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.     

Alkylated poly(styrene-divinylbenzene) monolithic columns for mu-HPLC and CEC separation of phenolic acids

Macroporous poly(styrene-divinylbenzene) monolithic columns were prepared in fused silica capillaries of 100 microm id by in-situ copolymerization of styrene with divinylbenzene in the presence of propan-1-ol and formamide as the porogen system. The monoliths were subsequently alkylated with linear alkyl C-18 groups via Friedel-Crafts reaction to improve the retention and chromatographic resolution of strongly polar phenolic acids. A new thermally initiated grafting procedure was developed in order to shorten the time of the alkylation process. The grafting procedure was optimized with respect to the reaction temperature, time, the grafting reactant concentration, and the solvent used. The type of solvent and the grafting temperature are the most significant factors affecting the hydrodynamic properties, porosity, and efficiency of the columns. While the equivalent particle diameter of the grafted column increased, the capillary-like flow-through pore diameter decreased in comparison to non-alkylated monoliths. The hydrodynamic permeability of the monolith decreased, but the monolithic column still permitted fast micro-HPLC separations.     

One‐pot preparation of mercaptotetrazole‐silica hybrid monoliths by the thiol‐ene click reaction for mixed‐mode capillary liquid chromatography

A novel mercaptotetrazole‐silica hybrid monolithic column was prepared for capillary liquid chromatography, in which the thiol‐end mercaptotetrazole was mixed with hydrolyzed γ‐methacryloxypropyltrimethoxysilane and tetramethyloxysilane for the co‐polycondensation and thiol‐ene click reaction in a one‐pot process. The effects of the molar ratio of silanes, the amount of mercaptotetrazole, and the volume of porogen on the morphology, permeability and pore properties of the as‐prepared mercaptotetrazole‐silica hybrid monoliths were investigated in detail. A series of test compounds including alkylbenzenes, amides and anilines were employed for evaluating the retention behaviors of the mercaptotetrazole‐silica hybrid monolithic columns. The results demonstrated that the mercaptotetrazole‐silica hybrid monoliths exhibited hydrophobic, hydrophilic as well as ion‐exchange interaction. The run‐to‐run, column‐to‐column and batch‐to‐batch reproducibilities of the mercaptotetrazole‐silica hybrid monoliths were satisfactory with the relative standard deviations less than 1.4 (n  = 5), 3.9 (n  = 3) and 4.0% (n  = 5), respectively. In addition, the mercaptotetrazole‐silica hybrid monolith was further applied to the separation of sulfonamides, nucleobases and protein tryptic digests. These successful applications confirmed the promising potential of the mercaptotetrazole‐silica hybrid monolith in the separation of complex samples.     

Chemically modified chiral monolithic silica column prepared by a sol-gel process for enantiomeric separation by micro high-performance liquid chromatography

In this work a new type of chiral monolith silica column was developed for the chiral separation by micro high-performance liquid chromatography (micro-HPLC). The chiral monolith column with a continuous skeleton and a large through-pore structure was prepared inside a capillary of 100 microm I.D. by a sol-gel process, and chemically modified with chiral selectors, such as L-phenylalaninamide, L-alaninamide and L-prolinamide, on the surface of the monolithic silica column. Based on the principle of ligand exchange, these chiral monolithic columns were successfully used for the separation of dansyl amino acid enantiomers, as well as hydroxy acid enantiomers by micro-HPLC. The chromatographic conditions, the enantioselectivity and the performance of columns are discussed.     

Monolithic Silica Columns for HPLC,Micro‐HPLC,and CEC

Two types of monolithic silica columns derivatized to form an ODS phase, one prepared in a fused silica capillary (SR‐FS) and the other prepared in a mold and clad with an engineering plastic (poly‐ether‐ether‐ketone) (SR‐PEEK), were evaluated. The column efficiency and pressure drop were compared with those of a column packed with 5‐μm ODS‐silica particles and of an ODS‐silica monolith prepared in a mold and wrapped with PTFE tubing (SR‐PTFE). SR‐FS gave a lower pressure drop than a column packed with 5‐μm particles by a factor of 20, and a plate height of 20 μm at a linear velocity below 1 mm/s. SR‐PEEK showed higher flow‐resistance than the other monolithic silica columns, but they still showed a minimum plate height of 8–10 μm and a lower pressure drop than popular commercial columns packed with 5‐μm particles. The evaluation of SR‐FS columns in a CEC mode showed much higher efficiency than in a pressure‐driven mode.     

Preparation and characterization of molecularly imprinted silica monolith for screening sulfamethazine

In this work, a novel approach of preparing molecularly imprinted film‐derivatized silica monolith materials was developed by a two‐step procedure. The silica monolithic support was first prepared by the sol–gel method with tetramethoxysilane as the precursor. Subsequently, vinyl groups were introduced onto the surface of silica monolith by immobilization of γ‐methacryloxypropyltrimethoxysilane. The prepolymerization mixtures, consisting of methacrylic acid as a functional monomer, ethylene dimethacrylate as a crosslinker, sulfamethazine as a template molecule and an ionic liquid as porogen, were injected into the silica monolith immobilized vinyl groups to form the molecularly imprinted films on the surface of the vinyl functionalized silica monolith. The monolithic materials were characterized by SEM, Fourier transform IR and solid‐state reflection UV spectra. The resulted imprinted materials were evaluated under CEC and HPLC mode. The results indicated that there were enough recognition sites on the surface of the imprinted film‐derivatized monolithic materials for selectively recognizing sulfamethazine from the sulfonamide mixture. Ionic liquids, which was utilized as the porogens, could improve the flow‐through property and the imprinting effect of the molecularly imprinted film‐functionalized silica monolithic materials.     

Comprehensive study of the macropore and mesopore size distributions in polymer monoliths using complementary physical characterization techniques and liquid chromatography

Poly(styrene‐co‐divinylbenzene) monolithic stationary phases with two different domain sizes were synthesized by a thermally initiated free‐radical copolymerization in capillary columns. The morphology was investigated at the meso‐ and macroscopic level using complementary physical characterization techniques aiming at better understanding the effect of column structure on separation performance. Varying the porogenic solvent ratio yielded materials with a mode pore size of 200 nm and 1.5 μm, respectively. Subsequently, nano‐liquid chromatography experiments were performed on 200 μm id × 200 mm columns using unretained markers, linking structure inhomogeneity to eddy dispersion. Although small‐domain‐size monoliths feature a relatively narrow macropore‐size distribution, their homogeneity is compromised by the presence of a small number of large macropores, which induces a significant eddy‐dispersion contribution to band broadening. The small‐domain size monolith also has a relatively steep mass‐transfer term, compared to a monolith containing larger globules and macropores. Structural inhomogeneity was also studied at the mesoscopic level using gas‐adsorption techniques combined with the non‐local‐density‐function‐theory. This model allows to accurately determine the mesopore properties in the dry state. The styrene‐based monolith with small domain size has a distinctive trimodal mesopore distribution with pores of 5, 15, and 25 nm, whereas the monolith with larger feature sizes only contains mesopores around 5 nm in size.     

丙基脲硅胶毛细管整体柱的制备及性能研究

采用溶胶-凝胶技术在毛细管中原位合成硅胶整体柱,通过表面化学修饰技术制备了极性的丙基脲硅胶整体柱.对所制备的整体柱柱性能进行了评价,考察了极性物质在该整体柱上的保留行为,并对其可能的保留机理进行了探讨.研究表明,该柱在亲水作用电色谱模式下能有效分离苯酚类极性小分子化合物.     

Preparing titania aerogel monolithic chromatography columns using supercritical carbon dioxide

The search for a method to fabricate monolithic inorganic columns has attracted significant recent attention due to their unique ability in separation applications of various biomolecules. Silica and polymer based monolithic columns have been prepared, but titania and other metal oxide monoliths have been elusive, primarily due to their fragility. This article describes a new approach for preparing nanostructured titania based columns, which offer better performance over conventional particle packed columns for separating a wide variety of biomolecules including phosphopeptides. TiO₂ monolithic aerogels were synthesized in separation columns using in situ sol‐gel reactions in supercritical carbon dioxide (scCO₂) followed by calcination, and compared to those prepared in heptanes. The characterization results show that scCO₂ is a better solvent for the sol‐gel reactions, providing lower shrinkage with the anatase TiO₂ monolith composed of nanofibers with very high surface areas. The monolithic columns show the ability to isolate phosphopeptides with little flow resistance compared to conventional titania particle based microcolumns.     

Investigating retention characteristics of a mixed‐mode stationary phase and the enhancement of monolith selectivity for high‐performance liquid chromatography

The synthesis and chromatographic behavior of an analytical size mixed‐mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3‐cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance π‐type bonding within a single separation environment. Selectivity studies were undertaken using n‐alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the π‐type selectivities afforded by the two different ligands on the mixed‐mode column.     

牛血清白蛋白修饰毛细管整体柱的制备及组氨酸对映体分离

以甲基丙烯酸缩水甘油酯为单体, 乙二醇二甲基丙烯酸酯为交联剂, 环己醇和正十二醇混合溶液为致孔剂, 在最佳聚合条件下, 以偶氮二异丁腈为引发剂, 制备了毛细管整体柱基质, 并且研究了单体、交联剂及致孔剂对整体柱基质孔结构及渗透性的影响; 使用Epoxy方法在基质表面键合BSA, 制得BSA修饰的毛细管整体柱. 将此毛细管整体柱应用于毛细管电色谱中, 成功地分离出了组氨酸对映体, 分离度良好.     

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.     

Reduced-bore monolithic silica column modified with C8-TEOS for reversed-phase electrochromatography

Monolithic silica columns of 2.7 mm ID were prepared and derivatized with C8-TEOS and TEOS by on-column sol-gel reaction. These C8 large diameter monolithic silica columns gave 21 000 theoretical plates for aromatic hydrocarbons in 60% acetonitrile and 40% Tris-HCI buffer. The surface areas as well as the separation reproducibility were improved on coating by the sol-gel approach. Joule heating was greatly reduced by using monolithic columns to which fine quartz sand had been added during column preparation. Since this is a preliminary investigation on a monolithic column with such a large inner diameter, the separation efficiency was not so high as that presently achieved in normal capillary electrochromatography (CEC). However, use of the columns improved sample loadability and concentration detectability of electrochromatography, and semi-preparative separations could be performed.     

One‐step strategy for the synthesis of a derivatized cyclodextrin‐based monolithic column

Derivatized β‐cyclodextrin (β‐CD) functionalized monolithic columns were prepared by a “one‐step” strategy using click chemistry. First, the intended derivatized β‐CD monomers were synthesized by a click reaction between propargyl methacrylate and mono‐6‐azido‐β‐CD and then sulfonation or methylation was carried out. Finally, monolithic columns were prepared through a one‐step in situ copolymerization of the derivatized β‐CD monomer and ethylene glycol dimethacrylate. The sulfated β‐CD‐based monolith was successfully applied to the hydrophilic interaction liquid chromatography separation of nucleosides and small peptides, while the methylated β‐CD‐functionalized monolith was useful for the separation of nonpolar compounds and drug enantiomers in capillary reversed‐phase liquid chromatography. The structures of the monomers were characterized by Fourier transform infrared spectroscopy and mass spectrometry. The physicochemical properties and column performance of monoliths were evaluated by scanning electron microscopy and micro high performance liquid chromatography. This strategy has considerable prospects for the preparation of other derivatized CD‐functionalized methacrylate monoliths.     

Silica‐based zwitterionic monolithic stationary phase for separation of neutral and ionized solutes using pressurized CEC

A porous zwitterionic monolith was prepared by in situ covalent attachment of lysine to a γ‐glycidoxypropyltrimethosysilane‐modified silica monolith. The prepared column was used to perform neutral and ionized solutes separations by pressurized (pCEC). Due to the zwitterionic nature of the resulting stationary phase, the monolithic column provided both electrostatic attraction and repulsion sites for electrochromatographic retention for ionized solutes. Separation of several nucleotides was investigated on the monolithic column. It was shown that the nucleotides could be separated based on hydrophilic and electrostatic interactions between the stationary phase and analyte. Besides, the separation property of the zwitterionic silica monolith was compared with the use of diamine‐bonded silica monolith as stationary phase. As expected, the lysine monolith exhibited a lower retention for the five nucleotides, which was due to the dissociation of the external carboxylic acid groups, leading to electrostatic repulsion with negatively charged solutes. Under the same experimental conditions, separation of the five nucleotides on the zwitterionic column was in less than 8 min, while that on the diamine column was in approximately 60 min.     

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.     

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)。     

Comparison of monolithic silica and polymethacrylate capillary columns for LC

Organic polymer monolithic capillary columns were prepared in fused-silica capillaries by radical co-polymerization of ethylene dimethacrylate and butyl methacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in the presence of various amounts of porogenic solvent mixtures and different concentration ratios of monomers and 1-propanol, 1,4-butanediol, and water. The chromatographic properties of the organic polymer monolithic columns were compared with those of commercial silica-based particulate and monolithic capillary and analytical HPLC columns. The tests included the determination of H-u curves, column permeabilities, pore distribution by inversed-SEC measurements, methylene and polar selectivities, and polar interactions with naphthalenesulphonic acid test samples. Organic polymer monolithic capillary columns show similar retention behaviour to chemically bonded alkyl silica columns for compounds with different polarities characterized by interaction indices, Ix, but have lower methylene selectivities and do not show polar interactions with sulphonic acids. The commercial capillary and analytical silica gel-based monolithic columns showed similar selectivities and provided symmetrical peaks, indicating no significant surface heterogeneities. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra-column contributions. With 0.3 mm ID capillary columns, corrections for extra-column volume contributions are sufficient, but to obtain true information on the efficiency of 0.1 mm ID capillary columns, the experimental bandwidths should be corrected for extra-column contributions to peak broadening.     

Novel monolithic poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethylsilane) capillary columns for biopolymer separation

Hydrophobic organo-silane based monolithic capillary columns were prepared by thermally initiated free radical polymerisation within the confines of 200 microm i.d. fused silica capillaries. A novel crosslinker, namely bis(p-vinylbenzyl)dimethylsilane (BVBDMS), was copolymerised with p-methylstyrene (MS) in the presence of 2-propanol and toluene, using alpha,alpha'-azoisobutyronitrile (AIBN) as initiator. Monolithic capillary columns, differing in the total monomer, microporogen content and microporogen nature were fabricated and the chromatographic efficiency of each monolith, regarding the separation of proteins, peptides and oligonucleotides, was evaluated and compared. Changes in monolith morphology were monitored by scanning electron microscopy (SEM). Porosity and specific surface areas of the supports were studied by means of mercury intrusion porosimetry and BET measurements, respectively. Pressure drop vs. flow rate measurements proved the prepared poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethylsilane) (MS/BVBDMS) monoliths to be mechanically stable and swelling propensity (SP) factors of 0.78-1.10 indicate high crosslinking homogeneity.     

Divinylbenzene-based monolithic capillary columns in capillary liquid chromatography

The effect of the conditions of synthesis of divinylbenzene-based monolithic capillary columns on their chromatographic characteristics was studied. It was demonstrated that the porosity and permeability of the column change significantly even at small deviations from the optimum conditions of polymerization of the monolith in the column. By contrast, the minimum value of HETP proved to be only slightly sensitive to the conditions of synthesis, ranging within ～10–20 μm. The conditions of polymerization of the monolith were found to produce more pronounced effect on the slope of the right branch of the van Deemter curve (parameter C), with the flattest curve being observed for columns prepared under optimum conditions. The minimum value of HETP for polymer monolithic capillary columns was found to be similar to that for silica gel monolithic capillary columns, but the latter are characterized by C values approximately an order of magnitude lower.