Simultaneous separation of water‐ and fat‐soluble vitamins in isocratic pressure‐assisted capillary electrochromatography using a methacrylate‐based monolithic column

A method of simultaneous separation of water‐ and fat‐soluble vitamins using pressure‐assisted CEC with a methacrylate‐based capillary monolithic column was developed. In the proposed method, water‐soluble vitamins were mainly separated electrophoretically, while fat soluble‐ones were separated chromatographically by the interaction with a methacrylate‐based monolith. A mixture of six water‐soluble and four fat‐soluble vitamins was separated simultaneously within 20 min with an isocratic elution using 1 M formic acid (pH 1.9)/acetonitrile (30:70, v/v) containing 10 mM ammonium formate as a mobile phase. When the method was applied to a commercial multivitamin tablet and a spiked one, the vitamins were successfully analyzed, and no influence of the matrix contained in the tablet was observed.     

MnCl_2改性γ-Al_2O_3毛细管填充柱气相色谱分析氢同位素

针对常规气相色谱填充柱分析稳定氢同位素的柱效低、峰宽大、保留时间长等问题,采用MnCl_2改性γ-Al_2O_3填充的石英毛细管柱开展了系统性柱效分析及氢同位素分析技术研究。研究结果表明,使用MnCl_2对γ-Al_2O_3进行改性后,可大大改善单纯的γ-Al_2O_3表面有序度、孔结构和吸附性质,并将正氢(o-H_2)和仲氢(p-H_2)峰洗脱在单一谱峰区域内。制备的长1.0 m、内径0.53 mm的石英毛细填充柱与热导检测器(TCD)级联测试,在体积浓度1至10 m L/L范围内有较好的线性关系,对于低浓度样品检测的相对误差不大于5%。H_2、HD和D_2的保留时间可分别缩短至39、46和60 s,检出限可分别降低至0.046、0.067和0.072 m L/L。毛细管填充柱较常规填充柱具有峰形尖锐、相邻组分分离度高、保留时间短、检出限低等优点,可用于低浓度氢同位素快速测量及氢同位素在线分析。     

Hypercrosslinking: new approach to porous polymer monolithic capillary columns with large surface area for the highly efficient separation of small molecules

Monolithic polymers with an unprecedented surface area of over 600 m(2)/g have been prepared from a poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) precursor monolith that was swollen in 1,2-dichloroethane and hypercrosslinked via Friedel-Crafts reaction catalyzed by ferric chloride. Both the composition of the reaction mixture used for the preparation of the precursor monolith and the conditions of the hypercrosslinking reaction have been varied using mathematical design of experiments and the optimized system validated. Hypercrosslinked monolithic capillary columns contain an array of small pores that make the column ideally suited for the high efficiency isocratic separations of small molecules such as uracil and alkylbenzenes with column efficiencies reproducibly exceeding 80,000 plates/m for retained compounds. The separation process could be accelerated while also improving peak shape through the use of higher temperatures and a ternary mobile phase consisting of acetonitrile, tetrahydrofuran, and water. As a result, seven compounds were well separated in less than 2 min. These columns also facilitate separations of peptide mixtures such as a tryptic digest of cytochrome c using a gradient elution mode which affords a sequence coverage of 93%. A 65 cm long hypercrosslinked capillary column used in size exclusion mode with tetrahydrofuran as the mobile phase afforded almost baseline separation of toluene and five polystyrene standards.     

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

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

Octyl-functionalized hybrid silica monolithic column for reversed-phase capillary electrochromatography

Hybrid silica monolithic stationary phase functionalized with octyl groups was synthesized by a two-step acid/base-catalyzed hydrolysis/co-condensation of tetraethoxysilane (TEOS) and n-octyltriethoxysilane (C(8)-TEOS). The influences of determining factors in the sol-gel process such as the monomer ratio and water content on the monolith formation were systematically investigated. An increase in the TEOS/C(8)-TEOS ratio in the polymerization mixture shifted the pore size distribution towards smaller pore diameter with larger pore volume. The optimal TEOS/C(8)-TEOS volume ratio was found to be 90/50, under which condition the median pore diameter of the monolith was around 1.0 microm with pore volume of 3.25 cm(3)/g. The chromatographic characteristics of the monolithic column prepared with the optimized fabrication condition were studied. Some aromatic compounds including alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) and phenols were successfully separated on the octyl-functionalized silica monolithic column with high column efficiency up to 180,000 plates/m.     

Polymethacrylate-based monolithic capillary column with weak cation exchange functionalities for capillary electrochromatography

Polymethacrylate-based monolith with weak cation exchange functionalities was prepared in capillary column (i.d. 100 μm, o.d. 375 μm) by in situ polymerization of butyl methacrylate, ethylene dimethacrylate and N-methacryloyl-L-glutamic acid in presence of porogens. The porogen mixture included N,N-dimethyl formamide and phosphate buffer. The preparation procedure of stationary phase contained the synthesis of monomer, silanization of capillary inner wall and in situ polymerization. The use of amino acid based monomer for the monolith synthesis is one of the originalities of this novel approach. N-methacryloyl-L-glutamic acid has two carboxyl functionalities. The separation of the solutes were performed at different acetonitrile/phosphate buffer and acetonitrile/sodium hydroxide contents. The applied voltage for the alkyl benzenes was changed between +5 and +30 kV. CEC separations of alkyl benzenes, acidic, basic, phenolic and some polycylic aromatic compounds were succesfully performed under capillary-electrochromatography mode with cathodic electroosmotic flow.     

Incorporation of carbon nanotubes in porous polymer monolithic capillary columns to enhance the chromatographic separation of small molecules

Multiwalled carbon nanotubes have been entrapped in monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary columns to afford stationary phases with enhanced liquid chromatographic performance for small molecules in the reversed phase. While the column with no nanotubes exhibited an efficiency of only 1800 plates/m, addition of a small amount of nanotubes to the polymerization mixture increased the efficiency to over 15,000 and 35,000 plates/m at flow rates of 1 and 0.15 μL/min, respectively. Alternatively, the native glycidyl methacrylate-based monolith was functionalized with ammonia and, then, shortened carbon nanotubes, bearing carboxyl functionalities, were attached to the pore surface through the aid of electrostatic interactions with the amine functionalities. Reducing the pore size of the monolith enhanced the column efficiency for the retained analyte, benzene, to 30,000 plates/m at a flow rate of 0.25 μL/min. Addition of tetrahydrofuran to the typical aqueous acetonitrile eluents improved the peak shape and increased the column efficiency to 44,000 plates/m calculated for the retained benzene peak.     

Photografted methacrylate‐based monolithic columns coated with cellulose tris(3,5‐dimethylphenylcarbamate) for chiral separation in CEC

A chiral capillary monolithic column for enantiomer separation in capillary electrochromatography was prepared by coating cellulose tris(3,5‐dimethylphenylcarbamate) on porous glycidyl methacrylate‐co‐ethylene dimethacrylate monolith in capillary format grafted with chains of [2(methacryloyloxy)ethyl] trimethylammonium chloride. The surface modification of the monolith by the photografting of [2(methacryloyloxy)ethyl] trimethylammonium chloride monomer as well as the coating conditions of cellulose tris(3,5‐dimethylphenylcarbamate) onto the grafted monolithic scaffold were optimized to obtain a stable and reproducible chiral stationary phase for capillary electrochromatography. The effect of organic modifier (acetonitrile) in aqueous mobile phase for the enantiomer separation by capillary electrochromatography was also investigated. Several pairs of enantiomers including acidic, neutral, and basic analytes were tested and most of them were partially or completely resolved under aqueous mobile phases. The prepared monolithic chiral stationary phases exhibited a good stability, repeatability, and column‐to‐column reproducibility, with relative standard deviations below 11% in the studied electrochromatographic parameters.     

Capillary electrophoresis of neurotransmitters using in-line solid-phase extraction and preconcentration using a methacrylate-based weak cation-exchange monolithic stationary phase and a pH step gradient

A novel approach for in-line solid-phase extraction capillary electrophoresis (SPE-CE) for basic analytes was developed. The method is based on the use of a weak cation-exchange monolith synthesised in situ in the front end of the CE capillary via photoinitiated polymerization to form poly(methacrylic acid-co-ethylene glycol dimethacrylate), which was used to create the SPE phase in-line with the CE separation capillary. The monolithic SPE material exhibited a surface area of 23.1 m2/g and a capacity of 403 nM for dopamine. Adsorption of the analytes as protonated, cationic species onto the SPE phase was achieved using an electrolyte of 6 mM phosphate and 12 mM sodium ion, buffered at pH 7.0, which is above the pKa of the monolith but below the pKa of the analytes. Elution of the analytes from the SPE phase was achieved using an electrolyte with a pH below that of the pKa of the monolith, namely 12 mM phosphate and 12 mM sodium ion, buffered at pH 3.0. Due to the discontinuous electrolyte combination, analytes were simultaneously eluted and focused as the electrophoretically mobilised pH step boundary moved through the SPE monolith, after which the analytes were separated by conventional CZE in the remainder of the capillary. Quantitative extraction from a solution of 0.5 microg/ml dopamine and epinephrine was achieved when flushing up to 15 column volumes of sample through the capillary. The limits of detection (S/N=3) for dopamine and epinephrine were 3.7 and 4.3 ng/ml, and this method provided a sensitivity enhancement for dopamine of 462 times compared to CZE using hydrodynamic injection. The developed method was used to preconcentrate a test mixture of neurotransmitters comprising dopamine, epinephrine, 5-hydroxytryptamine, metanephrine and also histamine. The applicability of this approach to real life samples was demonstrated by using a urine sample from a healthy person to detect dopamine at sub-ppm levels.     

Determination of carbamate insecticides in Chinese medicinal herbs by gas chromatography with a nitrogen-phosphorus detector

A simplified method for determining carbamate insecticides (including metolcarb, isoprocarb, fenobucarb, carbofuran, pirimicarb, and carbaryl) in Chinese medicinal herbs (White Peony Alba, Red Peony Root, and Baical Skullcap Root) is described. Standards were fortified into Chinese medicinal herbs at 3 levels (0.05-0.5 mg/kg). The carbamates were extracted with dichloromethane in a Soxhlet apparatus and analyzed by gas chromatography with a nitrogen-phosphorus detector. The results showed average recoveries between 80.77 and 104.56%. The method evidenced good robustness, accuracy, and precision for monitoring carbamates in Chinese medicinal herb samples, and it is a suitable alternative to replace the currently dedicated analytical systems. The minimum detectable amount ranged from 3.0 x 10(-10) to 5.0 x 10(-10)g, and the limit of quantification was 0.05 mg/kg. The method is rapid, simple, sensitive, and reproducible, and it can be conveniently used as a low-cost, rapid method for measuring the carbamate insecticide contamination of Chinese medicinal herbs.     

Determination of four kinds of carbamate pesticides by capillary zone electrophoresis with amperometric detection at a polyamide-modified carbon paste electrode

In this paper, a polyamide-modified carbon paste electrode in capillary zone electrophoresis with amperometric detection (CZE-AD) was firstly applied to the determination of four carbamate pesticides: fenobucarb, isoprocarb, metolcarb and carbaryl. The four carbamates were hydrolyzed in alkalescent aqueous solutions, resulting in the formation of 2-sec-butylphenol, 2-isopropylphenol, m-cresol and α-naphthol, which could be determined by amperometry after capillary electrophoretic separation. Under the selected optimum conditions, the four analytes could be perfectly separated within 23 min. The linear ranges of 2-sec-butylphenol, 2-isopropylphenol and m-cresol were from 1.0 × 10⁻⁷ to 2.0 × 10⁻⁵ mol L⁻¹ and that of α-naphthol was from 2.0 × 10⁻⁷ to 2.0 × 10⁻⁵ mol L⁻¹ and their detection limits were 3.0 × 10⁻⁸, 3.0 × 10⁻⁸, 3.0 × 10⁻⁸ and 6.0 × 10⁻⁸ mol L⁻¹, respectively (S/N = 3). Fenobucarb, isoprocarb, metolcarb and carbaryl can be indirectly determined by this CZE-AD method with recovery of 105, 104, 110 and 98% and R.S.D. of 4, 3, 4 and 3%, respectively. Above results demonstrated that this method was of high sensitivity, good repeatability and could be used in the rapid determination of the pesticide residues.     

金属有机框架CPL-1填充柱气相色谱分析氢同位素

金属有机框架(MOFs)材料CPL-1的比表面积大、孔径均一,在低温条件下对氢同位素具有良好的量子筛分效应,是气相色谱固定相潜在的应用材料。采用CPL-1填充制备了长0.5 m、内径1 mm的微孔填充柱,借助单晶Al_2O_3颗粒间隙构建了色谱载气流通路径,在低温条件下探索研究了CPL-1填充柱的氢同位素分析性能。结果表明,在77 K时CPL-1对H_2和D_2的吸附量接近4 mmol/g,优于MnCl2/γ-Al_2O_3和γ-Al_2O_3,CPL-1填充柱在取样量0.25~2 mL范围内对低浓度氢同位素样品的检测具有良好的线性关系,检测的相对误差小于4%。CPL-1填充柱具有线性范围宽、重复性好、准确度高等优点,在氢同位素色谱分析中具有潜在的应用价值。     

Determination of Magnolol and Honokiol by Non-aqueous Capillary Electrophoresis

Introduction Capillaryelectrophoresis(CE),ahighefficiency separationtechnique,hasrapidlydevelopedsince1981[1].Notonlyhavedifferentmodesbeenrepor ted[1—3],butalsosomenon aqueouselectrophoresis media[4]havebeenapplied.Theimportantproperties ofnonaqueousmed…     

A silica monolithic column prepared by the sol-gel process for enantiomeric separation by capillary electrochromatography

A method for the preparation of a silica monolithic capillary electrochromatography (CEC) column for the separation of enantiomers has been developed. The porous silica monolith was fabricated inside a fused-silica capillary column by using the sol-gel process. After gelation for 24 h, hydrothermal treatment at 100 degrees C for 24 h was performed to prevent the sol-gel matrix from cracking. The prepared monolith was then coated with Chirasil-beta-Dex which represents a chiral polymer prepared by grafting permethyl-beta-cyclodextrin to polymethylsiloxane with an octamethylene spacer. Immobilization of Chirasil-beta-Dex was performed by heat treatment at 120 degrees C for 48 h to give a nonextractable coating. The column performance was evaluated by using racemic hexobarbital as a model compound. The efficiency of 9.2 x 10(4) theoretical plates/m for the first eluted enantiomer of hexobarbital was obtained at an optimal flow rate of the mobile phase. The effect of mobile phase composition on enantiomeric separation of hexobarbital was also investigated. The column proved to be stable for more than one hundreds of runs during a two-months period. The enantiomers of several neutral and negatively charged chiral compounds were baseline separated on this column.     

The synthesis of chloropropyl-functionalized silica hybrid monolithic column with modification of N,N-dimethyl-N-dodecylamine for capillary electrochromatography separation

A chloropropyl-functionalized silica (CP-silica) hybrid monolithic column was synthesized within the confines of a capillary via the sol–gel process using tetramethoxysilane (TMOS) and (3-chloropropyl)-trimethoxysilane (CPTMS) as the precursors. The resulting CP-silica hybrid monolith inside the capillary showed homogeneous macroporous morphology and was well attached to the inner wall of the capillary. The obtained CP-silica hybrid monolithic capillary column demonstrated the inherent hydrophobic property and could be applied as a reversed-phase stationary phase in CEC directly. Due to the great chemical reactivity of the incorporated chloro groups on the hybrid silica monolithic matrix, the chloropropyl moieties on the surface of the hybrid silica monolith matrix could be conveniently further modified by a tertiary amine of N,N-dimethyl-N-dodecylamine (DMDA) via the nucleophilic substitution reaction at 70 °C to introduce a dodecyl groups (C12) onto the CP-silica hybrid monolithic matrix. The resulting C12-silica hybrid monolithic column not only demonstrated the significantly enhanced hydrophobic property in the separation of alkylbenzenes in reversed-phase capillary electrochromatography (RP-CEC), but also the strong electroosmotic flow (EOF) in a wide pH range. Five alkylbenzenes could be baseline separated in 3 min with column efficiency ranging from 189 700 to 221 000 N/m with a 70% ACN running buffer in CEC.     

Surfactant-bound monolithic columns for separation of proteins in capillary high performance liquid chromatography

A surfactant-bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA–EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties (i.e., morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase.     

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)。     

Fast preparation of polystyrene-based monolith using microwave irradiation for micro-column separation

The conventional analytical HPLC was successfully developed for micro-column separation by using a simple eluate splitting system, a self-preparation of monolithic column and an on-capillary column detector in our laboratory. A typical polystyrene-based monolith was quickly prepared inside the fused-silica capillary, which in situ polymerization was carried out in 10 min by microwave irradiation. The reactant solution consisted of styrene (ST) as a functional monomer, divinylbenzene (DVB) as a cross-linking agent, toluene and isooctane as porogenic solvents, and azobisisobutyronitrile (AIBN) as an initiator. The monolith was proved to form in the center of the capillary and adhered to the column inner wall by the scanning electron micrograph. Its chromatographic behaviors were evaluated in detail by varying the flow rate and percentage of mobile phases, and under the optimal condition, baseline separation of the model analytes including thiourea, benzene, toluene, ethylbenzene was obtained with a highest theoretical plate number near 11,290 N/m by the developed capillary HPLC. Furthermore, the stability and porosity of the prepared monolith were systematically investigated by a simple flow method. Figure A polystyrene-based monolith was rapidly prepared inside the fused-silica capillary, which in situ polymerization was carried out about 10 min by microwave irradiation.     

含多面体低聚倍半硅氧烷杂化毛细管整体柱的制备及修饰

本文采用自由基聚合法原位制备了两种杂化毛细管整体柱。首先以含有一个甲基丙烯酸基团的多面体低聚倍半硅氧烷(POSS)试剂(Bu-POSS)为单体、以含有多个甲基丙烯酸基团的POSS试剂(POSS-MA)为交联剂在二元致孔剂(正丙醇/聚乙二醇400)和引发剂(偶氮二异丁腈)存在下发生热引发聚合,在毛细管中形成聚(Bu-POSS-co-POSS-MA)杂化整体柱;另外仅以POSS-MA为单体在相同条件下制备聚(POSS-MA)杂化整体柱,并将这两种杂化整体柱应用于小分子的毛细管液相色谱(cLC)分析。结果表明,含POSS杂化整体柱具有制备简单、重现性好以及稳定性高的特点。此外,利用聚(POSS-MA)杂化整体柱表面剩余的甲基丙烯酸基团,可以将功能单体(甲基丙烯酸硬脂酸酯等)化学键合到整体柱上,不但可以提高色谱柱效,而且使其具有不同的选择性。本文所发展的以POSS试剂为原料采用自由基聚合法制备杂化整体柱的方法为新型杂化整体柱的制备提供了一种新思路。     

Ti (IV) attached‐phosphonic acid functionalized capillary monolith as a stationary phase for in‐syringe‐type fast and robust enrichment of phosphopeptides

In this study, poly(vinylphosphonic acid‐co‐ethylene dimethacrylate), poly(VPA‐co‐EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 μm. The phosphopeptides from different sources were enriched on Ti (IV)‐attached poly(VPA‐co‐EDMA) monolith using a syringe‐pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of β‐casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA‐co‐EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of β‐casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal‐to‐noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA‐co‐EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.