Mixed-mode reversed-phase and ion-exchange monolithic columns for micro-HPLC

This paper describes the fabrication of RP/ion-exchange mixed-mode monolithic materials for capillary LC. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (gamma-MAPS), monoliths were formed by copolymerisation of pentaerythritol diacrylate monostearate (PEDAS), 2-sulphoethyl methacrylate (SEMA) with/without ethylene glycol dimethacrylate (EDMA) within 100 microm id capillaries. In order to investigate the porous properties of the monoliths prepared in our laboratory, mercury intrusion porosimetry, SEM and micro-HPLC were used to measure the monolithic structures. The monolithic columns prepared without EDMA showed bad mechanical stability at high pressure, which is undesirable for micro-HPLC applications. However, it was observed that the small amount (5% w/w) of EDMA clearly improved the mechanical stability of the monoliths. In order to evaluate their application for micro-HPLC, a range of neutral, acidic and basic compounds was separated with these capillaries and satisfactory separations were obtained. In order to further investigate the separation mechanism of these monolithic columns, comparative studies were carried out on the poly(PEDAS-co-SEMA) monolithic column and two other monoliths, poly(PEDAS) and poly(PEDAS-co-2-(methacryloyloxy)ethyl-trimethylammonium methylsulphate (METAM)). As expected, different selectivities were observed for the separation of basic compounds on all three monolithic columns using the same separation conditions. The mobile phase pH also showed clear influence on the retention time of basic compounds. This could be explained by ion-exchange interaction between positively charged analytes and the negatively charged sulphate group.     

手性有机聚合物毛细管电色谱整体柱研究进展

毛细管电色谱具有高分离效率、多种保留机制和高选择性的优点。近年来,利用毛细管电色谱进行对映异构体的手性拆分受到了广泛关注。相对于传统的填充柱和开管柱,整体柱在手性拆分方面具有显著优势。与手性硅基整体柱相似,手性有机聚合物整体柱由于具有大孔,可产生较高的流速而压降较小。该文综述了近十年手性有机聚合物整体柱制备方法的研究进展,将手性有机聚合物整体柱的制备方法分为"原位聚合法"和"手性修饰法"两种,虽然前者制备简单并广泛应用于早期研究,但聚合混合液成分的微小改变即可引起最终聚合物的形态变化,并且大部分带丙烯基的手性选择剂较难从市场购买。因此,手性修饰法因作为手性选择剂基质的整体柱制备且优化只需进行一次的优势而受到普遍关注。亲核取代、杂环开环和点击化学是常用的修饰手段。该文总结了这两种制备方法的应用,同时对未来的研究方向提出参考性意见。     

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.     

Affinity monolithic capillary columns for glycomics/proteomics: 1. Polymethacrylate monoliths with immobilized lectins for glycoprotein separation by affinity capillary electrochromatography and affinity nano-liquid chromatography in either a single column or columns coupled in series

In this report, microcolumn separation schemes involving monolithic capillary columns with immobilized lectins, and relevant to nanoglycomics/nanoproteomics were introduced. Positive and neutral monoliths based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) were designed for achieving lectin affinity chromatography (LAC) by nano-LC and CEC. The positive monoliths (i.e., monoliths with cationic sites) afforded relatively high permeability in nano-LC but lack predictable EOF magnitude and direction, while neutral monoliths provided a good compromise between reasonable permeability in nano-LC and predictable EOF in CEC. Lectin affinity nano-LC permitted the enrichment of classes of different glycoproteins having similar N-glycans recognized by the immobilized lectin, whereas lectin affinity CEC provided the simultaneous capturing and separation of different glycoproteins due to differences in charge-to-mass ratio. Also, this investigation demonstrated for the first time the coupling of lectin capillary columns in series (i.e., tandem columns) for enhanced separation of glycoproteins by LAC using the CEC modality. Furthermore, in the coupled columns format, glycoforms of a given glycoprotein were readily separated.     

Recent progress in polar stationary phases for CEC

This review summarizes most of the recent developments in the preparation and application of polar stationary phases for CEC covering the literature published since the year 2004. These polar stationary phases have been adopted for separation of analytes by the modes of packing column CEC, open-tubular CEC (o-CEC) and monolithic column CEC. Currently, development of o-CEC using biomolecules, such as protein and DNA, as the immobilized ligands is highlighted partly due to the simplicity of preparation. Furthermore, monolithic columns have been extended quickly, particularly inorganic materials-based monoliths, such as silica, zirconia, hafnium, etc., as an alternative to packed columns have been developed quickly.     

Stellan Hjertén's contribution to the development of monolithic stationary phases

This overview is presented to celebrate the birthday of one of the luminaries of the separation science and my friend - Stellan Hjertén. He made significant contributions to a variety of areas in separation science such as electrophoresis, LC, and CEC to name just a few. Since the scope of his work was enormous, this review will focus only on a single aspect of his scientific activities, the design and applications of monolithic materials. During the years starting from 1989, Stellan Hjertén published many excellent papers concerning the preparation of acrylamide chemistry-based monoliths and their use in both micro-HPLC and CEC. The following text details his works in the field.     

Performance limits of monolithic and packed capillary columns in high-performance liquid chromatography and capillary electrochromatography

A method is proposed for the comprehensive characterization and comparison of columns in the high-performance liquid chromatographic (HPLC) and capillary electrochromatographic (CEC) modes. Using this approach, column parameters such as the number of plates, the eddy-diffusion and mass-transfer contributions to peak broadening, the permeability, and the analysis time are incorporated in a single graph and a comparison in terms of efficiency and speed is obtained. The chromatographic performance of silica-based and polymer-based monolithic capillary columns is discussed and a comparison is made with the performance of packed columns. Also, the potential of ultra-high-pressure liquid chromatography is discussed in this context. In the HPLC mode, the best results were obtained with silica monoliths; in the CEC mode, the low-density methacrylate-ester-based monoliths showed the best performance.     

Influence of the hydrothermal treatment on the chromatographic properties of monolithic silica capillaries for nano-liquid chromatography or capillary electrochromatography

In the last decade, silica monolithic capillaries have focused more and more attention on miniaturized separation techniques like capillary electrochromatography (CEC), nano-liquid chromatography (nano-LC) and chip electrochromatography owing to their unique chromatographic properties and their simplified preparation compared with packed columns. They are synthesized according to a sol-gel multi-step process that includes, after a gelation step at 40 degrees C leading to the formation of the macropores network and the silica skeleton, a post-gelation step (hydrothermal treatment at 120 degrees C in basic medium) that allows to tailor the mesopores and finally a calcination or a washing step to remove remaining polymers. In order to reduce the synthesis time, the number of synthesis steps and above all the temperature synthesis, to adapt the synthesis of such silica monoliths in polymeric microsystem devices, we extensively studied the influence of the hydrothermal treatment and its duration on textural (pore size distribution) and chromatographic properties (retention, efficiency) of in situ-synthesized capillary monoliths in nano-LC and CEC. This study was performed on pure silica and octyl chains grafted silica monoliths. Untreated monoliths show small pores (<6 nm), whereas hydrothermally treated monoliths exhibit medium and large mesopores (8-17 nm). It was demonstrated that the hydrothermal treatment at 120 degrees C was not necessary for pure silica monolithic capillaries dedicated to normal phase liquid chromatography or hydrophilic interaction liquid chromatography (HILIC) and electrochromatography: the suppression of the hydrothermal treatment did not impair efficiencies in CEC and in nano-LC but contributed to increase in retention factors. Minimal plate heights of ca. 5 microm in CEC and 6 microm in nano-LC were obtained with or without hydrothermal treatment with bare silica. In the same way, the hydrothermal treatment was not necessary for grafted silica monoliths only dedicated to CEC. However, the results clearly indicate that the hydrothermal treatment becomes essential before grafting in order to preserve the efficiency of the monolithic silica capillaries dedicated to nano-LC: in this particular case, the suppression of the hydrothermal treatment leads approximately to a loss of a factor two in efficiency.     

Extending the array of crosslinkers suitable for the preparation of polymethacrylate-based monoliths

The in situ preparation of monolithic capillary columns comprising copolymers of butyl methacrylate with ethylene dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and pentaerythritol tetraacrylate using thermal polymerization within 250 microm ID capillaries and their application for micro-HPLC separations of proteins has been studied. For all crosslinkers, optimization of the porogenic mixture consisting of 1-propanol and 1,4-butanediol yielded monoliths with pore sizes above 1 microm suitable for rapid separations at low back pressure. Very good separations were achieved for a protein mixture consisting of ribonuclease A, cytochrome c, myoglobin, and ovalbumin with all tested columns.     

Optimized preparation of poly(styrene-co- divinylbenzene-co-methacrylic acid) monolithic capillary column for capillary electrochromatography

Preparation of a poly(styrene-co-divinylbenzene-co-methacrylic acid) monolithic stationary phase for the use in capillary electrochromatography (CEC) has been improved by optimizing the polymerization conditions. It is observed that the reaction time strongly affects column efficiency, while the proportion of isooctane in porogen influences peak symmetry of some solutes seriously. The lifetime of the monolithic columns prepared mainly depends on the pH of buffers used. Reproducibility of electroosmotic flow (EOF) from batch to batch columns are lower than 2.8% relative standard deviation. Unlike other types of capillary electrochromatographic monoliths, a pH-dependent EOF was observed on this type of column. Separation of various types of compounds including aromatic hydrocarbons, hormones, anilines, basic pharmaceuticals, and peptides was achieved. The facile preparation and wide application of this monolithic column may make styrene-based polymer a potential stationary phase in CEC.     

Voltage-assisted capillary LC of peptides using monolithic capillary columns prepared by ring-opening metathesis polymerization

We examined the use of monolithic capillary columns prepared via ring-opening metathesis polymerization (ROMP) for peptide separation in voltage-assisted capillary LC (voltage-assisted CLC). In order to demonstrate their potential for peptide separation, ROMP-derived monoliths with RP properties were prepared. The preparation procedure of monoliths was transferred from ROMP monoliths optimized for CLC. ROMP monoliths were synthesized within the confines of 200 microm id fused-silica capillaries with a length of 37 cm. After optimization of the chromatographic conditions, the separation performance was tested using a well-defined set of artificial peptides as well as two peptidic mixtures resulting from a tryptic digest of BSA as well as a collagenase digest of collagen. ROMP monoliths showed comparable performance to other monolithic separation media in voltage-assisted CLC published so far. Therefore, we conclude that by optimizing the composition of the ROMP monoliths as well as by using the controlled manner of their functionalization, ROMP monoliths bear a great potential in CLC and CEC.     

Recent advances in polymeric monolithic stationary phases for electrochromatography in capillaries and chips

This review article summarizes the advances made over the last two years in polymeric monoliths for capillary electrochromatography (CEC). It covers the scientific literature in the period extending form the second half of 2002 until the end of first half of 2004. Currently, there is an increasing interest in monolithic stationary phases in CEC as an alternative to particulate packed capillary columns due in major part to the simplicity of the in situ preparation of monolithic stationary phases and the availability of a wide chemistry for surface ligands, which allow for tailoring the chromatographic sorbent needed for solving a given separation problem(s). The various approaches, formats, and chemistries used for the preparation of monolithic stationary phases are described.     

毛细管电色谱中整体式高聚物毛细管柱技术的进展

对近期毛细管电色谱（CEC）中利用高分子聚合反应制备整体式（monolithic)毛细管柱的技术，特别对各种不同类型的整体式毛细管的制备方法进行了详细介绍；对该类毛细管用于不同样品的分离情况进行了总结。     

Theoretical and nomenclatural considerations of capillary electrochromatography with monolithic stationary phases

During the past decade, CEC has been one of the few novel achievements in the field of separation science attracting a wide interest. The technology progress permitted the realization of the long-sought idea to employ an electroosmotically driven flow through the columns improving the separations in terms of both resolution and efficiency. The early practical obstacles related to the use of conventional bead-packed columns have been solved by the introduction of continuous beds, also known as monoliths. Hitherto, various synthesis approaches have been successfully developed producing monolithic beds in situ in capillary columns, sharing similar physical structure built up of tiny particles (in the sub-microm range) that are covalently linked together and to the capillary wall. Parallel with the practical column technology studies, the theory of electrochromatography has been continuously developed, focusing on such basic issues as EOF characterization, separation efficiency, and peak dispersion effects. This review provides a short introduction to the theory of CEC with special attention to monolithic separation beds. The paper also summarizes the latest achievements in CEC and discusses the nomenclature, EOF characteristics, and some specific advantages of monolithic column technology.     

Novel preparation of organic polymer-based monolithic column by microwave irradation

Microwave irradiation was firstly attempted for the preparation of organic-based monoliths of poly（styrene-divinylbenzene- methacrylic acid）, which single step in situ polymerization was carried out during 15 min. The colunm permeability, electrophoretic and chromatographac behaviors were comparatively evaluated using pressure-assisted CEC, GEC and low pressure-driven separation modes. The largest theoretical plates for the preparing column could be close to 18,0000 plates/m for thiourea in the mode of p-CEC. It provided a viable alternative to traditional initiation means for the perparation of monolithic capillary columns.     

Alkylated poly(styrene-divinylbenzene) monolithic columns for mu-HPLC and CEC separation of phenolic acids

Macroporous poly(styrene-divinylbenzene) monolithic columns were prepared in fused silica capillaries of 100 microm id by in-situ copolymerization of styrene with divinylbenzene in the presence of propan-1-ol and formamide as the porogen system. The monoliths were subsequently alkylated with linear alkyl C-18 groups via Friedel-Crafts reaction to improve the retention and chromatographic resolution of strongly polar phenolic acids. A new thermally initiated grafting procedure was developed in order to shorten the time of the alkylation process. The grafting procedure was optimized with respect to the reaction temperature, time, the grafting reactant concentration, and the solvent used. The type of solvent and the grafting temperature are the most significant factors affecting the hydrodynamic properties, porosity, and efficiency of the columns. While the equivalent particle diameter of the grafted column increased, the capillary-like flow-through pore diameter decreased in comparison to non-alkylated monoliths. The hydrodynamic permeability of the monolith decreased, but the monolithic column still permitted fast micro-HPLC separations.     

Preparation and characterization of hexyl methacrylate monolithic columns for CEC

The preparation of hexyl methacrylate (HMA) monolithic columns for CEC separations has been investigated with two initiation systems: (i) ammonium peroxodisulphate and TEMED to activate the polymerization reaction, and (ii) by thermal initiation with AIBN. For both initiators, the influence of composition of porogenic solvent on morphological and chromatographic properties of monoliths was investigated. Two porogenic solvent systems, aqueous and non-aqueous media, were also studied for monolithic beds polymerized with AIBN. Under optimal conditions, low minimum plate heights (9.6 mum for peroxodisulphate, 8.4 and 10.0 mum for AIBN in aqueous and non-aqueous porogenic solvents, respectively) were obtained. A comparison in terms of chromatographic performance of HMA monoliths with butyl methacrylate columns polymerized with both initiators was also performed. The resulting HMA-based stationary phases also exhibited a good repeatability and column-to-column reproducibility, with RSD values below 5.6% in the studied electrochromatographic parameters. The potential of HMA-based columns was demonstrated by the analysis of complex mixtures of polyaromatic hydrocarbons and anabolic steroids.     

Practical aspects of using methacrylate-ester-based monolithic columns in capillary electrochromatography

Methacrylate-ester-based monoliths containing quaternary ammonium groups were prepared in situ in capillary columns and in simultaneous experiments in vials, employing thermal initiation. The chromatographic properties of the monoliths were determined with capillary electrochromatography (CEC), and their morphology was studied with mercury-intrusion porosimetry on the bulk materials. Materials with different, well repeatable pore-size distributions could be prepared. A satisfactory column-to-column and run-to-run repeatability was obtained for the electro-osmotic mobility, the retention characteristics (k-values) and the efficiency on the columns prepared and tested in the CEC mode. A relatively high electro-osmotic flow was observed in the direction of the positive electrode. The electro-osmotic mobility was found to be influenced only marginally by mobile-phase parameters such as the pH, ionic strength, and acetonitrile content. The retention behavior of the monolithic columns was similar to that of columns packed with C18-modified silica particles. Columns could be prepared with optimum plate heights ranging from 6 microm for unretained compounds to 20 microm for well retained (k=2.5) polyaromatic hydrocarbons. However, for specific analytes a - still unexplained - lower chromatographic column efficiency was observed.     

Acrylamide-based monoliths as robust stationary phases for capillary electrochromatography

A method is described for the synthesis of rigid, macroporous polymers (monoliths) to be used as stationary phases in capillary electrochromatography (CEC). The procedure reproducibly results in columns with good mechanical and chemical stability. Once the procedure was optimized, it yielded the desired CEC columns in nearly 100% of the cases. The batch-to-batch standard deviation of the migration of the electroosmotic flow (EOF) marker for nine randomly chosen columns was 5%. The polymerization is carried out inside the capillary, an aqueous phase is used as solvent. Monomers based on acrylamides with varying hydrophilicity were used to introduce the interactive moieties together with piperazine diacrylamide as cross-linker and vinylsulfonic acid as provider of the charged, EOF-producing moieties. The pore size of the monoliths was adjusted by adding varying amounts of ammonium sulfate to the reaction mixture. In this manner, the average pore size of a given monolith could be reproducibly adjusted to values ranging from 50 nm to 1.3 microm. The procedure was optimized for four particular types of monoliths, which differed in hydrophobicity. The latter was adjusted by introducing suitable co-monomers, such as alkyl chain-bearing molecules, into the monolithic structure. Attempts to systematically investigate the chromatographic behavior of the monolithic stationary phases were made, using a model mixture of aromatic compounds as sample. The standard deviations for the run-to-run reproducibility of the retention times for unretained and retained analytes were <1.5%. Flat Van Deemter curves were measured even at elevated flow-rates (2 mm/s). Plate heights between 10 and 15 microm were measured in this range. The retention order was taken as the principal indication for the chromatographic mode. The separation was found to be governed neither by pure reversed-phase nor by pure normal-phase chromatography, even on monoliths, where large amounts of C6 ligands had been introduced.