Biocompatible polymeric monoliths for protein and peptide separations

The concept of biocompatibility with reference to chromatographic stationary phases for separation of biomolecules (including proteins and peptides) is introduced. Biocompatible is a characteristic that indicates resistance to nonspecific adsorption of biomolecules and preservation of their structures and biochemical functions. Two types of biocompatible polymeric monoliths [i. e., polyacrylamide‐ and poly(meth)acrylate‐based monoliths] used for protein and peptide separations are reviewed in detail, with emphasis on size exclusion, ion exchange, and hydrophobic interaction chromatographic modes. Biocompatible monoliths for enzyme reactors are also included. The two main synthetic approaches to produce biocompatible monoliths are summarized, i. e., surface modification of a monolith that is not inherently biocompatible and direct copolymerization of hydrophilic monomers to form a biocompatible monolith directly. Integration of polyethylene glycol into the poly(meth)acrylate monolith network is becoming popular for reduction of non‐specific protein interactions.     

Affinity processes realized on high-flow-through methacrylate-based macroporous monoliths

The technology for preparation of rigid macroporous polymers suggested in the late 1980s has become a powerful instrument for the development of a novel scientific and practical field. At present, monolithic stationary phases are widely used in the processes of bioseparation (chromatography), bioconversion (enzyme reactors) as well as in other processes based on interphase mass distribution (for example, solid phase peptide and oligonucleotide synthesis). Bioaffinity modes of suggested dynamic methods are very promising for their use in different analytical processes (immunological, ecological, medical and other types of analytical monitoring), preparative isolation of blood proteins such as myoglobin, hemoglobin, immunoglobulins, etc. and also recombinant products directly from cell supernatants or lysates. For the first time, it has been shown that bioaffinity pairing with participation of immobilized on carefully designed rigid supports is very fast and the whole process of affinity separation can be realized within second time scale. The principle of bioaffinity recognition is generaly at the construction of biological reactors (for example, enzyme reactors). Improved kinetics of biocatalized reactions is explained by a minimal influence on the surface of the used sorbent. Very perspective field is the use of discussed monoliths for solid phase chemical synthesis of fragments of biological macromolecules (peptides and oligonucleotides). Several examples of these applications will be presented and discussed.     

Affinity monolith chromatography

The combined use of monolithic supports with selective affinity ligands as stationary phases has recently given rise to a new method known as affinity monolith chromatography (AMC). This review will discuss the basic principles behind AMC and examine the types of supports and ligands that have been employed in this method. Approaches for placing affinity ligands in monoliths will be considered, including methods based on covalent immobilization, biospecific adsorption, entrapment, and the formation of coordination complexes. Several reported applications will then be presented, such as the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, and biomimetic chromatography. Other applications that will be discussed are chiral separations and studies of biological interactions based on AMC.     

Use of monolithic supports in proteomics technology

An overview on the utilization of monoliths in proteomics technology will be given. Both silica- and polymer-based monoliths have broad use for microseparation of tryptic peptides in reversed-phase (RP) mode before identification by mass spectrometry (MS) or by MS/MS. For two-dimensional (2D) LC separation of peptides before MS or MS/MS analysis, a combination of ion-exchange, usually cation-exchange (CEX) chromatography with RP chromatography on monolithic supports can be employed. Immobilized metal ion affinity chromatography monoliths with immobilized Fe3+-ions are used for the isolation of phosphopeptides. Monoliths with immobilized affinity ligands are usually applied to the rapid separation of proteins and peptides. Miniaturized reactors with immobilized proteolytic enzymes are utilized for rapid on- or offline digestion of isolated proteins or protein mixtures prior to identification by LC-MS/MS. Monoliths also have broad potential for application in sample preparation, prior to further proteomic analyses. Monolithic supports with large pore sizes can be exploited for the isolation of nanoparticles, such as cells, organelles, viruses and protein aggregates. The potential for further adoption of monolithic supports in protein separation and enrichment of low abundance proteins prior to proteolytic digestion and final LC-MS/MS protein identification will be discussed.     

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.     

Monolithic enantiomer-selective stationary phases for capillary electrochromatography

Monolithic materials have become a well-established format for stationary phases in the field of capillary electrochromatography. Four types of monoliths, namely particle-fixed, silica-based, polymer-based, and molecularly imprinted monoliths, have been utilized as enantiomer-selective stationary phases in CEC. This review summarizes recent developments in the area of monolithic enantiomer-selective stationary phases for CEC. The preparative procedure and the characterization of these columns are highlighted. In addition, the disadvantages and limitations of different monolithic enantiomer-selective stationary phases in CEC are briefly discussed.     

Development of an affinity silica monolith containing alpha1-acid glycoprotein for chiral separations

An affinity monolith based on silica and containing immobilized alpha(1)-acid glycoprotein (AGP) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of AGP in the silica monolith was 18% higher than that obtained with silica particles and 61% higher than that measured for a GMA/EDMA monolith. The higher surface area of the silica monolith gave materials that contained 1.5- to 3.6-times more immobilized protein per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the AGP silica monolith were evaluated by injecting two chiral analytes onto this column (i.e., R/S-warfarin and R/S-propranolol). In each case, the AGP silica monolith gave higher retention plus better resolution and efficiency than AGP columns containing silica particles or a GMA/EDMA monolith. The AGP silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with AGP as a chiral stationary phase.     

Preparation of a Polymer Monolithic Column Using Ionic Liquid as Porogen and Its Application in Separations of Proteins and Small Molecules

A polymer-based monolithic column was prepared for high-performance liquid chromatography (HPLC), using ionic liquids as porogen within the confines of a stainless steel column (50 × 4.6 mm i.d.). In the process, 1-butyl-3-methylimidazolium chloride and dodecyl alcohol were used as bi-porogens, vinyl ester resin as the monomer, ethyleneglycol dimethacrylate as the crosslinker, CCl₄ as the initiator, and FeCl₂ as the catalytic agent to prepare the polymer-based monolithic column. Scanning electron microscopy, nitrogen adsorption–desorption instrument, and mercury intrusion porosimetry were used to assay the characteristics of the monolith, respectively. The optimized monolith showed uniform structure and good permeability. Then, the column was used as the stationary phase of HPLC to separate standard proteins and human plasma with gradient elution. Besides, the monolith was used to separate aromatic compounds from the mixture. The results showed that the addition of IL could effectively improve the structure of monoliths prepared by atom transfer radical polymerization technique. The results also suggested that this kind of monolith could be used as a simple, cheap and effective stationary phase for HPLC.     

Analyses of synthetic antioxidants by capillary electrochromatography using poly(styrene-divinylbenzene-lauryl methacrylate) monolith

In this study, several organic polymer-based monoliths prepared by single step in situ copolymerization of styrene- and methacrylate ester-based monomers (styrene (S), divinylbenzene (DVB) and lauryl methacrylate (LMA)) were developed as stationary phases of capillary electrochromatography (CEC) for the analyses of synthetic antioxidants. These monoliths were characterized by examining the SEM image, IR spectrum, and measuring the pore size, surface area, conversion yield, and thermal decomposition temperature. The polymerization procedure was optimized by varying the reaction temperature, the reaction time, and the LMA-styrene ratio. The LMA-styrene ratio had the most significant influence on the peak symmetry of butylated hydroxyanisole (BHA) and 2, 6-di-tert-butyl-4-methyl phenol (BHT), the latter being greatly affected by excessive peak tailing in the poly(S-DVB) monolith. It showed that the interaction between the poly(S-DVB) monolith and the antioxidant (BHT or BHA) was significantly altered by the insertion of LMA. Compared with the best HPLC and CE methods previously reported, this proposed CEC method provides a comparable separation ability for the five antioxidants analyzed. This study demonstrates that the potentiality of poly(S-DVB-LMA) monolith as stationary phase, especially for CEC system, because of high thermal stability and good column reproducibility.     

Porous Monoliths: Stationary Phases of Choice for High Performance Liquid Chromatography in Various Formats

 Modern porous monoliths have been conceived as a new class of stationary phases for high performance liquid chromatography (HPLC) in classical columns in the early 1990s and later extended to the capillary format. These monolithic materials are prepared using simple processes carried out in an external mold (inorganic monoliths) or within the confines of the column (organic monoliths and all capillary columns). These methods afford 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, the convection considerably accelerates mass transport within the monolithic separation medium and improves the separations. As a result, the monolithic columns perform well even at very high flow rates. The applications of monolithic capillary columns are demonstrated on numerous separations in the HPLC mode.     

Porous polymer monoliths for extraction: diverse applications and platforms

Polymer monoliths are becoming increasingly popular as sorbent materials, and, along with silica monoliths, they are sometimes touted as replacements for the particulate stationary phases used in HPLC. This critical and prospective review shows how polymer monoliths are in fact finding numerous extraction roles that do not resemble HPLC. They are showing great promise as extractors in a remarkable range of platforms, formats and hyphenated systems with functions ranging from chromatographic preconcentration to large-scale preparative extraction. Monolith surface chemistry, morphology and the approaches to monolith synthesis are discussed with regards to these emerging roles.     

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.     

蛋白质组学中的固定化酶反应器制备的研究进展

固定化酶反应器作为蛋白质组学研究中"bottom-up"策略重要的组件,具有酶解快速、酶解效率高、酶稳定性和活性高、简单易操作、能够与多种检测方式联用等优点,对于发展高效快速的蛋白质组学分析方法具有重要意义。本文就固定化酶反应器的制备方法及其在蛋白质组学中的应用做简单的概述,着重介绍酶的固定方法、固定化酶的载体、用于固定的酶的种类。近几年固定化酶反应器的研究集中于提高固酶量、保持酶活性、增加酶解效率、减小非特异性吸附等方面。研究结果表明,采用纳米材料、整体材料等新型载体,提高载体亲水性,采用多酶同时酶解等方法能够有效改善固定化酶反应器的性能,提高蛋白质的鉴定效率。     

Synthesis of zirconia monoliths for chromatographic separations

The aim of this work is to join the advantages of two different kinds of stationary phases: monolithic columns and zirconia-based supports. On the one hand, silica monolithic columns allow a higher efficiency with a lower back-pressure than traditional packed columns. On the other hand, chromatographic stationary phases based on zirconia have a higher thermal and chemical stability and specific surface properties. Combining these advantages, a zirconia monolith with a macroporous framework could be a real improvement in separation sciences. Two main strategies can be used in order to obtain a zirconia surface on a monolithic skeleton: coating or direct synthesis. The coverage by a zirconia layer of the surface of a silica-based monolith can be performed using the chemical properties of the silanol surface groups. We realized this coverage using zirconium alkoxide and we further grafted n-dodecyl groups using phosphate derivatives. Any loss of efficiency was observed and fast separations have been achieved. The main advance reported in this paper is related to the preparation of zirconia monoliths by a sol-gel process starting from zirconium alkoxide. The synthesis parameters (hydrolysis ratio, porogen type, precursor concentration, drying step, etc.) were defined in order to produce a macroporous zirconia monoliths usable in separation techniques. We produced various homogeneous structures: zirconia rod 2 cm long with a diameter of 2.3 mm, and zirconia monolith inside fused silica capillaries with a 75 microm I.D. These monoliths have a skeleton size of 2 microm and have an average through pore size of 6 microm. Several separations have been reported.     

Recent advances in the preparation and application of monolithic capillary columns in separation science

Novel column technologies involving various materials and efficient reactions have been investigated for the fabrication of monolithic capillary columns in the field of analytical chemistry. In addition to the development of these miniaturized systems, a variety of microscale separation applications have achieved noteworthy results, providing a stepping stone for new types of chromatographic columns with improved efficiency and selectivity. Three novel strategies for the preparation of capillary monoliths, including ionic liquid-based approaches, nanoparticle-based approaches and “click chemistry”, are highlighted in this review. Furthermore, we present the employment of state-of-the-art capillary monolithic stationary phases for enantioseparation, solid-phase microextraction, mixed-mode separation and immobilized enzyme reactors. The review concludes with recommendations for future studies and improvements in this field of research.     

Are we approaching a post‐monolithic era?

Thirty years after their introduction, monolithic stationary phases are an important member of chromatographic phases. When compared to conventional particulate materials, the continuous internal structure of both inorganic silica and organic polymer monoliths allows some hydrodynamic and analytical possibilities that are not provided by conventional particulate stationary phases. Polymer‐based monolithic stationary phases offer simple preparation and straightforward surface modification, which makes them very versatile materials that are applicable, for example, as chromatographic stationary phases, sample enrichment units, enzymatic reactors, and external trigger‐responding materials. On the other hand, current polymer monoliths cannot compete with efficiency provided by superficially porous and sub 2 µm particles. In this highlight article, I take advantage of the 30th anniversary of their introduction to discuss several concerns related to polymer‐based monolithic stationary phases. Particularly, I focus on preparation repeatability, porous properties, swelling of the polymers in organic solvents, column efficiency for small molecules, and heterogeneity of dominant flow‐through pores. In the end, I offer three possible approaches on how to overcome drawbacks related to stationary phases heterogeneity to further increase the applicability of polymer‐based monolithic stationary phases.     

Capillary electrochromatography with monolithic silica columns. IV. Electrochromatographic characterization of polar bonded monolithic stationary phases having surface-bound cyano functionalities

Two polar ligands, namely 3-hydroxypropionitrile and 1H-imidazole-4,5-dicarbonitrile (IDCN) were covalently attached to epoxy-activated silica-based monolithic capillary columns via an epoxide ring-opening reaction to yield CN-OH-Monolith and 2CN-OH-Monolith, respectively. The silica monolith was prepared by a sol-gel process, and the resulting "rod-like" stationary phase was subjected to pore tailoring with an alkaline solution to convert small pore domains to mesopore domains, thus yielding a monolith with bimodal pore structure consisting of flow through pores (i.e., flow channels for mobile-phase flow) and mesopores that provide most of the adsorption capacity of the monolith toward the separated solutes. The two polar monoliths, CN-OH-Monolith and 2CN-OH-Monolith, were evaluated in normal-phase CEC with organic-rich mobile phases less polar than the stationary phase. The 2CN-OH-Monolith bearing more polar functions than the CN-OH-Monolith exhibited more retention and improved selectivity toward model polar solutes.     

高效液相色谱整体柱在药物分离分析中的应用进展

本文对高效液相整体柱在药物分离分析方面的应用进行了综述.主要介绍了以烷氧基硅烷为主要原料,采用溶胶-凝胶法制备的硅胶整体柱,由于其具有微米级通孔结构和大的比表面积,他们在高效、快速分离小分子物质方面得到广泛地应用.对于聚合物整体柱,主要介绍了包括分子印迹聚合物在内的有机聚合物整体柱在药物分离、生物样品的处理等方面的应用.     

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.     

Silica gel-based monoliths prepared by the sol-gel method: facts and figures

There is a great deal of interest in continuous beds as stationary phases for both HPLC and CEC. There are various ways to prepare monoliths, by polymerization of organic species or by polymerization of silicon alkoxides. The former method has recently been reviewed, while silica based monoliths are now commercially available. The purpose of this paper is to deal with the problems associated with silica based monoliths. The most important problem is obviously the cracking and the shrinkage of the bed during drying. The second problem is monolith cladding. Much literature has been published but no definitive solution is available and thus a wide research area remains open. Monoliths are a compromise between loadability, permeability and mass transfer kinetics. Due to the better mass transfer properties of a monolithic skeleton over distinct particles, high flow rates and high speed separations are possible.