Preparation and evaluation of a neutral methacrylate-based monolithic column for hydrophilic interaction stationary phase by pressurized capillary electrochromatography

A polar and neutral polymethacrylate-based monolithic column was evaluated as a hydrophilic interaction capillary electrochromatography (HI-CEC) stationary phase with small polar–neutral or charged solutes. The polar sites on the surface of the monolithic solid phase responsible for hydrophilic interactions were provided from the hydroxy and ester groups on the surface of the monolithic stationary phase. These polar functionalities also attract ions from the mobile phase and impart the monolithic solid phase with a given zeta potential to generate electro-osmotic flow (EOF). The monolith was prepared by in situ copolymerization of a neutral monomer 2-hydroxyethyl methacrylate (HEMA) and a polar cross-linker with hydroxy group, pentaerythritol triacrylate (PETA), in the presence of a binary porogenic solvent consisting cyclohexanol and dodecanol. A typical HI-CEC mechanism was observed on the neutral polar stationary phase for both neutral and charged analytes. The composition of the polymerization mixture was systematically altered and optimized by altering the amount of HEMA in the polymerization solution as well as the composition of the porogenic solvent. The monoliths were tested in the pCEC mode. The resulting monoliths had different characteristics of hydrophilicity, column permeability, and efficiency. The effects of pH, salt concentration, and organic solvent content on the EOF velocity and the separation of nucleic acids and nucleosides on the optimized monolithic column were investigated. The optimized monolithic column resulted in good separation and with greater than 140,000 theoretical plates/m for pCEC.     

CEC with monolithic poly(styrene-divinylbenzene-vinylsulfonic acid) as the stationary phase

In this study, poly(styrene-divinylbenzene-vinylsulfonic acid) monolithic columns, prepared by singlestep in situ polymerization, were first used as separation columns for CEC in which vinylsulfonic acid was employed as the charge-bearing monomer. The effects of the polymerization conditions (compositions of monomer and porogenic solvent, and polymerization time) on the chromatographic property of the polystyrene-based monolithic column were examined. Furthermore, an increase in water content of the porogenic solvent markedly altered the retention behaviors of these analytes, where the monolithic column was able to be produced in a higher ratio of vinylsulfonic acid. Finally, the study demonstrated that acidic analytes could be successfully separated by the polystyrene-based monolithic column under an acidic mobile phase.     

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。     

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。     

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。     

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome and peptide profiling analysis by using a monolithic analytical capillary column

An automated nano-LC-MS/MS platform without trap column was established, which only used a 20 cm lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA) monolithic capillary column to allow preconcentration and separation of peptides. The monolithic column had the advantages of good permeability and low backpressure resulting in higher flow rates for capillary columns. Tryptic digests of bovine albumin and yeast protein extract were tested using the monolithic column system. High proteomic coverage using this approach were demonstrated in this study. Furthermore, peptide samples extracted from mouse liver were separated by using the monolithic column system combined with size-exclusion chromatography prefractionation. This monolithic column system might be a promising alternative for the automated system previously using a trap column for routine proteome and peptide profiling analysis.     

Flow-through pore characteristics of monolithic silicas and their impact on column performance in high-performance liquid chromatography

In order to elucidate the role of the flow-through characteristics with regard to the column performance in high-performance liquid chromatography (HPLC) native and n-octadecyl bonded monolithic silica rods and columns, respectively of 100 mm length and 4.6 mm ID with mesopores in the range between 10 and 25 nm and macropores in the range between 0.7 and 6.0 μm were examined by mercury intrusion/extrusion, scanning electron microscopy, image analysis and permeability. The obtained data of the flow-through pore sizes and porosity values as well as surface-to-volume ratio of the stationary phase skeleton enabled to predict their influence to the chromatographic separation efficiency. Our data demonstrate that mercury porosimetry is a reliable technique to obtain all the characteristic parameters of the flow-through pores of silica monoliths. An important result of our examination was that the surface-to-volume ratio of monolithic silica skeletons had more significant impact to the separation process, rather than the average flow-through pore sizes. We could also show the essential differences between the particulate and monolithic stationary phases based on theoretical computation. The results, obtained from other characterization methods also indicated the structural complexity of monolithic silica samples. Permeability of columns is a generally applicable parameter to characterize all chromatographic phases no matter the chemistry or format. The correlation coefficient obtained for mercury intrusion and permeability of water was 0.998, though our investigation revealed that the surface modification is more likely influencing the obtained results. Further, the assumption of the cylindrical morphology of flow-through pores is not relevant to the investigated monolithic silica columns. These results on the morphology of the flow-through pores and of the skeletons were confirmed by the image analysis as well. Our main finding is that the flow-through pore sizes are not relevant for the estimation of the chromatographic separation efficiency of monolithic silica columns.     

Analyses of benzophenones by capillary electrochromatography using methacrylate ester-based monolithic columns

In this study, eight benzophenones, which are commonly used as UV filters in various cosmetics and plastics, were analyzed by capillary electrochromatography with a methacrylate ester-based monolithic column. The effects of the composition and pH of mobile phase, porogenic solvent ratio, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) content on benzophenone separations were examined. For all benzophenones, separation performances were markedly improved in monolithic columns with larger 1-propanol ratio and higher AMPS content. Furthermore, a twofold increase in AMPS content almost reduced the separation time in half when a monolithic column had an adequately high surface area, i.e. monolithic column was produced in a higher ratio of 1-propanol. As well, the retention behaviors of these analytes in the monolithic column were strongly influenced by the level of acetonitrile in the mobile phase, and the pH of the mobile phase also had an apparent influence on separation resolution.     

以聚乙二醇/甲醇为二元致孔剂的新型磺酸甜菜碱型两性离子亲水毛细管整体柱的合成与色谱评价

使用新型二元致孔剂聚乙二醇(PEG)/甲醇,以N,N-二甲基-N-甲基丙烯酰胺基丙基-N,N-二甲基-N-丙烷磺酸内盐(SPP)为单体,季戊四醇三丙烯酸酯(PETA)为交联剂,偶氮二异丁腈(AIBN)为引发剂,通过原位聚合法制备磺酸甜菜碱型两性离子亲水毛细管整体柱。对各反应物的配比进行了优化。结果表明,当单体与致孔剂的质量比为1∶2.5,并且致孔剂中PEG与甲醇的质量比为1∶2,单体内部SPP与PETA的质量比为1∶1,AIBN为总质量的0.1%时为最优配比;PEG/甲醇二元致孔剂的加入实现了对整体柱内部孔径大小的调节,得到了结构更为均一,渗透性、机械稳定性良好的毛细管整体柱,并且理论塔板数与传统制备方法相比有显著提高,在毛细管液相色谱模式下最高可达2.4×105塔板/m。将制备的整体柱应用于毛细管液相色谱和加压毛细管电色谱分离酚类、核苷类等极性小分子混合物,得到了很好的分离效果。     

聚丙烯酸酯类毛细管整体柱的制备及其在加压毛细管电色谱中的应用

毛细管电色谱(CEC)是毛细管电泳(CE)和微径液相色谱(micro—HPLC)技术的结合,是集CE的电子迁移机制和micro-HPLC的分配分离机理发展起来的一种高效微分离技术．CEC以塞子流型的电渗流代替抛物线流型的压力流,具有CE的高效性,能够分离电中性化合物而具有HPLC的高选择性．     

Recent advances in micro-scale and nano-scale high-performance liquid-phase chromatography for proteome research

High-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS–MS) is regarded as one of the most powerful techniques for separation and identification of proteins. Recently, much effort has been made to improve the separation capacity, detection sensitivity, and analysis throughput of micro- and nano-HPLC, by increasing column length, reducing column internal diameter, and using integrated techniques. Development of HPLC columns has also been rapid, as a result of the use of submicrometer packing materials and monolithic columns. All these innovations result in clearly improved performance of micro- and nano-HPLC for proteome research.     

光聚合整体式咖啡因印迹毛细管柱的制备及分离性能

分子印迹技术作为一种制备对目标分子具有专一识别能力的功能高分子的方法 ,近年来在化学化工、生物化学与生物技术的许多领域中得到广泛应用 [1～ 4 ] .分子印迹技术与微分离方法 (包括微柱液相色谱、毛细管电泳、毛细管电色谱和芯片分离等 )结合已引起人们极大的兴趣和关注[5,6] .毛细管柱是毛细管电色谱和微柱液相色谱的关键部件 ,目前普遍使用的是烷基键合硅胶微粒的填充柱 ,存在制备时须烧塞和填充两大困难 ,以及使用时易产生气泡和易折断等缺点 .将含被识别分子 (印迹分子 )、交联剂、溶剂、功能单体和引发剂的混合液注入毛细管 ,经光…     

Preparation of phenylboronic acid-silica hybrid monolithic column with one-pot approach for capillary liquid chromatography of biomolecules

A phenylboronic acid-silica hybrid monolithic column for capillary liquid chromatography (cLC) was prepared through one-pot process by using 4-vinylphenylboronic acid (VPBA) and alkoxysilanes simultaneously. The effects of the molar ratio of tetramethyloxysilane/γ-methacryloxypropyltrimethoxysilane (TMOS/γ-MAPS), amount of VPBA, and the volume of diethylene glycol (DEG) on the morphologies, permeabilities and pore properties of the prepared VPBA-silica hybrid monolithic columns were studied in detail. A relatively uniform monolithic structure with high porosity was obtained with optimized ingredients. A series of cis-diol-containing compounds, alkylbenzenes, amides, and anilines were utilized to evaluate the retention behaviors of the VPBA-silica hybrid monolithic column. The result demonstrated that the prepared VPBA-silica hybrid monolithic column exhibited multiple interactions including hydrophobicity, hydrophilicity, as well as cation exchange apart from the expected affinity interaction. The run-to-run, column-to-column and batch-to-batch reproducibility of the VPBA-silica hybrid monolith were satisfactory with the relative standard deviations (RSDs) less than 1.63% (n = 5), 2.02% (n = 3) and 2.90% (n = 5), respectively, indicating the effectiveness and practicability of the proposed method. In addition, the VPBA-silica hybrid monolithic column was further applied to the separation of proteins and tryptic digest of bovine serum albumin (BSA), respectively. The successful applications suggested the potential of the VPBA-silica hybrid monolith in proteome analysis.     

Parameters affecting the separation of intact proteins in gradient-elution reversed-phase chromatography using poly(styrene-co-divinylbenzene) monolithic capillary columns

An experimental study was performed to investigate the effects of column parameters and gradient conditions on the separation of intact proteins using styrene-based monolithic columns. The effect of flow rate on peak width was investigated at constant gradient steepness by normalizing the gradient time for the column hold-up time. When operating the column at a temperature of 60 °C a small C-term effect was observed in a flow rate range of 1–4 μL/min. However, the C-term effect on peak width is not as strong as the decrease in peak width due to increasing flow rate. The peak capacity increased according to the square root of the column length. Decreasing the macropore size of the polymer monolith while maintaining the column length constant, resulted in an increase in peak capacity. A trade-off between peak capacity and total analysis time was made for 50, 100, and 250 mm long monolithic columns and a microparticulate column packed with 5 μm porous silica particles while operating at a flow rate of 2 μL/min. The peak capacity per unit time of the 50 mm long monolithic column with small pore size was superior when the total analysis time is below 120 min, yielding a maximum peak capacity of 380. For more demanding separations the 250 mm long monolith provided the highest peak capacity in the shortest possible time frame.     

Effects of the operation parameters on Hydrophilic Interaction Liquid Chromatography separation of phenolic acids on zwitterionic monolithic capillary columns

Strongly polar phenolic acids are weakly retained and often poorly separated in reversed-phase (RP) liquid chromatography. We prepared zwitterionic polymethacrylate monolithic columns for micro-HPLC by in situ co-polymerization in fused-silica capillaries. The capillary monolithic columns prepared under optimized polymerization conditions show some similarities with the conventional particulate commercial ZIC-HILIC silica-based columns, however have higher retention and better separation selectivity under reversed-phase conditions, so that they can be employed for dual-mode HILIC-RP separations of phenolic acids on a single column. The capillary polymethacrylate monolithic sulfobetaine columns show excellent thermal stability and improved performance at temperatures 60–80 °C. The effects of the operation conditions on separation were investigated, including the type and the concentration of the organic solvent in the aqueous-organic mobile phase (acetonitrile and methanol), the ionic strength of the acetate buffer and temperature. While the retention in the RP mode decreases at higher temperatures in mobile phases with relatively low concentrations of acetonitrile, it is almost independent of temperature at HILIC conditions in highly organic mobile phases. The best separation efficiency can be achieved using relatively high acetate buffer ionic strength (20–30 mmol L⁻¹) and gradient elution with alternately increasing (HILIC mode) and decreasing (RP mode) concentration of aqueous buffer in aqueous acetonitrile. Applications of the monolithic sulfobetaine capillary columns in alternating HILIC-RP modes are demonstrated on the analysis of phenolic acids in a beer sample.     

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).     

Fast fabrication of a hybrid monolithic column containing cyclic and aliphatic hydrophobic ligands via photo‐initiated thiol‐ene polymerization

Three monomers, octakis (3‐mercaptopropyl) octasilsesquioxane, 1,2,4‐trivinylcyclohexane and isophytol were employed to synthesize a novel monolithic stationary phase via photo‐initiated thiol‐ene click polymerization for reversed‐phase liquid chromatography. Several factors such as porogenic system, reaction time and the molar ratio of functional groups were investigated in detail. The resulting poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column exhibited suitable permeability for fast separation and outstanding thermal stability. Five alkylbenzenes were employed to evaluate the ability of chromatographic separation of the resulting monolithic columns at different flow rates, and showed the highest column efficiencies of 90,200–93,100 N/m (corresponding to 10.4–10.6 μm of plate height) at a velocity of 0.41 mm/s. The baseline separations of five anilines and eight phenols further proved the applicability of poly(POSS‐co‐TVCH‐co‐isophytol) monolithic column in the separation of small molecules.     

Siliceous mesocellular foam for high-performance liquid chromatography: Effect of morphology and pore structure

Spherical siliceous mesocellular foam (MCF) particles with an average particle size of 4.8 μm have been successfully prepared. These spherical particles were tailored in pore sizes and surface areas. They were functionalized with C₈ or C₁₈ groups, and applied towards reversed phase high-performance liquid chromatography (HPLC) column separations. Their high surface areas gave rise to very good retention characteristics, as illustrated in the separation of a series of alkylbenzene solutes with increasing chain length. The highly interconnected porous structure and ultralarge pore size of MCF allowed the columns to be used at high flow rates without much loss in column efficiency. The column efficiency and peak symmetry were further improved by eliminating the micropores of the stationary phase. The reversed phase column packed with C₁₈-modified spherical MCF particles provided for excellent separation of different deoxynucleosides, illustrating the broad applicability of these materials due to their controlled pore size.     

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.     

Separation of parabens in capillary electrochromatography using poly(styrene-divinylbenzene-methacrylic acid) monolithic column

In this study, a series of poly(styrene-divinylbenzene-methacrylic acid) monolithic capillaries was used as the separation column of CEC for the analyses of parabens in commercial pharmaceutical and cosmetic products. The results showed that the chromatographic characteristics of these analytes were strongly affected by the preparation condition of the monolithic column including monomer content, porogenic solvent composition, and polymerization time. Baseline separations were markedly sped up by lowering the polymerization time without any obvious loss of resolution. Furthermore, mobile-phase composition (pH, ACN, and electrolyte concentration) was also able to effectively improve the separation behavior. Similar to the influence of lowering the polymerization time, retention times for all analytes were significantly shortened in the CEC method by decreasing the electrolyte concentration in the mobile phase.