Open-tubular capillary columns with a porous layer of monolithic polymer for highly efficient and fast separations in electrochromatography

Open-tubular columns for CEC separations having inner-wall coated with a thin layer of porous monolithic polymer have been studied. A two-step process including (i) UV-initiated polymerization leading to a layer of porous poly(butyl methacrylate-co-ethylene dimethacrylate), and (ii) UV-initiated grafting of ionizable monomers appear to be well suited for the preparation of these columns. The thickness of the porous polymer layer is controlled by the percentage of monomers in the polymerization mixture and/or length of the irradiation time. The layer thickness significantly affects retention, efficiency, and resolution in open-tubular CEC. Under optimized conditions, column efficiencies up to 400,000 plates/m can be achieved. Use of higher temperature and application of pressure enables a significant acceleration of the open-tubular CEC separations.     

Preparation and HPLC applications of rigid macroporous organic polymer monoliths

Rigid porous polymer monoliths are a new class of materials that emerged in the early 1990s. These monolithic materials are typically prepared using a simple molding process carried out within the confines of a closed mold. For example, polymerization of a mixture comprising monomers, free-radical initiator, and porogenic solvent affords macroporous materials with large through-pores that enable applications in a rapid flow-through mode. The versatility of the preparation technique is demonstrated by its use with hydrophobic, hydrophilic, ionizable, and zwitterionic monomers. Several system variables can be used to control the porous properties of the monolith over a broad range and to mediate the hydrodynamic properties of the monolithic devices. A variety of methods such as direct copolymerization of functional monomers, chemical modification of reactive groups, and grafting of pore surface with selected polymer chains is available for the control of surface chemistry. Since all the mobile phase must flow through the monolith, the convection considerably accelerates mass transport within the molded material, and the monolithic devices perform well, even at very high flow rates. The applications of polymeric monolithic materials are demonstrated mostly on the separations in the HPLC mode, although CEC, gas chromatography, enzyme immobilization, molecular recognition, advanced detection systems, and microfluidic devices are also mentioned.     

Capillary electrochromatography with zwitterionic stationary phase on the lysine-bonded poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column

A polymer-based neutral monolithic capillary column was prepared by radical polymerization of glycidyl methacrylate and ethylene dimethacrylate in a 100 mum id fused-silica capillary, and the prepared monolithic column was subsequently modified based on a ring opening reaction of epoxide groups with 1 M lysine in solution (pH 8.0) at 75 degrees C for 10 h to produce a lysine chemically bonded stationary phases in capillary column. The ring opening reaction conditions were optimized so that the column could generate substantial EOF. Due to the zwitterionic functional groups of the lysine covalently bonded on the polymer monolithic rod, the prepared column can generate cathodic and anodic EOF by varying the pH values of running buffer during CEC separation. EOF reached the maximum of -2.0 x 10(-8) m2v(-1)s(-1) and 2.6 x 10(-8) m2v(-1)s(-1) with pH of the running buffer of 2.25 and 10, respectively. As a consequence, neutral compounds, ionic solutes such as phenols, aromatic acids, anilines, and basic pharmaceuticals were all successfully separated on the column by CEC. Hydrophobic interaction is responsible for separation of neutral analytes. In addition, the electrostatic and hydrophobic interaction and the electrophoretic migration play a significant role in separation of the ionic or ionizable analytes.     

Separation of basic, acidic and neutral compounds by capillary electrochromatography using uncharged monolithic capillary columns modified with anionic and cationic surfactants

A mode of capillary electrochromatography (CEC), based on the dynamical adsorption of surfactants on the uncharged monolithic stationary phases has been developed. The monolithic stationary phase, obtained by the in situ polymerization of butyl methacrylate with ethylene dimethacrylate, was dynamically modified with an ionic surfactant such as the long-chain quaternary ammonium salt of cetyltrimethylammonium bromide (CTAB) and long-chain sodium sulfate of sodium dodecyl sulfate (SDS). The ionic surfactant was adsorbed on the surface of polymeric monolith by hydrophobic interaction, and the ionic groups used to generate the electroosmotic flow (EOF). The electroosmotic mobility through these capillary columns increased with increasing the content of ionic surfactants in the mobile phase. In this way, the synthesis of the monolithic stationary phase with binary monomers can be controlled more easily than that with ternary monomers, one of which should be an ionic monomer to generate EOF. Furthermore, it is more convenient to change the direction and magnitude of EOF by changing the concentration of cationic or anionic surfactants in this system. An efficiency of monolithic capillary columns with more than 140000 plates per meter for neutral compounds has been obtained, and the relative standard deviations observed for to and retention factors of neutral solutes were about 0.22% and less than 0.56% for ten consecutive runs, respectively. Effects of mobile phase composition on the EOF of the column and the retention values of the neutral solutes were investigated. Simultaneous separation of basic, neutral and acidic compounds has been achieved.     

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.     

微电渗流毛细管原位柱的制备及性能考察

 采用二元致孔剂原位聚合的方法制备了一种新型微电渗流毛细管原位柱。与三元致孔剂制柱方法相比 ,具有制备过程简单、重复性好、能够方便地通过改变致孔剂配比来改变柱床的孔径和孔结构的特点。得到的毛细管柱内部结构均匀 ,通透性好。通过对改变不同致孔剂配比所制备的原位柱的孔结构特征及电渗流情况考察 ,及对柱长和柱径与电渗流的关系的探讨 ,发现制备的原位柱在较高 pH值和较高的有机改性剂浓度条件下 ,电渗流均能保持在较低值 ,可以适应不同电泳分离模式的需要。     

Preparation and characterization of molecularly imprinted monolithic column based on 4-hydroxybenzoic acid for the molecular recognition in capillary electrochromatography

A novel prepared method of molecularly imprinted monolithic polymers (MIPs) using 4-hydroxybenzoic acid (4-HBA) as templates for capillary electrochromatography (CEC) was developed. A strategy of high concentration of monomers in the pre-polymerization mixture was used to fulfil the solubility of polar imprinted molecule and reduction of the interference during complex formation. The imprinted polymer capillary monolithic column was synthesized by an in situ therm-initiated copolymerization of methacrylic acid (MAA) and ethylene glycol dimethacrylate with a mixture of toluene-isooctane as a porogenic solvent in the presence of a polar model imprinting molecule, 4-HBA. On the resultant MIP monolithic column, the effect of parameter of CEC on electroosmotic flow (EOF) and the retention of 4-HBA was investigated. The column efficiency of the imprinted molecule, 4-HBA, was 13,000 plates/m. The resolution of isomers of HBA was 5.0 and good molecular recognition was achieved for 4-HBA.     

Preparation and application of hydrophobic hybrid monolithic columns containing polyhedral oligomeric silsesquioxanes for capillary electrochromatography

Hydrophobic organic-inorganic hybrid monolithic columns were synthesized via thermally initiated free radical polymerization with the confines of 75 μm id capillary using a polyhedral oligomeric silsesquioxane (POSS) reagent containing eight or more methacrylate groups as the crosslinker. Three organic functional monomers, butyl methacrylate (BuMA), lauryl methacrylate (LMA) and methacrylic acid (MAA), were selected and copolymerized with the POSS in the presence of 1-propanol and 1,4-butanediol to prepare the poly(POSS-co-BuMA), poly(POSS-co-LMA), and poly(POSS-co-MAA) monoliths, respectively. The 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) was copolymerized as ionizable monomer into the poly(POSS-co-BuMA) and poly(POSS-co-LMA) for the generation of EOF in capillary electrochromatography (CEC). A hybrid poly(POSS-co-LMA-co-MAA) monolith was also similarly prepared by copolymerizing ternary monomers of POSS, LMA, and MAA, and compared with poly(POSS-co-BuMA), poly(POSS-co-LMA), and poly(POSS-co-MAA) monoliths. The resulting four kinds of POSS-contained hybrid monoliths exhibited good permeability and mechanical stability. Their column efficiencies were evaluated by the separation of alkylbenzene homologues and polar compounds in CEC. The results indicated that the highest efficiencies of 194,100 and 102,100 theoretical plates per meter for thiourea and benzene were obtained on the poly(POSS-co-LMA-co-MAA) monolith. Additionally, the poly(POSS-co-LMA-co-MAA) monolith exhibited better selectivity for separation of polar compounds than those of other hybrid monoliths.     

Preparation and evaluation of the highly cross‐linked poly(1‐hexadecane‐co‐trimethylolpropane trimethacrylate) monolithic column for capillary electrochromatography

In this paper, a novel highly cross‐linked porous monolithic stationary phase having a long alkyl chain ligand (C16) was introduced and evaluated in CEC. The monolithic stationary phase was prepared by in situ copolymerization of 1‐hexadecene, trimethylolpropane trimethacrylate, and 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid (AMPS) in the presence of ternary porogenic solvent (cyclohexanol/1,4‐butanediol/water). In preparing monoliths, the ternary cross‐linker trimethylolpropane trimethacrylate was usually applied to preparing molecularly imprinted polymers or molecularly imprinted solid‐phase extraction, instead of binary cross‐linker ethylene dimethacrylate. 1‐Hexadecene was introduced to provide the non‐polar sites (C16) for chromatographic retention, while AMPS was used to generate the EOF for transporting the mobile phase through the monolithic capillary. Monolithic columns were prepared by optimizing proportion of porogenic solvent and AMPS content in the polymerization solution as well as the cross‐linkers. The monolithic stationary phases could generate a strong and stable EOF in various pH values and exhibit an RP‐chromatographic behavior for neutral compounds. For charged compounds, the separation was mainly based on the association of hydrophobic, electrostatic and electrophoretic interaction.     

Development of acrylate-based monolithic stationary phases for electrochromatographic separations

Organic monolithic stationary phases were synthesized in fused-silica capillaries. They were prepared by in situ polymerization under UV irradiation of various alkyl acrylates, 1,3-butanediol diacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid in a ternary porogenic solvent. The resulting stationary phases were tested in CEC. The influence of UV irradiation energy on the resulting separative performances of the monoliths was studied. It was thus demonstrated that the use of hexyl acrylate rather than butyl acrylate and lauryl methacrylate gives highly efficient monoliths (more than 300 000 plates per meter) with optimized EOF. It was also confirmed that the mobile phase ionic strength may affect significantly the separation efficiency. The influence of the nature of the mobile phase organic modifier (ACN or methanol) on EOF, retention, efficiency, and selectivity was studied and differences were observed. Finally, the performances of monolithic stationary phases developed and optimized for CEC separations were evaluated in nanoLC.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

Preparation and evaluation of rigid porous polyacrylamide-based strong cation-exchange monolithic columns for capillary electrochromatography

A CEC monolithic column with strong cation-exchange (SCX) stationary phase based on hydrophilic monomers was prepared by in situ polymerization of acrylamide, methylenebisacrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a complete organic binary porogenic solvent consisting of DMSO and dodecanol. The sulfonic groups provided by the monomer AMPS on the surface of the stationary phase generate an EOF from anode to cathode, and serve as an SCX stationary phase at the same time. The monolithic stationary phase exhibited normal-phase chromatographic behavior for neutral analytes. For charged analytes, electrostatic interaction/repulsion with the monolith was observed. The strong SCX monolithic column has been successfully employed in the electrochromatographic separation of basic drugs, peptides, and alkaloids extracted from natural products.     

Acrylic-based high internal phase emulsion polymeric monolith for capillary electrochromatography

The use of high internal phase emulsion polymers (polyHIPEs) for CEC applications has remained relatively unexplored. A few reports exist in the literature for the preparation of similar structures. In this study, polyHIPEs having high porosity, and interconnected open-cell structure, were introduced and evaluated as stationary phase for CEC. The polyHIPE monolithic columns were prepared by the in situ polymerization of isodecylacrylate (IDA) and divinylbenzene (DVB) in the continuous phase of a high internal phase emulsion (HIPE). Due to its well-defined polyHIPE structure with interconnected micron size spherical voids, the columns synthesized with different initiator concentrations were successfully used for the separation of alkylbenzenes. Furthermore, the columns indicated a strong electroosmotic flow (EOF) without any additional EOF generating monomer probably due to the presence of ionizable sulfate groups coming from the water-soluble initiator used in the preparation of polyHIPE matrix. The best chromatographic performance in the separation of alkylbenzenes was achieved by using 70% ACN in the mobile phase with high column efficiency (up to 200 000 plates/m).     

Monolithic columns with a gradient of functionalities prepared via photoinitiated grafting for separations using capillary electrochromatography

Stationary phases for capillary electrochromatography with a longitudinal gradient of functionalities have been prepared via photoinitiated grafting of polymer chains onto the pore surface of a porous polymer monolith. In order to achieve the desired retention and electroosmotic flow, the hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) monolith with optimized porous properties was grafted with a layer of ionizable polymer, poly(2-acrylamido-2-methyl-1-propanesulfonic acid). A moving shutter and a neutral density filter were used to control the dose of UV light received at different locations along the monolith in order to create the longitudinal gradient of functionalities. Formation of the desired gradients was confirmed using electron probe microanalysis of different locations along the column. The preparation technique significantly affects performance in the CEC mode as demonstrated on the separations of a model mixture using columns both with homogeneous distribution of grafts and with a gradient of functionality. Columns grafted with the gradient of functionalities were found superior to those functionalized uniformly. A comparison of the performance of the gradient column with another containing evenly distributed functionalities showed the performance benefits of the "gradient" column.     

Preparation and characterization of a molecularly imprinted monolithic column for pressure‐assisted CEC separation of nitroimidazole drugs

A polymethacrylate‐based molecularly imprinted monolithic column bearing mixed functional monomers, using non‐covalent imprinting approach, was designed for the rapid separation of nitroimidazole compounds. The new monolithic column has been prepared via simple in situ polymerization of 2‐hydroxyethyl methacrylate, dimethylaminoethyl methacrylate and ethylene dimethacrylate, using (S)‐ornidazole ((S)‐ONZ) as template in a binary porogenic mixture consisting of toluene and dodecanol. The composition of the polymerization mixture was systematically altered and optimized by altering the amount of monomers as well as the composition of the porogenic solvent. The column performance was evaluated in pressure‐assisted CEC mode. Separation conditions such as pH, voltage, amount of organic modifier and salt concentration were studied. The optimized monolithic column resulted in excellent separation of a group of structurally related nitroimidazole drugs within 10 min in isocratic elution condition. Column efficiencies of 99 000, 80 000, 103 000, 60 000 and 99 000 plates/m were obtained for metronidazole, secnidazole, ronidazole, tinidazole and dimetridazole, respectively. Parallel experiments were carried out using molecularly imprinted and non‐imprinted capillary columns. The separation might be the result of combined effects including hydrophobic, hydrogen bonding and the imprinting cavities on the (S)‐ONZ‐imprinted monolithic column.     

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.     

丁基胺丙基硅胶毛细管整体柱的制备及其电色谱性能研究

采用溶胶-凝胶技术制备了丁基胺丙基硅胶毛细管整体柱,此整体固定相表面同时含有能产生阳极的电渗流的仲胺官能团和产生疏水作用的正丁基和丙基官能团。对所制备的整体柱电色谱性能进行了详细的表征和分析。考察了流动相pH值对电渗流的影响;对烷基苯同系物、有机酸酸性化合物和苯胺类碱性化合物保留行为进行了研究,并对其可能的保留机理进行了探讨。实验结果表明,对于中性化合物的保留机理主要基于反相作用;而对于酸性化合物的保留行为则是基于混合模式作用机理,即除了电泳作用外,还包括阴离子交换和疏水作用。碱性化合物在丁基胺丙基硅胶毛细管整体柱上的峰形较好,没有明显的峰拖尾现象。     

Preparation of high efficiency and highly retentive monolithic silica capillary columns for reversed-phase chromatography by chemical modification by polymerization of octadecyl methacrylate

High efficiency and highly retentive monolithic silica capillary columns were obtained by polymerization of octadecyl methacrylate using alpha,alpha'-azobis-isobutyronitrile (AIBN) as a free radical initiator. Hybrid type monolithic silica columns (25 cm total length x 200 microm I.D.) prepared from a mixture of tetramethoxysilane and methyltrimethoxysilane were used as a support. The effects of the monomer and the radical initiator concentrations in the reaction mixture were examined. The performance of the columns was tested in terms of column efficiency and retention behavior by using alkylbenzenes and a few other compounds as solutes and compared with that of hybrid monolithic silica columns modified with octadecylsilyl-(N,N-diethylamino)silane (ODS-DEA). Highly retentive monolithic silica columns were obtained by polymerization at high monomer concentrations. Although a decrease in column efficiency was observed with the increase in the monomer concentration in a feed mixture, an improvement in efficiency was achieved (a plate height value lower than 10 microm) by increasing an initiator concentration without significant variations in column retention properties. Results obtained by polymerization using other monomers are also presented to demonstrate the applicability of the preparation method.     

The use of derivatized cyclodextrins as solubilizing agents in the preparation of macroporous polymers employed as amphiphilic continuous beds in capillary electrochromatography

Employing solubilization by complexation with CDs, new mixed-mode monolithic stationary phases for CEC and micro-LC were synthesized. Free radical copolymerization was performed in aqueous solution with a CD-solubilized hydrophobic monomer, a water-soluble crosslinker (piperazinediacrylamide), and a charged monomer (vinylsulfonic acid). Different hydrophobic methacrylate monomers (isobornyl, adamantyl, cyclohexyl, and phenyl methacrylate) were investigated. Chromatographic properties of the synthesized monoliths were studied with aqueous and nonaqueous mobile phases with hydrophobic and polar analytes. Due to the amphiphilic nature of the polymers synthesized, the elution orders obtained correspond to the RP mode and to the normal-phase mode dependent on the polarity of the mobile phase. However, observations made with polar solutes and polar mobile phase can only be explained by a mixed-mode retention mechanism. The influence of the total monomer concentration (%T) on the chromatographic properties and on the specific permeability was elucidated. Run-to-run, day-to-day, and capillary-to-capillary reproducibility of electroosmotic mobility and retention factors were determined. Comparison of retention data with those of a commercial octadecyl silica gel HPLC column reveals that the methylene selectivity of the monolithic capillaries prepared in this study is very similar to that of routinely used octadecyl silica gels.     

A novel ionic liquid monolithic column and its separation properties in capillary electrochromatography

A novel ionic liquid (IL) monolithic capillary column was successfully prepared by thermal free radical copolymerization of IL (1-vinyl-3-octylimidazolium chloride, ViOcIm⁺Cl⁻) together with lauryl methacrylate (LMA) as the binary functional monomers and ethylene dimethacrylate (EDMA) as the cross-linker in binary porogen. The proportion of monomers, porogens and cross-linker in the polymerization mixture was optimized in detail. The resulting IL-monolithic column could not only generate a stable reversed electroosmotic flow (EOF) in a wide pH range (2.0–12.0), but also effectively eliminate the wall adsorption of the basic analytes. The obtained IL-monolithic columns were examined by scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR). These results indicated that the IL-monolithic capillary column possessed good pore properties, mechanical stability and permeability. The column performance was also evaluated by separating different kinds of compounds, such as alkylbenzenes, thiourea and its analogues, and amino acids. The lowest plate height of ∼6.8 μm was obtained, which corresponded to column efficiency (theoretical plates, N) of ∼147,000 plates m⁻¹ for thiourea. ILs, as a new type of functional monomer, present a promising option in the fabrication of the organic polymer-based monolithic columns in CEC.