Polymethacrylate-type monoliths functionalized with chiral amino phosphonic acid-derived strong cation exchange moieties for enantioselective nonaqueous capillary electrochromatography and investigation of the chemical composition of the monolithic polymer

In situ prepared monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) (poly(GMA-co-EDMA)) capillary columns were activated to reactive thiol-monoliths and subsequently functionalized with (S)-N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutanephosphonic acid as chiral selector by radical addition to afford enantioselective strong cation exchanger (SCX) capillary columns (100 microm inner diameter (ID)). These monolithic capillaries were devised for the enantioseparation of chiral bases by nonaqueous and aqueous capillary electrochromatography (CEC) and the results obtained for mefloquine and its tert-butylcarbamate as test compounds were compared to those obtained with particulate silica-based analogs (packed columns). Despite abolishment of nonspecific ionic interactions between the cationic solutes and residual silanols that may diminish separation factors of the silica-based chiral SCX particles, the poly(GMA-co-EDMA)-supported SCX monolith did not, as expected, show better enantioselectivities, which was assumed to be due to detrimental nonspecific interactions between the analytes and the lipophilic polymer backbone. In order to minimize these unfavorable contributions, less lipophilic monoliths were developed by copolymerization of different amounts of the hydrophilic monomer 2-hydroxyethyl methacrylate (HEMA) with GMA and EDMA, leading to GMA-co-HEMA-co-EDMA-terpolymeric monoliths. By this increase of the hydrophilicity of the monolithic support the enantioselectivity of the resultant SCX stationary phase could be enhanced and reached values comparable to the packed silica-based enantioselective SCX capillaries. Additionally, the mobile phase composition and other variables were examined and it could be shown that the separation factors are considerably affected by diverse parameters such as acetonitrile-methanol ratio and type and concentration of the counterion. Mefloquine enantiomers could be separated with alpha-values up to 1.56 and a maximum plate count of ca. 60,000 m(-1) could be achieved.     

Advances in capillary electrochromatography and micro-high performance liquid chromatography monolithic columns for separation science

Over the last decade, monoliths or continuous beds have emerged as an alternative to traditional packed-bed columns for use in capillary electrochromatography (CEC) and micro-high performance liquid chromatography (micro-HPLC). Monolithic columns can be divided into two categories: silica-based monolithic columns and rigid organic polymer-based monolithic columns resulting from the polymerization of acrylamide, styrene, acrylate or methacrylate monomers. In this paper, the chemistry and most recent applications of these various types of monoliths in both CEC and micro-HPLC are presented.     

Amino-Functionalized Monolithic Poly(glycidyl methacrylate-<Emphasis Type="Italic">co</Emphasis>-divinylbenzene) Ion-Exchange Stationary Phases for the Separation of Oligonucleotides

Monolithic capillary columns were prepared by thermal initiated copolymerization of glycidyl methacrylate (GMA) and divinylbenzene (DVB) inside silanized 200 µm i.d. fused silica capillaries. Polymerization mixtures containing different amounts of porogen (1-decanol and tetrahydrofuran (THF)) and different ratios of monomer and crosslinker were used for synthesis. For characterization the pore size distribution profiles of the resulting monoliths were determined by mercury intrusion porosimetry. The morphology of the copolymer was investigated by scanning electron micrographs (SEM). A high linear dependence between flow rate and pressure drop was achieved which indicates that the polymer is pressure-stable even at high flow rates. After characterization the produced GMA-DVB monoliths, which contain reactive epoxide groups, were modified by reaction with diethylamine to obtain a poly(3-diethylamino-2-hydroxypropyl methacrylate-co-divinylbenzene) ion-exchange monolithic stationary phase. The synthesized monoliths contain ionizable amino groups that are useful for anion-exchange chromatography (AEC). Poly(3-diethylamino-2-hydroxypropyl methacrylate-co-divinylbenzene) monolithic columns allowed a fast and highly efficient separation of a homologous series of phosphorylated oligothymidylic acids [d(pT)_12-18]. Since durability is an important parameter of chromatographic column characterization, the separation performance for d(pT)_12-18 in a freshly produced capillary column and on the same column after 100 chromatographic runs was compared.     

Preparation and evaluation of hydrophilic C18 monolithic sorbents for enhanced polar compound retention in liquid chromatography and solid phase extraction

Hydrophilic C18 monolithic polymer sorbents were synthesized for use in solid phase extraction (SPE) and in capillary liquid chromatography (LC). The approach involved incorporating both hydrophobic and hydrophilic monomers into a monolithic material, by copolymerization of stearyl methacrylate (SMA), poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) and ethylene dimethacrylate (EDMA) in the presence of selected porogens, to produce translucent mesoporous monolithic materials in bulk (SPE) or white macroporous monoliths inside fused silica capillary columns (capillary LC). A capillary column containing one of the hydrophilic C18 monoliths (i.e. poly(SMA-co-PEGMEMA-co-EDMA) with 15% (w/w) PEGMEMA) demonstrated nearly 35% reduction in retention of polycyclic aromatic compounds and greater than 40% increase in retention of phenols compared to a hydrophobic C18 monolithic column. In addition, the hydrophilic monolith demonstrated significantly improved resolution of phenols. Similar monolithic materials prepared in bulk were ground and sieved to obtain 45-65 μm particles with desired rigidity for SPE. To achieve optimum extraction performance for phenols, several parameters, including sample pH and volume, and eluent type and volume, were investigated. Under optimized experimental conditions, the method demonstrated good sensitivity (1.6 ng/mL LOD) and linearity (R(2)>0.97 for 10-200 ng/mL). Again, incorporation of 15% (w/w) PEGMEMA in the monolith increased the extraction efficiency of phenols in water from approximately 20% to 67-92% compared to a hydrophobic C18 monolithic material. Increased wettability of the sorbent by the aqueous sample matrix and the presence of hydrogen-bonding interactions are responsible for the improved retention of polar compounds.     

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.     

Optimization of binary porogen solvent composition for preparation of butyl methacrylate monoliths in capillary liquid chromatography

Butyl methacrylate monolithic columns in 320 microm i.d. fused silica capillaries for reversed-phase capillary liquid chromatography were prepared by radical polymerization initiated thermally with azobisisobutyronitrile (AIBN). Polymerization mixture contained butyl methacrylate (BMA) as the function monomer and ethylene dimethacrylate (EDMA) as the crosslinking agent with 1,4-butanediol and 1-propanol as a binary porogen solvent. Ratio of 1,4-butanediol to 1-propanol in the porogen solvent was optimized regarding the monolithic column efficiency and performance. Total porosity, column permeability, separation impedance, Walters hydrophobicity index, retention factors, peak asymmetry factors, height equivalents to a theoretical plate and peak resolutions were used for characterization of the prepared monolithic columns. The polymerization mixture consisting of 17.8% of BMA, 21.8% of EDMA, 18.0% of 1,4-butanediol, 42.0% of 1-propanol and 0.4% AIBN generated monolithic columns of the best performance having a sufficient permeability and the lowest separation impedance. It was also demonstrated that monolithic columns of this composition exhibited good preparation reproducibility and an excellent pressure resistance when applied in capillary liquid chromatography.     

Preparation and Evaluation of Long Chain Alkyl Methacrylate Monoliths for Capillary Chromatography

This work describes the fabrication of long chain alkyl methacrylate monolithic materials for use as stationary phases in capillary liquid chromatography. After capillary inner wall surface activation with 3-(trimethoxysilyl)propyl methacrylate, monoliths were formed by copolymerization of either lauryl or stearyl methacrylate (LMA or SMA) with ethylene dimethacrylate (EDMA) as crosslinker, in the presence of azobisisobutyronitrile (AIBN) as initiator and a mixture of porogenic solvents including water, 1-propanol and 1,4-butanediol. The composition of the polymerization mixture was changed in terms of monomer, crosslinker and porogen ratio composition, in order to compare the influence of these parameters. The monoliths were prepared in 320 ??m i.d. and 200 mm length capillaries. The column morphology was characterized by optical microscopy and scanning electron microscopy (SEM). Total porosity and permeability of each column were calculated using uracil as unretained material by measuring the pressure drop across the columns as a function of linear velocity. The microglobule average size for each column was also determined using Hagen?CPoiseuille equation and compared with the SEM images. As expected, a decrease of the porogen to monomer ratio corresponded to smaller microglobules and a lower total porosity. The columns were then chromatographically evaluated; good results were obtained when these capillaries were used to separate mixtures of phenols, aromatics and drug compounds.     

Capillary electrochromatography with monolithic stationary phases: 1. Preparation of sulfonated stearyl acrylate monoliths and their electrochromatographic characterization with neutral and charged solutes

A novel monolithic stationary phase having long alkyl chain ligands (C17) was introduced and evaluated in capillary electrochromatography (CEC) of small neutral and charged species. The monolithic stationary phase was prepared by the in situ copolymerization of pentaerythritol diacrylate monostearate (PEDAS) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent consisting of cyclohexanol/ethylene-glycol/water. While AMPS was meant to support the electroosmotic flow (EOF) necessary for transporting the mobile phase through the monolithic capillary, the PEDAS was introduced to provide the nonpolar sites for chromatographic retention. Monolithic columns at various EOF velocities were readily prepared by conveniently adjusting the amount of AMPS in the polymerization solution as well as the composition of the porogenic solvent. The monolithic stationary phases thus obtained exhibited reversed-phase chromatography behavior toward neutral solutes and yielded a relatively strong EOF. For charged solutes (e.g., dansyl amino acids), nonpolar as well as electrostatic interaction/repulsion with the monoliths were observed in addition to electrophoretic migration. Therefore, for charged solutes, selectivity and migration can be readily manipulated by changing various parameters including the nature of the monolith and the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). Ultrafast separation on the time scale of seconds of 17 different charged and neutral pesticides and metabolites were performed using short capillary columns of 8.5 cm x 100 microm ID.     

Polyhedral oligomeric silsesquioxanes as functional monomer to prepare hybrid monolithic columns for capillary electrochromatography and capillary liquid chromatography

A simple approach to fabricate hybrid monolithic column within the confines of fused-silica capillaries (75 μm i.d.) was introduced. A polyhedral oligomeric silsesquioxanes (POSS) reagent containing a methacrylate group was selected as functional monomer, and copolymerized with bisphenol A dimethacrylate (BPADMA) or ethylene dimethacrylate (EDMA) in the presence of porogenic solvents via thermally initiated free radical polymerization. After optimization of the preparation conditions, two POSS-containing hybrid monoliths were successfully prepared and exhibited good permeability and stability. By comparison of the separation efficiencies of the resulting poly(POSS-co-BPADMA) and poly(POSS-co-EDMA) monoliths in capillary electrochromatography (CEC) and capillary liquid chromatography (cLC), it was indicated the former has better column efficiencies for alkylbenzenes, phenols, anilines and PAHs in CEC and cLC than the latter. Particularly, the hybrid poly(POSS-co-BPADMA) monolith is more suitable for separation of PAHs due to π–π interaction between the analytes and aromatic rings in the surface of monolithic stationary phase.     

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.     

Single-step preparation and characterization of polymeric monolith for pressurized capillary electrochromatography of typical homologs

A monolithic stationary phase was prepared in a single step by in situ copolymerization of iso-butyl methacrylate (IBMA), ethylene dimethacrylate (EDMA), and N,N-dimethylallylamine (DMAA) in a binary porogenic solvent consisting of N,N-dimethylformamide (DMF) and 1,4-butanediol. As the frame structures of monoliths, the amino groups are linked to support the EOF necessary for driving the mobile phase through the monolithic capillary, while the hydrophobic groups are introduced to provide the nonpolar sites for the chromatographic retention. To evaluate the column performance, separations of typical kinds of neutral or charged homologs, such as alkylbenzenes, phenols (including isomeric compounds of hydroquinone, resorcin, and catechol), and anilines (including isomeric compounds of o-phenylenediamine and 1,4-phenylenediamine), were performed, respectively on the prepared column under the mode of pressurized pCEC. Effects of the buffer pH and the mobile phase composition on the linear velocity of mobile phase and the retention factors of these compounds were investigated. It was found that the retention mechanism of charged solutes could be attributed to a mixed mode of hydrophobic interaction and electrophoresis, while an RP chromatographic behavior on the monolithic stationary phases was exhibited for neutral solutes. Especially, basic compounds such as anilines were well separated on the monolithic columns in the "counterdirectional mode," which effectively eliminated the electrostatic adsorption of basic analytes on the charged surface of the stationary phases.     

反相毛细管整体柱的制备及其在多肽混合物分离中的应用

采用甲基丙烯酸月桂酯为基础功能单体,乙二醇二甲基丙烯酸酯为交联剂,正十二醇、1,4-丁二醇及二甲基亚砜为致孔剂,在内径为75 μm的石英毛细管内制备了具有良好机械性能及化学稳定性的反相毛细管整体柱。考察了致孔剂的种类、比例以及交联剂在单体混合物中的比例对柱压和分离效果的影响;以单体15%、交联剂15%、致孔剂70%(均为质量分数)作为优化配方,在70 ℃条件下反应24 h;并对所合成的毛细管整体柱进行了电镜表征,测试了流速、柱长与柱压的关系。结果表明,毛细管整体柱的通透性良好,可通过延长柱长的方法提高分离效果。将所制备的毛细管整体柱装于纳升级高效液相色谱仪上进行牛血清白蛋白及血浆样本的胰蛋白酶酶切液的分离,获得了比较理想的分离效果。     

Preparation of Monolithic Capillary Chromatographic Columns Using Supercritical Fluid as a Porogen Solvent

Monolithic polymeric beds were synthesized in fused silica capillaries using either trimethylolpropane trimethacrylate (TRIM) or a mixture of butyl methacrylate (BMA) with ethylene glycol dimethacrylate (EDMA) as monomers. Carbon dioxide at temperature and pressure conditions above its critical values was used as a porogen solvent. The purpose of using the supercritical carbon dioxide was to have the possibility of changing the solvation power (and thus the porosity of the resulting monolith) of the porogen by pressure and temperature changes instead of changing the porogen composition. The experiments were performed using a special setup consisting of a stainless steel high-pressure reactor to which the fused silica capillary was connected. The synthesized monoliths underwent liquid chromatographic evaluation. The polyTRIM capillary monoliths were characterized by different permeability, which depended on the pressure of the synthesis. BMA/EDMA columns were applied for separation of alkylbenzenes and a model mixture of proteins.     

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

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

Novel method to prepare polystyrene-based monolithic columns for chromatographic and electrophoretic separations by microwave irradiation

Microwave irradiation can provide a viable alternative to the traditional means such as ultraviolet light and thermal initiation for the preparation of monolithic capillary columns. Polystyrene-based monolithic stationary phases were prepared in situ in fused-silica capillaries and simultaneously in vials. The column permeability, electrophoretic and chromatographic behavior were evaluated using pressure-assisted capillary electrochromatography (pCEC), capillary electrochromatography (CEC) and low pressure liquid chromatography (LPLC). With an optimal monolithic material, the largest theoretical plates for preparing the column could be close to 18,000 plates/m for thiourea in the mode of pCEC. Furthermore, the influence of the composition of the porogenic solvents (toluene/isooctane) on the morphology of organic-based monoliths [poly(styrene-divinylbenzene-methacrylic acid)] was systematically studied with mercury intrusion porosimetry and scanning electron microscopy. The monoliths which were prepared with a high content of isooctane had a bigger pore size and better permeability, and hence resulted in a faster separation.     

Rapid Preparation of C18 Monoliths for Micro‐column Separation Using Ultraviolet and Microwave Irradiation

An organic‐based monolith with long alkyl chain ligands was prepared by UV photo‐initiation using (a) 1‐octadecene as a functional monomer, (b) ethylene glycol dimethacrylate (EDMA) as the cross‐linking agent, (c) 1‐propanol, 1,4‐butanediol and dimethylformamide as triporogenic solvents, and (d) Irgacure 1800 as the initiator. The monoliths containing a high fraction of 1‐octadecene possessed a better total porosity, improved permeability, and result in faster separation. Similar monolithic capillary was quickly fabricated in 3 min by microwave irradiation using azobisisobutyronitrile as the thermal initiator. Conventional polyimide‐coated capillaries were used instead of expensive UV‐transparent capillaries in both methods.     

Iminodiacetic acid functionalised organopolymer monoliths: application to the separation of metal cations by capillary high-performance chelation ion chromatography

Lauryl methacrylate-co-ethylene dimethacrylate monoliths were polymerised within fused silica capillaries and subsequently photo-grafted with varying amounts of glycidyl methacrylate (GMA). The grafted monoliths were then further modified with iminodiacetic acid (IDA), resulting in a range of chelating ion-exchange monoliths of increasing capacity. The IDA functional groups were attached via ring opening of the epoxy group on the poly(GMA) structure. Increasing the amount of attached poly(GMA), via photo-grafting with increasing concentrations of GMA, from 15 to 35 %, resulted in a proportional and controlled increase in the complexation capacity of the chelating monoliths. Scanning capacitively coupled contactless conductivity detection (sC⁴D) was used to characterise and verify homogenous distribution of the chelating ligand along the length of the capillaries non-invasively. Chelation ion chromatographic separations of selected transition and heavy metals were carried out, with retention factor data proportional to the concentration of grafted poly(GMA). Average peak efficiencies of close to 5,000 N/m were achieved, with the isocratic separation of Na, Mg(II), Mn(II), Co(II), Cd(II) and Zn(II) possible on a 250-mm-long monolith. Multiple monolithic columns produced to the same recipes gave RSD data for retention factors of <15 % (averaged for several metal ions). The monolithic chelating ion-exchanger was applied to the separation of alkaline earth and transition metal ions spiked in natural and potable waters.     

New vinylester‐based monoliths as a new stationary phase for capillary electrochromatography

Vinyl ester‐based monoliths are proposed as a new group of stationary phase for CEC. The capillary monolithic columns were prepared by using two vinyl ester monomers, vinyl pivalate (VPV), and vinyl decanoate (VDC) by using ethylene dimethacrylate (EDMA) as the cross‐linking agent, and 2‐acrylamido‐2‐methylpropane sulfonic acid as the charge‐bearing monomer. The monoliths with different pore structures and permeabilities were obtained by varying the type and composition of the porogen mixture containing isoamyl alcohol and 1,4‐butanediol. The electrochromatographic separation of alkylbenzenes was successfully performed by using an acetonitrile/aqueous buffer system as the mobile phase in a CEC system. Vinyl ester monoliths with short alkyl chain length (i.e. poly(VPV‐co‐EDMA) exhibited better separation performance compared with the monolith with long alkyl chain length (i.e. poly(VDC‐co‐EDMA). In the case of VPV‐based monoliths, the theoretical plate numbers higher than 250 000 plates/m were achieved by using a porogen mixture containing 33% v/v of isoamyl alcohol. For both VDC and VPV‐based monoliths, the column efficiency was almost independent of the superficial velocity in the range of 2–12 cm/min.     

Preparation of monolithic columns with target mesopore-size distribution for potential use in size-exclusion chromatography

The main factors affecting the mesopore porosity of methacrylate-ester based monolithic columns were investigated. We prepared 40 monolithic capillary columns with porosity controlled by varying the proportions of butyl methacrylate (BMA) and ethylene dimethacrylate (EDMA) monomers and of 1,4-butanediol (BUT) and 1-propanol (PROP) as the porogen solvent in the polymerisation mixtures by thermally initiated in situ polymerisation in fused-silica capillaries. Using mixture design software, we systematically varied the composition of the polymerisation mixtures to find significant factors affecting mesopore formation. Multivariate analysis of the experimental data obtained for the fabricated columns yielded a model for prediction of the mesopore porosity in monolithic beds as a function of the composition of the polymerisation mixture used to prepare polymethacrylate monolithic capillary columns. The mean absolute deviation of predicted porosities is 0.029 for most of the columns, with only eight columns showing deviations exceeding 0.050. The main factor affecting the mesopore porosity proved to be the combination of the concentration of hydrophobic monomer (BMA) and the concentration of the less-polar solvent, 1-propanol, in the porogen mixture. The proportion of mesopores in the monolithic capillary columns increases with increasing concentration of 1-propanol and with decreasing concentration ratios of the cross-linker (EDMA) to monomer (BMA) and of BUT to PROP porogenic solvents.     

微波聚合快速制备分子印迹毛细管电色谱整体柱

以甲基丙烯酸为功能单体、己二醇二甲基丙烯酸酯为交联剂、 对羟基苯甲酸为模板分子, 采用微波辐射聚合的方式快速制备了分子印迹毛细管电色谱整体柱, 并取得了较好的印迹效果. 分子印迹材料的原位制备5 min即可完成, 大大快于国内外传统的方法.