of the resulting capillary columns were tested in capillary electrochromatography (CEC) mode using a mixture of thiourea and eight aromatic compounds. Plate number as high as 210 000 plates/m were found for a capillary column with optimized porous properties. The monolithic columns were also able to separate mixtures of peptides.     

Less common applications of monoliths. III. Gas chromatography

Porous polymer monoliths emerged about two decades ago. Despite this short time, they are finding applications in a variety of fields. In addition to the most common and certainly best known use of this new category of porous media as stationary phases in liquid chromatography, monolithic materials also found their applications in other areas. This review article focuses on monoliths in capillaries designed for separations in gas chromatography.     

Review on recent and advanced applications of monoliths and related porous polymer gels in micro-fluidic devices

This review critically summarises recent novel and advanced achievements in the application of monolithic materials and related porous polymer gels in micro-fluidic devices appearing within the literature over the period of the last 5 years (2005-2010). The range of monolithic materials has developed rapidly over the past decade, with a diverse and highly versatile class of materials now available, with each exhibiting distinct porosities, pore sizes, and a wide variety of surface functionalities. A major advantage of these materials is their ease of preparation in micro-fluidic channels by in situ polymerisation, leading to monolithic materials being increasingly utilised for a larger variety of purposes in micro-fluidic platforms. Applications of porous polymer monoliths, silica-based monoliths and related homogeneous porous polymer gels in the preparation of separation columns, ion-permeable membranes, preconcentrators, extractors, electrospray emitters, micro-valves, electrokinetic pumps, micro-reactors and micro-mixers in micro-fluidic devices are discussed herein. Procedures used in the preparation of monolithic materials in micro-channels, as well as some practical aspects of the micro-fluidic chip fabrication are addressed. Recent analytical/bioanalytical and catalytic applications of the final micro-fluidic devices incorporating monolithic materials are also reviewed.     

整体柱用于糖蛋白质分离富集研究新进展

蛋白质糖基化是最常见、最重要的蛋白质翻译后修饰之一,由糖链和多肽链以多种形式共价修饰而成.糖蛋白在生物体内种类繁多,分布广泛,具有重要的生理功能.复杂生物体系中糖蛋白的绝对丰度低,高丰度非糖蛋白质的存在对低丰度糖蛋白质产生夹带、包覆以至掩盖的作用,以现有的技术难以发现并分离鉴定大多数低丰度糖蛋白,已成为当前糖蛋白质组学...     

新型整体材料的制备及其在蛋白质组学中的应用

蛋白质组学是后基因时代生命科学研究的核心内容之一,其中发展高分辨、高灵敏度、高准确性和高通量的分析技术至关重要。整体材料具有制备简单、传质速度快、色谱柱反压低、表面易修饰等优点,因此已被广泛用于蛋白质组学研究中。本文对各种整体材料(包括有机聚合物整体材料、硅胶整体材料、有机-无机杂化整体材料)的制备方法及其在蛋白质组学研究(如蛋白质的酶解、蛋白质和肽段的分离以及在线酶解-分离-鉴定的高通量分析)中的应用进行了系统综述。     

Monolithic capillary columns based on pentaerythritol acrylates for molecular‐size‐based separations of synthetic polymers

Monolithic capillary columns based on pentaerythritol triacrylate and pentaerythritol tetraacrylate were synthesized using different compositions of polymerization mixtures and different polymerization conditions. The impact of porogen type and porogen/monomer ratio on the porosity of synthesized monoliths was investigated. Porogen type appears to be the main factor influencing the separating properties of the monolithic sorbent. Using optimal polymerization conditions (porogen type, porogen/monomer ratio, reaction temperature, time etc.) monoliths with a porous structure optimized for polymer separations can be obtained. The monolithic capillary columns containing porous sorbents with optimized porosity are capable of separating 10 to 12 polystyrene standards in one chromatographic run utilizing both size exclusion chromatography and hydrodynamic chromatography separation mechanisms.     

Preparation of monolithic polymers with controlled porous properties for microfluidic chip applications using photoinitiated free‐radical polymerization

A broad variety of monolithic macroporous polymers with both controlled chemistry and porous properties was prepared using UV‐initiated free‐radical polymerization. The chemistry of the monoliths is defined by the composition of the monomer mixture used for the polymerization. The use of functional methacrylate monomers such as glycidyl methacrylate, 2‐hydroxyethyl methacrylate, butyl methacrylate, 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid, and [2‐(methacryloyloxy) ethyl] trimethylammonium chloride enabled the preparation of monoliths with reactive, hydrophilic, hydrophobic, and ionizable functionalities, respectively. The porous properties of these monoliths were mainly affected by the choice of the porogenic solvent system. Because the UV polymerization was carried out at room temperature, even low molecular weight alcohols and other low boiling point solvents could safely be used to create a versatile series of binary porogenic mixtures. Monoliths were prepared in spatially defined positions using the photolithographic technique within a fused silica capillary and on microfluidic chips, and the former was demonstrated with the separation of derivatized amines by means of capillary electrochromatography in the reversed‐phase mode. Similarly, a monolith prepared in the microchip format was used to demonstrate a microextraction with enrichment of a solution of green fluorescent protein by a factor of 1000. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 755–769, 2002; DOI 10.1002/pola.10155     

聚合物整体固定相的制备及条件优化

采用无搅动原位聚合模式,在聚醚醚酮柱管中直接制备了聚合物整体固定相。通过扫描电镜观察到该整体固定相的孔径分布呈双峰模式,且孔结构均匀。用压汞法测定了该固定相的孔径分布、孔隙率及比表面积等参数,考察了致孔剂组成、聚合温度及交联剂含量等参数对固定相孔结构的影响,并对制备条件进行了优化。测定了流速与柱前压的关系,实验表明此整体固定相具有良好的通透性。通过对山羊血清和低聚核苷酸的分离分析,证明了所制备的整体固定相适合用于生物大分子的分离纯化。     

Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate-containing fluids and cell culture applications

Macroporous gels (MGs) with a broad variety of morphologies are prepared using the cryotropic gelation technique, i. e. gelation at subzero temperatures. These highly elastic hydrophilic materials can be produced from practically any gel-forming system with a broad range of porosity extending from elastic and porous gels with pore sizes up to 1.0 microm to elastic and sponge-like gels with pore sizes up to 100 microm. The versatility of the cryogelation technique is demonstrated by use of different chemical reactions (hydrogen bond formation, chemical cross-linking of polymers, free radical polymerization) mainly in an aqueous medium. Appropriate control over solvent crystallization (formation of solvent crystals) and rate of chemical reaction during the cryogelation allows the reproducible preparation of cryogels with tailored properties. Different approaches, such as chemical modification of reactive groups, grafting of the pore surface with an appropriate polymer, or direct copolymerization with functional monomers are used for control of the surface chemistry of MGs. Typically, MGs with pore sizes up to 1.0 microm are produced in the shape of beads and MGs with pore size up to 100 microm are prepared as monoliths, discs, and sheets. The difference in porous structure of MGs defines the main applications of these porous materials. Elastic beaded MGs are mostly used as carriers for cell and enzyme immobilization or for capture of low-molecular weight targets from particulate-containing fluids in expanded-bed mode. However, the elastic and sponge-like MG monoliths with interconnected pores measuring hundreds of mum have been successfully used as monolithic columns for chromatography of particulate-containing fluids (crude cell homogenates, viruses, whole cells, wastewater effluents) and as three-dimensional scaffolds for mammalian cell culture applications.     

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.     

Recent development of monolithic stationary phases with emphasis on microscale chromatographic separation

The column technologies play a crucial role in the development of new methods and technologies for the separation of biological samples containing hundreds to thousands compounds. This review focuses on the development of monolithic technology in micro-column formats for biological analysis, especially in capillary liquid chromatography, capillary electrochromatography and microfluidic devices in the past 5 years (2002-2007) since our last review in 2002 on monoliths for HPLC and CEC. The fabrication and functionalization of monoliths were summarized and discussed, with the aim of presenting how monolithic technology has been playing as an attractive tool for improving the power of existing chromatographic separation processes. This review consists of two parts: (i) the recent development in fabrication of monolithic stationary phases from direct synthesis to post-functionalization of the polymer- and silica-based monoliths tailoring the physical/chemical properties of porous monoliths; (ii) the application of monolithic stationary phases for one- and multi-dimensional capillary liquid chromatography, fast separation in capillary electro-driven chromatography, and microfluidic devices.