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.     

A model of flow-through pore formation in methacrylate ester-based monolithic columns

The main factors affecting the porosity of methacrylate-ester based monolithic columns were investigated. We prepared 23 monolithic capillary columns with porosity controlled by varying the proportions of butyl methacrylate and ethylene dimethacrylate monomers and of 1,4-butanediol and 1-propanol as the porogen solvent in the polymerization mixtures by thermally initiated in-situ polymerization in fused-silica capillaries. Using mixture design software, we systematically varied the composition of the polymerisation mixtures to find significant factors affecting flow-through pore formation. Multivariate analysis of the experimental data obtained for the fabricated columns yielded a model for prediction of the flow-through porosity in monolithic beds as a function of the composition of the polymerization mixture used to prepare polymethacrylate monolithic capillary columns. The mean error of prediction was lower than 8% for eight columns prepared independently of the original set of 15 columns used to derive the flow-through model. The flow-through porosity increases with increasing concentration of the binary porogen solvent mixture, the concentration of 1,4-butanediol being the main factor enhancing flow-through pore formation. On the other hand, increasing concentrations of the hydrophobic monomer butyl methacrylate and increasing concentrations of 1-propanol have a negative effect on flow-through pore formation. The capillary columns prepared with a high proportion of flow-through pores and a minimum amount of mesopores can be used for fast gradient separations of both low-molecular weight compounds and biopolymers.     

Methacrylate monolithic columns of 320 microm I.D. for capillary liquid chromatography

Monolithic capillary columns (320 microm I.D.) were prepared for capillary liquid chromatography (CLC) by radical polymerization of butylmethacrylate (BMA) and ethylenedimethacrylate (EDMA) in the presence of a porogen solvent containing propan-1-ol, butane-1,4-diol and water. The influence of the contents of the porogen solvent and EDMA in the polymerization mixture on the monolith porosity and column efficiency was investigated. The composition of the polymerization mixture was optimized to attain a minimum HETP of the order of tens of microm for test compounds with various polarities. The separation performance and selectivity of the most efficient monolithic column prepared was characterized by van Deemter curves, peak asymmetry factors and Walters hydrophobicity and silanol indices. It was demonstrated that the 320-microm I.D. monolithic column exhibited CLC separation performance similar to that observed for 100- and 150-microm I.D. monolithic columns reported in the literature; moreover, the 320-microm I.D. column was easier to operate in CLC and exhibited a higher sample loadability.     

Octyl-type monolithic columns of 530 microm i.d. for capillary liquid chromatography

A novel monolithic capillary column (530 microm i.d.) was prepared for capillary liquid chromatography (CLC) by in situ copolymerization of octyl methacrylate (MAOE) and ethylene dimethacrylate (EDMA) in the presence of a porogen solvent containing 1-propanol, 1,4-butanediol, and water with azobisisobutyronitrile as the initiator. The influences of the contents of the porogen solvent, EDMA and the various concentration ratios of 1-propanol to 1,4-butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of monolithic capillary columns were characterized by SEM and mercury intrusion porosimetry, respectively. Chromatographic evaluations of the columns were performed under CLC mode. The results showed that good permeability and stability can be obtained under optimal experimental conditions. The separation results of some acid, neutral and basic analytes demonstrated the hydrophobicity and low affinity to basic analytes of the new column. Three metal ions, i.e. Mg(II), Zn(II) and Cd(II) were also separated under ion-pair mode on the new monolithic capillary column and the results were acceptable.     

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.     

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography

Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 microm. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.     

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

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

Preparation 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.     

Rapid Separation of Proteins by Capillary HPLC on a Short Polymethacrylate-based Strong Cation-exchange Monolithic Column

A polymethacrylate‐based strong cation‐exchange capillary monolithic column was prepared by in‐situ copolymerization for the fast separation of proteins. Glycidyl methacrylate (GMA) was used as monomer, ethylenedimethacrylate (EDMA) as cross link agent and the mixture of 1‐propanol, 1,4‐butanediol and water as porogen solvent. The monolith was sulfonated using 1 mol/L Na₂SO₃ based on a ring opening of epoxides. The influences of the contents of the porogen solvent and GMA and the various concentration ratios of 1‐propanol to 1,4‐butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of the monolith were characterized by SEM and mercury intrusion porosimetry, respectively. Using only 1.5 cm length of this monolithic capillary column, four kinds of proteins, trypsin, cytochrome C, lysozyme (egg white) and egg albumin, were successfully separated from each other in 5 min at a high flow rate of 110 mm/s.     

Characterization of polymer monolithic stationary phases for capillary HPLC

Monolithic capillary columns have been prepared in fused‐silica capillaries by radical co‐polymerization of ethylene dimethacrylate and butyl methacrylate in the presence of porogen solvent mixtures containing various concentration ratios of 1‐propanol, 1,4‐butanediol, and water with azobisisobutyronitrile as the initiator of the polymerization reaction. The through pores in organic polymer monolithic columns can be characterized by “equivalent permeability particle size”, and the mesopores with stagnant mobile phase by “equivalent dispersion particle size”. Increasing the concentration of propanol in the polymerization mixture diminishes the pore volume and size in the monolithic media and improves the column efficiency, at a cost of decreasing permeability. Organic polymer monolithic capillary columns show similar retention behaviour to packed alkyl silica columns for compounds with different polarities characterized by interaction indices, I_x, but have different methylene selectivities. Higher concentrations of propanol in the polymerization mixture increase the lipophilic character of the monolithic stationary phases. Best efficiencies and separation selectivities were found for monolithic columns prepared using 62–64% propanol in the porogen solvent mixture. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra‐column contributions.     

Effect of multi-walled carbon nanotubes incorporation into benzyl methacrylate monolithic columns in capillary liquid chromatography

This work describes the preparation of polymer based monolithic materials and their use as stationary phases in capillary liquid chromatography. Multi-walled carbon nanotubes (MWCNT) were incorporated into a mixture containing benzyl methacrylate (BMA) and ethylene dimethacrylate (EDMA) as co-monomers. The optimized porogenic mixture was a ternary solution composed of cyclohexanol, 1,4-butandiol and butanol which resulted in a stable and homogeneous suspension. Six capillary columns with increasing amounts of MWCNT, from 0 to 0.4 mg mL(-1), were prepared by thermal polymerization in 0.32 mm (i.d.) and 150 mm length fused silica tubing. The chromatographic evaluation showed that the synthesized monolithic beds were mechanically stable while their porosity and permeability increased with the MWCNT content. The prepared capillary columns were tested for the separation of mixtures of ketones and phenols at an optimum flow rate of 2 μL min(-1). The results showed that incorporation of MWCNT slightly affected the retention while it enhanced the column efficiency by increasing the column efficiency by a factor of up to 9. This effect corresponded also to an improved resolution and full separation of the solutes.     

聚十二烷基甲基丙烯酸酯整体柱的制备及其在毛细管电色谱法分离肌红蛋白酶解产物中的应用

以十二烷基甲基丙烯酸酯(LMA)为功能单体,乙叉二甲基丙烯酸酯(EDMA)为交联剂,正丙醇、1,4-丁二醇和水为三元致孔剂,以及2-丙烯酰胺-2-甲基丙磺酸(AMPS)为电渗流产生剂,制备了聚十二烷基甲基丙烯酸酯整体柱。系统考察了AMPS含量和单体-致孔剂比例对柱性能的影响。结果表明,单体溶液和致孔剂的最佳聚合溶液质量比为35:65,其中单体溶液组成为59.5%（质量分数,下同）LMA、40%EDMA和0.5%AMPS,致孔剂溶液组成为60%正丙醇、30%1,4-丁二醇和10%水。在优化的流动相条件下应用制备的整体柱采用毛细管电色谱法成功地分离了肌红蛋白酶解产物。     

Preparation and evaluation of a monolithic molecularly imprinted polymer for the chiral separation of neurotransmitters and their analogues by capillary electrochromatography

A monolithic molecularly imprinted polymer (MIP) column was prepared as the stationary phase for the capillary electrochromatographic (CEC) separation of a group of structurally related compounds including dopamine (DA), (±)-epinephrine (EP), (-)-isoproterenol (ISO), (±)-norepinephrine (NE), (±)-octopamine (OCT), and (±)-synephrine (SYN). Here, (-)-NE was used as the template. Either methacrylic acid (MAA) or itaconic acid (IA) together with a mixture of ethylene glycol dimethacrylate (EDMA) and α,α'-azobis(isobutyronitrile) (AIBN) in N,N-dimethylformamide (DMF) was introduced into a pre-treated, silanised, fused-silica capillary by a thermal non-covalent polymerisation procedure. Optimised conditions for the polymerisation reaction were assessed by the separation efficiency of the template. Both the template/monomer/cross linker molar ratio and the compositions of the functional monomer, cross-linker, and porogen affected polymerisation. The optimum in situ polymerisation reaction was performed at 65 °C for 17 min. By varying CEC parameters like eluent composition and pH, we observed that the addition of SDS to the eluent clearly improved the CEC separations. With a mobile phase of citrate buffer (10 mM, pH 3)/SDS (40 mM)/acetonitrile (2/2/1, v/v/v) solution and an applied voltage of 10 kV, the six related structures of the template and their enantiomeric mixtures were satisfactorily separated at 30 °C.     

Capillary Electrochromatography on Methacrylate Based Monolithic Columns: Evaluation of Column Performance and Separation of Polyphenols

Fused-silica capillary columns (100 μm I.D.) englobing a porous monolithic stationary phase were prepared by in situ copolymerization of 2-ethylhexyl methacrylate, ethylene glycol dimethacrylate and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) in the presence of a porogenic mixture containing 1-propanol, 1,4 butanediol and water. The influence of the monomers ratio and the porogen solvent composition as well as the content of AMPS in the polymerization mixture on column total porosity and efficiency was investigated to attain minimum HETP values for the reversed-phase capillary electrochromatography separation of bioflavonoids. For the most promising column, the van Deemter plots, in both μ-HPLC and CEC, were also evaluated. In CEC the reduced plate height was found almost constant (1.6–2.0) within the range of linear mobile phase velocity between 0.2–2.0 mm s⁻¹. The chemical and mechanical stabilities of the monolithic column over a wide range of buffer pH (2-10) and time were satisfactory. Furthermore, the effects of mobile phase parameters, such as buffer concentration and organic modifier content, on the electroosmotic flow were studied systematically. CEC separations of standard mixtures of polyphenols, including flavonols, flavanones and flavanones-7-O-glycosides, were accomplished in less than 8 min. The CEC separation of the major flavanone glycoside constituents in the extract from a freshly squeezed grapefruit juice was also reported.     

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

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

胃蛋白酶亲和有机聚合物毛细管整体柱的制备及性能考察

以热引发原位聚合方法制备了聚（甲基丙烯酸缩水甘油酯（glycidyl methacrylate,GMA）-乙二醇二甲基丙烯酸酯（ethyleneglycol dimethacrylate,EDMA））毛细管整体柱,对整体柱的性能进行了表征。结果表明,柱内部结构均匀、渗透性好;整体柱能够实现苯等中性小分子化合物的分离,具有反相色谱特征,重现性和稳定性良好。利用整体柱环氧基团的活性,采用间接法,以戊二醛为连接臂制备胃蛋白酶亲和手性整体柱。在毛细管电色谱模式下进行了柱分离性能研究,并对缓冲液pH值和运行电压等分离条件进行了考察。结果表明,亲和整体柱对4种碱性手性药物（奈福泮、氨氯地平、西酞普兰、扑尔敏）有拆分效果,奈福泮、氨氯地平、西酞普兰能达到基线分离。本文为蛋白质亲和毛细管电色谱整体柱的制备和应用提供了新的思路和方法。     

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.     

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。     

光聚合法快速制备甲基丙烯酸酯类毛细管整体柱

采用甲基丙烯酸正丁酯(BMA)为功能单体, 乙二醇二甲基丙烯酸酯(EDMA)为交联剂, 正丙醇、1,4-丁二醇和水为致孔剂, Irgacure 1800为引发剂, 在毛细管内采用光引发原位聚合150 s快速制备了有机聚合物整体柱. 分别采用电色谱(CEC)、加压电色谱(p-CEC)和低压色谱(LPLC)模式对所制备的整体柱进行了性能评价, 基线分离了硫脲、甲苯、萘和联苯, 在加压电色谱(p-CEC)模式下硫脲的最低理论塔板高度达到了8.0 μm. 扫描电镜结果表明, 整体材料在毛细管柱中形成并与毛细管内壁结合紧密.     

Enantioseparation of 2-aryloxypropionic acids on chiral porous monolithic columns by capillary electrochromatography. Evaluation of column performance and enantioselectivity

The enantioseparation of 2-aryloxypropionic acids by capillary electrochromatography was tested on columns with a monolithic stationary phase prepared from silanized fused-silica capillaries (100 microm I.D.) by in situ copolymerization of glycidyl methacrylate, ethylene glycol dimethacrylate and methyl methacrylate in the presence of formamide and 1-propanol as the porogen solvents. The porous chiral monolithic stationary phases were prepared by reaction of the epoxy-groups at the surface of the monolith with (+)-1-(4-aminobutyl)-(5R,8S,10R)-terguride. To attain the minimum HETP values for the enantiodiscrimination of 2-phenoxypropionic acid, the influence of the composition of polymerization solution on column total porosity and efficiency was investigated. Optimum mobile phase conditions were found for all analytes tested using acetonitrile-methanol mixtures containing triethylamine and acetic acid as the buffer components. Furthermore, the chemical and mechanical stabilities of the columns were satisfactory, allowing hundreds of analyses.