Fabrication of large‐sized silica monolith exceeding 1000 mL with high structural homogeneity

Reproducible fabrication of the hierarchically porous monolithic silica in a large volume exceeding 1000 mL has been established. By the hydrothermal enlargement of the fully accessible small pores to exceed 50 nm in diameter, the capillary force emerged on solvent evaporation was dramatically reduced, which allowed the preparation of crack‐free monoliths with evaporative solvent removal under an ambient pressure. The local temperature inhomogeneity within a reaction vessel in a large volume was precisely controlled to cancel the heat evolved by the hydrolysis reaction of tetramethoxysilane and that consumed to melt ice cubes dispersed in the solution, resulting in large monolithic silica pieces with improved structural homogeneity. Homogeneity of the pore structure was confirmed, both on macro‐ and mesoscales, using SEM, mercury intrusion, and nitrogen adsorption/desorption measurements. Furthermore, the deviations in chromatographic performance were examined by evaluating multiple smaller monolithic columns prepared from the monolithic silica pieces cut from different parts of a large monolith. All the daughter columns thus prepared exhibited comparable performances to each other to prove the overall homogeneity of the mother monolith. Preliminary results on high‐speed separation of peptides and proteins by the octadecylsilylated silica monolith of the above production have also been demonstrated.     

Properties of a Non-Aromatic Epoxy Polymer-Based Monolithic Capillary Column for μ-HPLC

We report the chromatographic properties of a new type of epoxy polymer-based monolithic capillary column, the Tetrad-C column. The column was prepared by a completely new method—reaction of a tetra-functional epoxy monomer, 1,3-bis(N,N-diglycidylaminomethyl)cyclohexane (trade name Tetrad-C), with a diamine, 4-[(4-aminocyclohexyl)methyl)cyclohexane (BACM). This polymer monolith has no aromatic functional groups reducing chromatographic performance. The columns were carefully observed by scanning electron microscopy and evaluated chromatographically by use of a μ-HPLC system. It was found that morphological control of the epoxy polymer-based monolith was possible simply by changing the polymerization conditions, i.e., the polymerization temperature and/or the relative amounts of porogenic solvent and BACM. Another advantage was that volumetric shrinkage of the tetra-functional epoxy-based monolith during the polymerization reaction was much less than for the tri-functional epoxy-based monolithic (TEPIC) column reported in our previous paper. A Tetrad-C column 200 mm long afforded up to 10,000 plates for alkylbenzenes in reversed-phase-mode. This column can also work in HILIC mode, although the hydrophobicity of the column was greater than that of the TEPIC column. Heat treatment (160 °C for 2 h; to eliminate residual, unreacted, functional groups) had a negligible effect on column performance, indicating the columns were thermally stable.     

双苯基杂化整体柱的制备及毛细管电色谱性能研究

以苯基三乙氧基硅烷(PTES)和1,4-双三乙氧基硅基苯(BTEB)为反应单体,分别采用盐酸和十二胺为催化剂,通过两步酸碱催化的溶胶-凝胶法制备了新型双苯基杂化硅胶毛细管电色谱整体柱。分别采用扫描电镜、红外光谱和压汞法对材料的结构特性进行了表征。结果表明,所制备的材料具有特定的网络结构特征,孔径主要分布在3.4μm左右,材料的总孔容为3.5 cm3/g,比表面积为145 m2/g。新型整体柱能够很好地分离烷基取代苯、稠环芳烃、取代苯胺和硝基苯酚异构体。整体柱重复性实验表明,6种苯的同系物保留时间和容量因子的相对标准偏差分别小于0.61%和0.30%(n=8)。不同批次的整体柱的保留时间和容量因子的相对标准偏差分别小于7.2%和5.6%(n=3)。     

Improvement of proteome coverage using hydrophobic monolithic columns in shotgun proteome analysis

Monolithic columns are widely used in shotgun proteome analysis. However, it is difficult to increase the separation capability and proteome coverage by using conventionally organic polymer-based monolithic column due to the difficulty of controlling homogeneity of the overall pore structure (both pores and microglobules), which leads to relatively low column efficiency. Therefore, we studied the effect of constitute and percentage of porogenic solvent, functional monomer, column length, and separation gradient on the peak capacity and proteome coverage by methacrylate-based reversed phase monolithic columns. It was demonstrated that the porous property of the hydrophobic monolith, which was mainly determined by the porogenic solvent, was crucial to the proteome coverage when similar methacrylate monomer was utilized and a ternary porogenic solvent was adopted to prepare C12 monolithic column with relatively homogeneous overall pore structure. It was also shown that high proteome coverage could be reliably obtained with online multidimensional separation using totally monolithic columns system with the length of analytical column at 85 cm and reversed phase separation gradient at 210 min.     

Comparison of different types of outlet frits in slurry‐packed capillary columns

Fused‐silica capillary columns for high‐performance liquid chromatography with 320 and 250 μm inner diameter were prepared by slurry packing with 5 and 3 μm Nucleosil C₁₈ stationary phase. Different types of mechanical and monolithic outlet frits were used and their influence on the resulting column performance was evaluated. Columns with quartz wool exhibited symmetrical peaks and low theoretical plate height, and the preparation time was short. The performance of monolithic frits varied based on type of monolith, length of the frit, and silanization procedure. The best frit performed similarly to the quartz wool ones, but the preparation took several hours. Their main advantage lies in the possibility of on‐column detection, because the detection window can be burnt immediately behind the frit.     

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。     

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.     

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.     

纤维素键合型手性整体柱的制备及其在快速手性分离中的应用

采用N-丙烯酰琥珀酰亚胺(NAS)为基质单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,原位聚合制备聚(NAS-co-EDMA)毛细管整体柱,并通过化学键合法将自合成的纤维素-三(4-甲基苯甲酸酯)(CTMB)共价键合到整体柱上,制备用于快速手性分离的纤维素键合型手性整体柱.优化了整体柱制备和衍生化条件;通过对固定相红...     

Monolithic capillary columns based on ethylene glycol dimethacrylate for separation of polymers by molecular mass

Polar monolithic capillary columns for the molecular-mass separation of polystyrene standards are synthesized on the basis of ethylene glycol dimethacrylate. The monolith structure is optimized through variation in the type of porogen because variation in other synthesis parameters (the time and temperature of polymerization, the amount of monomer in the initial feed) is inefficient. The separation of polymers on monolithic sorbents proceeds via a combined exclusion-hydrodynamic mechanism. In terms of a model that allows for contributions of both mechanisms to retention, calibration plots are drawn for the synthesized columns. Monolithic columns with the optimum monolith structure make it possible to use up to 60% of the column free volume for the efficient separation of polymers in a broad molecular-mass range.     

Evaluation of a methacrylate-bonded cyclodextrins as a monolithic chiral stationary phase for capillary electrochromatography (CEC)-UV and CEC coupled to mass spectrometry

Glycidyl methacrylate-bonded β-cyclodextrin (GMA-β-CD) is synthesized as a new chiral monomer by direct chemical bonding with GMA using a fast and simple alternative procedure. Next, rigid and homogenous monolithic columns were prepared by polymerization of GMA-β-CD monomer with ethylene dimethacrylate (EDMA), in the presence of commonly used porogens and a charged achiral monomer to form a versatile chiral monolith. This is the first report in which a preparation procedure for a methacrylate-bonded CD is introduced for chiral separations in CEC. The degree of substitution of GMA-β-CD monomer and mobile-phase parameters were optimized to achieve the highest enantioselectivity and plate number. To evaluate the GMA-β-CD monolithic column, different classes of chiral compounds were screened. Under the optimized β-CD monolith phase and the optimum mobile-phase conditions, 30 neutral and basic chiral compounds and two acidic compounds could be separated. The high chemical and mechanical stability, homogenous microflow and no loss of material at the interface allows for the first time the feasibility of applying this polymer-based monolithic column for CEC coupled to ESI-MS. Compared with CEC-UV, CEC-ESI-MS showed higher sensitivity and lower resolution. However, resolution greater than 1.0 can still be obtained for majority of the select tested compound in CEC-ESI-MS with at least three out of seven compound providing Rs≥1.5. The results reinforce the potential of GMA-β-CD monolithic columns for chiral separations with high sensitivity in CEC-ESI-MS. Finally, using hexobarbital as the model chiral analyte, the monolithic column demonstrated excellent stability and reproducibility of retention time and enantioselectivity.     

大孔型疏水整体柱的制备及其表征

利用液体致孔剂正庚烷,将双甲基丙烯酸乙二醇酯(EDMA)和甲基丙烯酸缩水甘油酯(GMA)聚合成含有环氧基团的大孔型整体柱,用正丁胺修饰制备成疏水性整体柱。压汞法分析表明,疏水整体柱的孔隙率为60.2%,500nm以上的大孔占疏水整体柱孔隙率的65.7%。在2890cm/h的高流速下,疏水整体柱的背压只有10.9MPa。以牛血清白蛋白(BSA)为研究对象,动态吸附容量为14.4mg/g介质,在一定范围内,分离效果不受操作流速的影响。此外,在1445cm/h的流速下,3min内即可对细胞色素C、RNase A、溶菌酶和鸡卵清蛋白进行基线分离。结果表明,大孔型疏水整体柱可用于蛋白质的快速分离和分析。     

Production of novel polymer monolithic columns, with stationary phase gradients, using cyclic olefin co-polymer (COC) optical filters

Polymer monolithic columns with controlled surface ligand density, providing stationary phase gradients within monolithic capillary columns, have been developed using photo-grafting through optical filters. Utilising commercially available cyclic olefin co-polymer (COC) films, the production of an optical filter capable of attenuating UV irradiation, in a tailored manner, was investigated. This novel optical filter was successfully applied to the surface modification of poly(BuMA-co-EDMA) monolithic columns in a multi-step grafting procedure. Fabricated columns were subjected to scanning capacitively coupled contactless conductivity (sC(4)D), to determine the distribution of the grafted functional groups, axially along the column. Further modification to produce a chelating stationary phase gradient of iminodiacetic acid (IDA) was demonstrated. To demonstrate the distribution of the IDA sites, a metal cation (Cu(2+)) was complexed to the IDA forming a chelate. Upon the formation of a complex of IDA with Cu(2+), an overall drop in conductive response was observed. The COC optical filter was also used in the fabrication of a grafted gradient of strong cation exchanger (SCX), sulphopropyl methacrylate (SPM) upon a polymer monolith, demonstrating the broader applicability of such a filter.     

Preparation and characterization of monolithic column by grafting pH-responsive polymer

A novel modified monolithic column with pH-responsive polymer chains was prepared by grafting methacrylic acid onto the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith. The grafting polymerization was achieved in an in situ manner which was performed by pumping methacrylic acid directly through an acidic hydrolysis monolithic column using potassium peroxydisulfate initiated free-radical polymerization. The grafted monolithic column was demonstrated to be the pH-responsive to the pore structure and the chromatographic characterization. The permeability of the column and the retention factors of five benzene homologues decreased due to the conformational changes of the polymer chains when the pH of mobile phase increased from 4.5 to 7.5. Furthermore, the modified monolithic column was used as the pH-responsive stationary phase and exhibited an excellent separation of four basic proteins.     

Porous structure of the monolithic sorbent and separation properties of monolithic capillary columns in gas and liquid chromatography

Macroporous polymer based on polydivinylbenzene was used for the preparation of monolithic capillary columns with the diameter from 0.01 to 0.53 mm for separations by gas and liquid chromatography. The separation properties of the columns were studied by analysis of model systems of aromatic (in liquid chromatography) and light (in gas chromatography) hydrocarbons. The permeability was determined and the C parameter of the Van-Deemter equation was found for each column. The permeability of the majority of columns determined by gas chromatography is independent of the column diameter. The permeability of the same columns in liquid chromatography is also almost constant for the columns 0.53–0.1 mm in diameter; however, the permeability decreases sharply on going to columns of smaller diameter. In gas chromatography the value of the C parameter reflecting the effect of the mass transfer of the sorbate between the mobile and stationary phases on the smearing of a chromatographic peak in the column approximately the same for all columns. In liquid chromatography the value of the C coefficient in the Van-Deemter equation for the same capillary columns changes with a change in the column diameter and reaches a minimum for the columns 0.1 mm in diameter. The differences observed for the characteristics of the columns in gas and liquid chromatography are due to different structures of the macroporous monolith formed in columns of different diameter and to the effect of solvation of the monolith by the mobile phase under the conditions of liquid chromatography.     

Adamantyl-functionalized polymer monolith for capillary electrochromatography

An adamantyl (ADM)-functionalized monolithic stationary phase was newly synthesized by a single-step copolymerization of 1-adamantyl-(α-trifluoromethyl) acrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid in order to prevent the peak tailing of basic solutes in capillary electrochromatography and was compared with butyl methacrylate (BMA)-based one. The ADM structure shields the negatively charged groups on the surface of monolith from basic solutes, resulting in better peak shapes than BMA-based monolithic stationary phase. As the monomers ratio decreased, the monolithic column had lower retention and higher column efficiency which was likely due to lower phase ratio and smaller globule size of monolith, respectively. The ADM-functionalized monolithic columns exhibited a good repeatability and reproducibility of column preparation with relative standard deviation values below 9% in the studied chromatographic parameters.     

Preparation of Organic-Silica Hybrid Monolith with Anion Exchange/Hydrophilic Interaction Mixed-Mode Via Epoxy–Amine Ring-Opening Polymerization Using Polyethylenimine as Functional Monomer

Polyethylenimine (PEI) and 2,4,6,8-tetramethyl-2,4,6,8-tetrakis(propyl glycidyl ether)cyclotetrasiloxane (POSS–epoxy) were used as precursors for the preparation of organic-silica hybrid monolithic columns (PEI–POSS monolith) via epoxy–amine ring-opening polymerization (ROP). The high density of amine groups in PEI provides rich chromatographic interaction sites for the polar or acidic analytes in hydrophilic interaction (HILIC) and weak anion exchange (WAX) mechanisms. The column preparation conditions, such as the porogens, solvent and reaction temperature, were systematically investigated according to the morphology, permeability and column efficiency. The separation mechanisms of HILIC and WAX were evaluated with neutral polar compounds and halogen benzoic acids. Owing to the existence of reactive amine groups on the matrix surface, the PEI–POSS monolith is also an ideal starting material for the preparation of HILIC or strong anionic exchange (SAX) stationary phases by modification. The modification of PEI–POSS monoliths with iodomethane or bromoacetic acid via the nucleophilic substitution reaction could achieve the retention mechanisms of SAX or zwitterionic HILIC, respectively.

     

Separation of polystyrenes by means of open tubular capillary chromatography

Simulating polymer separation in flow-through channels of monolithic columns, separation of a mixture of polystyrene standards was investigated using open tubular capillary column of 2 μm inner diameter. High column efficiency was observed for polymers of molar mass ranged from few tens to few hundred kDas. Column efficiency significantly decreased for polymers with molar mass larger than 500 kDa nevertheless preserving value of few tens of thousands theoretical plates. Calibration curve observed for open capillary column is rather steep and can be well described by simple equation without quadratic term. In spite of low selectivity, capillary columns were able in separating wide range of polystyrene standards due to column high efficiency and in such a way supported an idea of hydrodynamic mechanism of polymer separation in flow-through channel of monolithic packings.     

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.     

Silica-based monolithic columns with mixed-mode reversed-phase/weak anion-exchange selectivity principle for high-performance liquid chromatography

This article describes the synthesis, chromatographic characterization, and performance evaluation of analytical (100 x 4.6 mm id) and semipreparative (100 x 10 mm id) monolithic silica columns with mixed-mode RP/weak anion-exchange (RP/WAX) surface modification. The monolithic RP/WAX columns were obtained by immobilization of N-(10-undecenoyl)-3-aminoquinuclidine onto thiol-modified monolithic silica columns (Chromolith) by a radical addition reaction. Their chromatographic characterization by Engelhardt and Tanaka tests revealed slightly lower hydrophobic selectivities than C-8 phases, as well as higher polarity and also improved shape selectivity than RP-18e silica rods. The surface modification enabled separation by both RP and anion-exchange chromatography principles, and thus showed complementary selectivities to the RP-18e monoliths. The mixed-mode monoliths have been tested for the separation of peptides and turned out to be particularly useful for hydrophilic acidic peptides, which are usually insufficiently retained on RP-18e monolithic columns. Compared to a corresponding particulate RP/WAX column (5 microm, 10 nm pore diameter), the analytical RP/WAX monolith caused lower system pressure drops and showed, as expected, higher efficiency (e.g. by a factor of about 2.5 lower C-term for a tetrapeptide). The upscaling from the analytical to semipreparative column dimension was also successful.