Preparation of a long‐alkyl‐chain‐based hybrid monolithic column with mixed‐mode interactions using a “one‐pot” process for pressurized capillary electrochromatography

A simple “one‐pot” approach for the preparation of a new vinyl‐functionalized organic–inorganic hybrid monolithic column is described. In this improved method, the hydrolyzed alkoxysilanes of tetramethoxysilane and triethoxyvinylsilane were used as precursors for the synthesis of a silica‐based monolith, while 1‐hexadecene and sodium ethylenesulfonate were used as vinyl functional monomers along with azobisisobutyronitrile as an initiator. The effects of reaction temperature, urea content, and composition of organic monomers on the column properties (e.g. morphology, mechanical stability, and chromatographic performance) were investigated. The monolithic column was used for the separation of neutral solutes by reversed‐phase pressurized capillary. Furthermore, the monolith can separate various aromatic amines, which indicated its excellent cation‐exchange capability and hydrophobic interactions. The baseline separation of the aromatic amines was obtained with a column efficiency of up to 78 000 plates/m.     

Silica‐particle‐supported zwitterionic polymer monolith for microcolumn liquid chromatography

A silica‐particle‐supported zwitterionic polymeric monolithic column, shortened as supported column (S‐column), was prepared by the in situ polymerization of methacrylic acid, ethylene dimethacrylate, and 2‐(dimethylamino)ethyl methacrylate in the presence of a ternary porogenic solvent containing water, methanol, and cyclohexanol in a 250 μm id fused‐silica capillary prepacked with 5 μm bare silica particles. In the S‐column, a thin layer of the polymers was formed around the silica particles in the form of nanoglobules, leaving the interstitial spaces between the particles free for liquid flow. The effects of the preparation conditions on the morphology of the monolith were investigated by scanning electron microscopy and backpressure measurements. The selected volumetric ratio of porogens, monomer concentration, polymerization time, and temperature are 1:1:8 (water/methanol/cyclohexanol), 25% v/v, 5 h, and 60°C, respectively. The S‐column was evaluated by comparison with its conventional organic counterpart in terms of morphology, mechanical stability, permeability, swelling–shrinking behavior, capacity, and efficiency. The results demonstrate that the S‐column is superior to its counterpart in all the terms with the exception of permeability. The above merits and zwitterionic property of the S‐column were further confirmed by separate separations of four inorganic anions and three organic cations.     

Preparation of chitosan functionalized monolithic silica column for hydrophilic interaction liquid chromatography

A novel cationic hydrophilic interaction monolithic stationary phase based on the chemical modification of carboxymethyl chitosan (CMCH) to the monolithic silica skeleton using carbodiimide as an activation reagent was prepared for performing capillary liquid chromatography. The amino and hydroxy moieties of CMCH functioned as both the ion-exchange sites and polar providers. The performance of the column was studied by the separation of polar acidic compounds. The chitosan functionalized monolithic silica column showed good selectivity for nucleosides, nucleotides, aromatic acids and aliphatic acids. The mechanism for the separation of these compounds was also studied. The results showed that these compounds were separated primarily based on the hydrophilic interaction mechanism.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.     

Preparation of a monolithic column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction for capillary liquid chromatography

A monolithic capillary column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in‐situ copolymerization of a homemade monomer N,N‐dimethyl‐N‐acryloxyundecyl‐N‐(3‐sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed‐mode stationary phase. It was demonstrated that the mixed‐mode stationary phase displayed typic dual retention mechanisms of reversed‐phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.     

Quinine‐modified polymer monolithic column with reversed‐phase /strong anion‐exchange mixed‐mode for pressurized capillary electrochromatography

Via the facile ring‐opening reaction of epoxy groups with quinine, a novel polymer monolith with quaternary ammonium for reversed‐phase/strong anion‐exchange mixed‐mode has been fabricated for pressurized capillary electrochromatography (pCEC). Optimization on the preparation of quinine‐modified monoliths has been investigated, and characteristics including morphology, permeability, mechanical stability, reproducibility, and column performance have been also studied. Active quaternary ammonium groups were conveniently produced to generate cationic action sites and stable anodic electroosmotic flow. Multiple interactions including reversed‐phase, strong anion‐exchange, electrostatic repulsion and π–π stacking interactions were obtained. Satisfactory separation capability of various analytes such as alkylbenzenes, polycyclic aromatic hydrocarbons, benzoic acid and its homologs, and β₂‐receptor excitants has been achieved. Applied to the real sample, the good resolution of three alkaloids in Corydalis yanhusuo were achieved by pCEC with the quinine‐modified monolith. The results light a potential access to facilely fabricating quaternary ammonium‐functionalized polymer monolith with multiple interactions for efficient electrochromatography profiling of various compounds.     

Preparation of a monolithic cation‐exchange material with hydrophilic external layers by two‐step reversible addition‐fragmentation chain transfer polymerization

In recent years, the efficient analysis of biological samples has become more important due to the advances of life science and pharmaceutical research and practice. Because biological sample pretreatment is the bottleneck for fast process, material development for efficient sample process in the high‐performance liquid chromatography analysis is highly desirable. In this research, a cation‐exchange restricted access monolithic column was synthesized by a reversible addition‐fragmentation chain transfer polymerization method. Utilizing the controlled/living property of the reversible addition‐fragmentation chain transfer method, a monolithic column of cross‐linked poly(sulfopropyl methacrylate) was prepared first and then linear poly(glycerol mono‐methacrylate) was immobilized covalently on the surface of the polymer. The monolithic material has both functionalities of cation‐exchange and protein exclusion. Protein recovery of 94.6% was obtained after grafting of poly(glycerol mono‐methacrylate) while the cation‐exchange property of the column is still retained. In the study, the relation between the synthetic conditions and properties of the materials was studied. The synthesis conditions including the porogen, monomer concentration, and ratio of monomers/initiator/reversible addition‐fragmentation chain transfer agent were optimized. The study provided a method for the preparation of restricted access monolithic columns: a bifunctional material by reversible addition‐fragmentation chain transfer polymerization method.     

Study of triacontyl-functionalized monolithic silica capillary column for reversed-phase capillary liquid chromatography

A novel stationary phase triacontyl-functionalized monolithic silica capillary column was successfully prepared for reversed-phase capillary liquid chromatography. The performance of the monolithic silica capillary column coated with triacontyl chain for the separation of alkylbenzenes, xylene isomers, polycyclic aromatic hydrocarbons, and mixture of α- and β-carotenes was studied, which was compared to that using the monolithic silica capillary column coated with octadecyl chain. The comparison results showed that triacontyl-functionalized monolithic silica capillary column would be a promising media to be used for the separation of isomeric solutes with long chain in reversed-phase capillary liquid chromatography.     

Sulfoalkylbetaine‐based monolithic column with mixed‐mode of hydrophilic interaction and strong anion‐exchange stationary phase for capillary electrochromatography

A novel monolithic stationary phase with mixed mode of hydrophilic and strong anion exchange (SAX) interactions based on in situ copolymerization of pentaerythritol triacrylate (PETA), N,N‐dimethyl‐N‐methacryloxyethyl N‐(3‐sulfopropyl) ammonium betaine (DMMSA) and a selected quaternary amine acrylic monomer was designed as a multifunctional separation column for CEC. Although the zwitterionic functionalities of DMMSA and hydroxy groups of PETA on the surface of the monolithic stationary phase functioned as the hydrophilic interaction (HI) sites, the quaternary amine acrylic monomer was introduced to control the magnitude of the EOF and provide the SAX sites at the same time. Three different quaternary amine acrylic monomers were tested to achieve maximum EOF velocity and highest plate count. The fabrication of the zwitterionic monolith (designated as HI and SAX stationary phase) was carried out when [2‐(acryloyloxy)ethyl]trimethylammonium methylsulfate was used as the quaternary amine acrylic monomer. The separation mechanism of the monolithic column was discussed in detail. For charged analytes, a mixed mode of HI and SAX was observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(PETA‐co‐DMMSA‐co‐[2‐(acryloyloxy)ethyl]trimethylammonium methylsulfate) monolithic column. The optimized monolith showed good separation performance for a range of polar analytes including nucleotides, nucleic acid bases and nucleosides, phenols, estrogens and small peptides. The column efficiencies greater than 192 000 theoretical plates/m for estriol and 135 000 theoretical plates/m for charged cytidine were obtained.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Polydopamine‐functionalized monolithic silica column for mixed‐mode chromatography†

Dopamine is easy to self‐polymerize under alkaline conditions and the resultant polydopamine is easy to adhere to the surface of many organic and inorganic materials. Based on the characteristics of dopamine, in this paper, a new polydopamine functionalized monolithic silica column was successfully prepared for performing mixed‐mode chromatography. The performance of the column was evaluated by the separation of different types of samples including alkylbenzenes, polycyclic aromatic hydrocarbons, aromatic acids, phenols, and bases. The mechanism for the separation of these compounds was studied and appeared to involve the mixed interactions containing π?π, hydrophobic, electrostatic, and hydrophilic interactions.     

Histidine‐modified organic‐silica hybrid monolithic column for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography

A novel organic‐silica hybrid monolith was prepared through the binding of histidine onto the surface of monolithic matrix for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography. The imidazolium and amino groups on the surface of the monolithic stationary phase were used to generate an anodic electro‐osmotic flow as well as to provide electrostatic interaction sites for the charged compounds at low pH. Typical per aqueous chromatographic behavior was observed in water‐rich mobile phases. Various polar and hydrophilic analytes were selected to evaluate the characteristics and chromatographic performance of the obtained monolith. Under per aqueous conditions, the mixed‐mode mechanism of hydrophobic and ion‐exchange interactions was observed and the resultant monolithic column proved to be very versatile for the efficient separations of these polar and hydrophilic compounds (including amides, nucleosides and nucleotide bases, benzoic acid derivatives, and amino acids) in highly aqueous mobile phases. The successful applications suggested that the histidine‐modified organic‐silica hybrid monolithic column could offer a wide range of retention behaviors and flexible selectivities toward polar and hydrophilic compounds.     

Preparation of a hybrid monolithic stationary phase with allylsulfonate for the rapid and simultaneous separation of cations in capillary ion chromatography

A hybrid monolithic column with sulfonate functionality was successfully prepared for the simultaneous separation of common inorganic cations in ion‐exchange chromatographic mode through a simple and easy single‐step preparation method. The strong cation‐exchange moieties were provided directly from allylsulfonate, which worked as an organic monomer in the single‐step reaction. Inorganic cations (Li⁺, Na⁺, K⁺, NH₄⁺, Cs⁺, Rb⁺, Mg²⁺, Ca²⁺, and Sr²⁺) were separated satisfactorily by using CuSO₄ as the eluent with indirect UV detection. The allysulfonate hybrid monolith showed a better performance in terms of speed and pressure drop than the capillary packed column. The number of theoretical plates achieved was 19 017 plates/m (in the case of NH₄⁺ as the analyte). The relative standard deviations (n = 6) of both retention time and peak height were less than 1.96% for all the analyte cations. The allysulfonate hybrid monolithic column was successfully applied for the rapid and simultaneous separation of inorganic cations in groundwater and the effluent of onsite domestic wastewater treatment system.     

Preparation of phosphorylcholine‐based hydrophilic monolithic column and application for analysis of drug‐related impurities with capillary electrochromatography

A hydrophilic monolithic CEC column was prepared by thermal copolymerization of zwitterionic monomer 2‐methacryloyloxyethyl phosphorylcholine (MPC), pentaerythritol triacrylate (PETA), either methacrylatoethyl trimethyl ammonium chloride (META) or sodium 2‐methylpropene‐1‐sulfonate (MPS) in a polar binary porogen consisting of methanol and THF. A typical hydrophilic interaction LC retention mechanism was observed for low‐molecular weight polar compounds including amides, nucleotides, and nucleosides in the separation mode of hydrophilic interaction CEC, when high content of ACN (>60%) was used as the mobile phase. The effect of the electrostatic interaction between the analytes and the stationary phase was found to be negligible. The poly(MPC‐co‐PETA‐co‐META or MPS) monolithic columns have an average column efficiency of 40 000 plates/m and displayed with a satisfactory repeatability in terms of migration time and peak areas. Finally, the column was successfully applied to determine the impurities of a positively charged drug pramipexole which are often separated by ion pair RP chromatography due to their high hydrophilicity. All four components can be baseline separated within 5 min with BGE consisting of ACN/20 mM ammonium formate buffer (pH 3.0; 80/20).     

Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix‐assisted laser desorption/ionization‐based proteomic analysis

Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC‐HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC‐HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC‐HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC‐HILIC has better peptide fractionation ability. We further demonstrated that ZIC‐HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC‐HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Sudan dyes in chili pepper powder by online solid‐phase extraction with a butyl methacrylate monolithic column coupled to liquid chromatography with tandem mass spectrometry

A poly(butyl methacrylate‐co‐ethylene dimethacrylate) monolithic column was fabricated and used as a novel sorbent for online solid‐phase extraction coupled to liquid chromatography with tandem mass spectrometry for the simultaneous determination of Sudan I–IV in chili pepper powder. The prepared columns were characterized by scanning electron microscopy, nitrogen adsorption‐desorption, and pressure drop measurements. Online solid‐phase extraction was performed on the synthesized monolithic column using 10 mM ammonium acetate solution as the loading solution with the aid of an online cleanup chromatography system. The desorption of Sudan I–IV was achieved with acetonitrile as the eluting solution at the flow rate of 0.5 mL/min. The extracted analytes were subsequently eluted into a C₁₈ analytical column for chromatographic separation using a mixture of 10% acetonitrile/90% formic acid (0.5%) solution as the mobile phase. Under the optimized conditions, the developed method had linear range of 1.0–50 μg/kg, a detection limit of 0.3 μg/kg, and a quantification limit of 1.0 μg/kg for each analyte. The intraday and interday recoveries of Sudan I–IV in chili pepper powder samples ranged from 94.8 to 100.9% and 94.9 to 99.4%, respectively. The intraday and interday precision were between 3.37–7.01% and 5.01–7.68%, respectively.     

Two‐dimensional strong cation exchange/positively charged reversed‐phase liquid chromatography for alkaloid analysis and purification

Peak tailing and nonalkaloid coelution usually hinder alkaloid purification. In this study, a 2DLC, strong cation exchange (SCX) coupled with positively charged RP (PGRP) LC, was developed to overcome these problems. Ten compounds including basic and nonbasic compounds were analyzed. Nonbasic compounds, which are coeluted with basic compounds on RP or PGRP columns, were weakly retained on the SCX column. In addition, a symmetrical peak shape (tailing factors <1.2) of basic compounds can be obtained in the current system. Compared to two other 2D systems, the current system provided the highest orthogonality (R² = 0.045). Furthermore, the SCX coupled with PGRP system was applied for alkaloid purification from a traditional Chinese medicine. Nineteen alkaloids were obtained and one of them was identified as a novel compound. The overall results demonstrate that the proposed system is a powerful tool for alkaloid purification.     

一种聚甲基丙烯酸酯类亲水作用毛细管电色谱整体柱的制备与评价

以2-羟基乙基甲基丙烯酸酯(HEMA)为单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,制备了亲水分离模式的聚HEMA-co-EDMA新型毛细管电色谱(CEC)整体柱。考察了整体柱的结构特征及其CEC性能,研究了极性物质的保留行为,并对其可能的保留机理进行了探讨。所制备的整体柱稳定性好,固定相表面带有极性羟基功能团,不仅能提供亲水相互作用位点,且能吸附流动相中的阴离子产生阳极电渗流(EOF)。在流动相中乙腈含量较高(＞62%,体积分数)的条件下,整体柱表现出典型的亲水作用,实现了对核苷、碱基和苯胺类带有碱性的强极性化合物的高效分离,并成功分离了苯酚类、苯甲酸类等中性或酸性的极性化合物。     

Fabrication of single‐walled carbon nanohorns incorporated a monolithic column for capillary electrochromatography

Single‐walled carbon nanohorns have received great interest for their unique properties and diverse potential applications. Herein, we demonstrated the feasibility of single‐walled carbon nanohorns incorporated poly(styrene‐divinylbenzene) monolith as the stationary phase for capillary electrochromatography, which were prepared by one‐step in situ copolymerization. Single‐walled carbon nanohorns were dispersed in styrene to give a stable and homogeneous suspension. The monolithic column gave effective separation for a wide range of aromatic compounds, which was based on hydrophobicity and π–π electrostatic stacking of single‐walled carbon nanohorns. The precisions of migration time and peak area varied in the ranges of 1.4–1.9% for intraday trials and 1.7–3.5% for interday trials, and 3.2–6.7% for intraday trials and 4.1–7.4% for interday trials, and 3.6–7.2% for inter‐column trials and 5.2–21.3% for inter‐column trials, respectively, indicating the good reproducibility of single‐walled carbon nanohorns embedded monolithic columns.     

弱阳离子交换整体柱的制备及其对人血浆中硝苯地平的在线分析

以甲基丙烯酸(MAA)为功能单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,以色谱柱管为模具,通过原位聚合法制备了弱阳离子交换整体柱。该柱能去除血浆中的内源性物质,对生物样品中的药物有富集作用。将其作为固相萃取柱与C18色谱柱联用,在线分析了人血浆中的硝苯地平。流动相为甲醇-水(体积比为70∶30),流速1.0 mL/min,检测波长235 nm。结果表明,硝苯地平在5.0～75.0 μg/L范围内线性关系良好(r=0.9993),方法的回收率为90.0%～99.0%,日内、日间相对标准偏差均小于5.0%。该方法精密度高,重现性良好,避免了繁琐的样品预处理过程,且弱离子整体柱可多次重复使用,为检测血浆中的痕量药物提供了一种快速、经济、有效的新方法。