Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography

Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.     

硅胶电色谱分离机理的研究

对硅胶电色谱柱的性能进行了考察,发现在水／有机溶剂流动相条件下,几乎不存在气泡问题,流动相的组成在有机溶剂浓度、电解质浓度、ＰＨ值等方面可以在较大范围变化,选用５种典型样品,对硅胶电色谱的分离机理进行了系统研究,发现有反相分离机理、正相吸附机理、离子交换机理以及电泳机理参与作用。同时考察了有机溶剂浓度、电解质浓度、ＰＨ等对分离的影响。此外,还首次提出了一种全新的电色谱模式－动态改性硅胶电色谱。     

Separation of basic, acidic and neutral compounds by capillary electrochromatography using uncharged monolithic capillary columns modified with anionic and cationic surfactants

A mode of capillary electrochromatography (CEC), based on the dynamical adsorption of surfactants on the uncharged monolithic stationary phases has been developed. The monolithic stationary phase, obtained by the in situ polymerization of butyl methacrylate with ethylene dimethacrylate, was dynamically modified with an ionic surfactant such as the long-chain quaternary ammonium salt of cetyltrimethylammonium bromide (CTAB) and long-chain sodium sulfate of sodium dodecyl sulfate (SDS). The ionic surfactant was adsorbed on the surface of polymeric monolith by hydrophobic interaction, and the ionic groups used to generate the electroosmotic flow (EOF). The electroosmotic mobility through these capillary columns increased with increasing the content of ionic surfactants in the mobile phase. In this way, the synthesis of the monolithic stationary phase with binary monomers can be controlled more easily than that with ternary monomers, one of which should be an ionic monomer to generate EOF. Furthermore, it is more convenient to change the direction and magnitude of EOF by changing the concentration of cationic or anionic surfactants in this system. An efficiency of monolithic capillary columns with more than 140000 plates per meter for neutral compounds has been obtained, and the relative standard deviations observed for to and retention factors of neutral solutes were about 0.22% and less than 0.56% for ten consecutive runs, respectively. Effects of mobile phase composition on the EOF of the column and the retention values of the neutral solutes were investigated. Simultaneous separation of basic, neutral and acidic compounds has been achieved.     

Enantioselective reversed-phase and non-aqueous capillary electrochromatography using a teicoplanin chiral stationary phase

Enantiomeric separation of chiral pharmaceuticals is carried out in aqueous and non-aqueous packed capillary electrochromatography (CEC) using a teicoplanin chiral stationary phase (CSP). Capillaries were slurry packed with 5 microm 100-A porous silica particles modified with teicoplanin and initially evaluated using a non-aqueous polar organic mode system suitability test for the separation of metoprolol enantiomers (Rs = 2.3 and 53000 plates m(-1)). A number of pharmaceutical drugs were subsequently screened with enantioselectivity obtained for 25 racemic solutes including examples of neutral, acidic and basic molecules such as coumachlor (Rs = 3.0 and 86000 plates m(-1)) and alprenolol (Rs = 3.3 and 135000 plates m(-1)) in reversed-phase and polar organic mode, respectively. A statistical experimental design was used to investigate the effects of non-aqueous polar organic mobile phase parameters on the CEC electroosmotic flow, resolution and peak efficiency for two model solutes. Results primarily indicated that higher efficiency and resolution values could be attained at higher methanol contents which is similar to findings obtained on this phase in liquid chromatography.     

Enantiomeric separation by capillary electrochromatography. I. Chiral separation of dansyl amino acids and organochlorine pesticides on a diol-silica dynamically coated with hydroxypropyl-beta-cyclodextrin

In this work, a commercially available diol-silica stationary phase was converted in situ to a chiral stationary phase by dynamically coating it with hydroxypropyl-beta-cyclodextrin (HP-beta-CD). This stationary phase was shown useful for the capillary electrochromatography (CEC) separation of neutral and anionic enantiomers such as some organochlorine pesticides and dansyl amino acids, respectively. The inclusion of HP-beta-CD in the mobile phase to produce the in situ chiral stationary phase allowed the rapid separation of the anionic dansyl amino acid enantiomers at relatively low electroosmotic flow (EOF). The formation of host-guest complexes between the dansyl amino acids and the neutral HP-beta-CD in the mobile phase lowered the actual charge-to-mass ratios of the anionic solutes, thus speeding up their transport by the EOF across the packed capillary column. Several parameters affecting enantioseparation were investigated, including the concentration of HP-beta-CD, ionic strength, pH, and organic modifier content of the mobile phase.     

混合固定相电中溶质的输运特征

 以离子交换和反相固定相构成的混合固定相电中 ,溶质迁移在受到疏水、离子交换作用的同时 ,对于带电溶质而言 ,还受到电泳迁移的影响。根据离子独立迁移原理 ,结合过程中的多种相互作用 ,得到了描述溶质表观迁移速率与其各形态迁移速率、各种相互作用之间相互关系的理论表达式 ;讨论了混合模式电中流动相的 pH及其中的有机调节剂浓度、混合固定相配比等对电渗流的影响及不同形态溶质在柱内的输运特征。结果表明 ,在电中采用混合固定相可以在较大的 pH和有机调节剂浓度范围内得到较强且稳定的电渗流。     

Enantiomer separation by strong anion-exchange capillary electrochromatography with dynamically modified sulfated beta-cyclodextrin

A novel mode of capillary electrochromatography (CEC) based on a dynamically modified stationary phase was presented for chiral separation. The capillary column was packed with strong anion-exchange (SAX) stationary phase packing; the sulfated beta-cyclodextrin (S-CD), which was added to the mobile phase, was dynamically adsorbed to the packing surface. Separation of enantiomers was achieved by their different abilities to form an inclusion complex with the adsorbed S-CD. The enantiomers of tryptophan, praziquantel, atropine, metoprolol, and verapamil were successfully separated in this system with a column efficiency of 36000-412000 plates/m. The resolution value obtained for atropine was as high as 11.23. The superiority of CEC with a dynamically modified stationary phase over that with a physically adsorbed stationary phase was demonstrated. The influence of ionic strength, S-CD concentration, and methanol content on separation was also studied.     

Capillary electrochromatography with physically and dynamically absorbed stationary phases

Adsorption is always considered a troublesome effect in capillary electrophoresis (CE) and capillary electrochromatography (CEC). However, the adsorption effect can also be exploited to prepare or optimize the stationary phase in CEC. Compared with the chemical synthesis of new stationary phase materials for CEC, this method is simpler and more convenient. This review is focused on CEC with physically and dynamically adsorbed stationary phases. Separation of some acidic, basic and neutral solutes as well as enantiomers in CEC with dynamically adsorbed stationary phases are presented. The theory for the migration of charged solutes and the stationary phases currently used in CEC are also briefly reviewed.     

Preparation of a neutral porous monolith and its evaluation in pressurized capillary electrochromatography with neutral and charged solutes

A neutral, nonpolar monolithic capillary column was evaluated as a hydrophobic stationary phase in pressurized CEC system for neutral, acidic and basic solutes. The monolith was prepared by in situ copolymerization of octadecyl methacrylate and ethylene dimethacrylate in a binary porogenic solvent consisting of cyclohexanol/1,4‐butanediol. EOF in this hydrophobic monolithic column was poor; even the pH value of the mobile phase was high. Because of the absence of fixed charges, the monolithic capillary column was free of electrostatic interactions with charged solutes. Separations of neutral solutes were based on the hydrophobic mechanism with the pressure as the driving force. The acidic and basic solutes were separated under pressurized CEC mode with the pressure and electrophoretic mobility as the driving force. The separation selectivity of charged solutes were based on their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase, and no obvious peak tailing for basic analytes was observed. Effects of the mobile phase compositions on the retention of acidic compounds were also investigated. Under optimized conditions, high plate counts reaching 82 000 plates/m for neutral compounds, 134 000 plates/m for acid compounds and 150 000 plates/m for basic compounds were readily obtained.     

Capillary electrochromatography with monolithic silica column: I. Preparation of silica monoliths having surface-bound octadecyl moieties and their chromatographic characterization and applications to the separation of neutral and charged species

Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 microm ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (> 2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160 000 plates/m, a value comparable to that obtained on columns packed with 3 microm C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio.     

非极性毛细管电色谱原位柱的阳离子表面活性剂动态修饰

毛细管电色谱是在毛细管中依靠电渗流来驱动流动相 ,同时溶质与固定相发生相互作用的一种色谱分离模式 ,它有高效液相色谱的高选择性 ,同时兼具毛细管电泳的高效性 [1] .传统电色谱柱是将HPLC填料装入毛细管 ,但由于装柱困难且易产生气泡而在一定程度上阻碍了电色谱的发展 [2～ 4 ] .通过柱内合成的方法直接在毛细管中制成连续床毛细管电色谱柱 ,可避免两端烧塞 . 1 995年 Svec等 [5,6]首次将连续床层色谱柱用于毛细管电色谱 ,此后 ,有关毛细管中原位合成连续床电色谱柱的方法得到了应用 [7～ 11] .为了使原位合成电色谱柱能产生电渗流 ,…     

Neutral octadecyl monolith for reversed phase capillary electrochromatography of a wide range of solutes

A neutral octadecyl monolithic (ODM) column for RP capillary electrochromatography (RP-CEC) has been developed. The ODM column was prepared by the in situ polymerization of octadecyl acrylate (ODA) as the monomer and trimethylolpropanetrimethacrylate (TRIM) as the crosslinker, in a ternary porogenic solvent containing cyclohexanol, ethylene glycol, and water. The ODM column exhibited cathodal EOF over a wide range of pH and ACN concentration in the mobile phase despite the fact that it was devoid of any fixed charges. It is believed that the EOF is due to the adsorption of ions from the mobile phase onto the surface of the monolith thus imparting to the neutral ODM column the zeta potential necessary to support the EOF required for mass transport across the monolithic column. Furthermore, the adsorption of mobile phase ions to the neutral monolith modulated solute retention and affected the separation selectivity. The wide applications of the neutral ODM column were demonstrated by its ability to separate a wide range of small and large solutes, both neutral and charged. While the separation of the neutral solutes was based on RP retention mechanism, the charged solutes were separated on the basis of their electrophoretic mobility and hydrophobic interaction with the C18 ligands of the stationary phase. As a typical result, the neutral monolithic column was able to separate peptides quite rapidly with a separation efficiency of nearly 200,000 plates/m, and this efficiency was exploited in tryptic peptide mapping of standard proteins, e. g., lysozyme and cytochrome C, by isocratic elution.     

毛细管电中中性溶质在氰基柱上分离机理的研究

 对中性溶质在氰基柱上的毛细管电色谱（CEC）分离特征进行了研究 ,讨论了流动相中有机调节剂种类及其体积分数、缓冲液种类等对溶质迁移速率的影响。通过对样品在氰基柱上的分离行为与其在反相ODS柱和正相SI柱上的分离行为的比较 ,说明中性溶质在氰基柱上的分离机理既具有部分反相特征又具有部分正相特征。受两种机理的影响 ,在不同的操作条件下极性不同的中性溶质在氰基柱上的迁移速率差别很大 ,较一般的正相和反相电中更易出现出峰顺序变化的现象 ,也说明这种分离模式更易于进行分离选择性调节。     

Phenylaminopropyl silica monolithic column for pressure assisted capillary electrochromatography

A novel stationary phase phenylaminopropyl silica (PhA-silica) monolith was successfully prepared for pressure assisted capillary electrochromatography (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by using [3-(phenylamino)propyl]trimethoxysilane as surface modification reagent to produce the phenylaminoporpyl function. The secondary amino groups on the surface of the monolithic stationary phase contributed to the generation of anodic electroosmotic flow (EOF) under acidic conditions. The phenyl group together with the spacer (-(CH(2))(3)-) in PhA-silica provides sufficient hydrophobic properties. To evaluate the column performance, effects of buffer pH and mobile phase composition on the mobile phase linear velocity and the retention factors of alkylbenzenes, phenols and anilines were investigated in pCEC mode. The monolithic stationary phases exhibit typical reversed-phase (RP) electrochromatographic behavior toward neutral solutes. Hydrophobic as well as electrophoretic migration process within the monoliths was observed for the separation of basic solutes such as anilines without peak tailing.     

吸附固定相电色谱和动态改性电色谱的手性分离

对动态改性电色谱手性分离进行了研究。电色谱柱填充强阴离子交换固定相（SAX0,添加在流动相中的磺化β－环糊精（S－CD）动态地吸陵于SAX填料表面,形成一层准手性固定相。色氨酸、阿托品和异博定对映体在本体系获得了很好的分离,它们的分离分别为2．06,10．1和1．96,对映体峰的柱效价于85,000塔板数／米和412,000塔板数／米之间。连续运行17次,死时间和色氨酸对映体的电色谱保留因子的相对标准偏差分别为0．53％,0．62％和0．69％。此外,以吸附于SAX填料的牛血清白蛋白和S－CD为手性固定相进行了电色谱手性分离的研究。在这两种体系下分离色氨酸对映体的分离度分别为3．86和2．97。吸附S－CD柱电色谱和动态改性电谱的重现性进行子比较,发现动态改性电色谱有更好的重现性。     

Monolithic silica-based capillary column with strong chiral cation-exchange type surface modification for enantioselective non-aqueous capillary electrochromatography

A silica-based monolithic stationary phase prepared by the sol-gel process in a 100 microm I.D. fused-silica (FS) capillary has been modified chemically with 3-mercaptopropyl trimethoxysilane followed by immobilization of a strong cation-exchange (SCX) type chiral selector, (S)-N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutane phosphonic acid, by radical addition reaction onto the reactive sulfhydryl surface. After a fine-tuning of the mobile phase composition, the enantioselective capillary column was evaluated for the separation of various chiral basic drugs by enantioselective non-aqueous capillary electrochromatography (CEC), in comparison to capillary column analogs packed with 3.5 microm silica particles having attached the same selector. The performance of the monolithic silica column was further compared to corresponding polymethacrylate-based organic polymer monoliths. The study indicated that strong counter-ions such as 2-aminobutanol or N,N,N',N'-tetramethylethylenediamine are needed, although they reduce the electroosmotic flow velocity and separation factors in comparison to less efficient counter-ions, in order to allow the elution of the oppositely charged solutes in the ion-exchange retention mode within reasonable run time and as sharp zones. In contrast, weak counter-ions such as N,N-diisopropylethylamine (Huenig base) provided stronger electroosmotic flow and much better separation factors, but relatively poor peak efficiencies. Overall, with the chemically functionalized monolithic silica column the high quality separations of packed column analogs could be approximated, with regards to both separation factors and peak performances. On the other hand, the monolithic capillary column certainly outperformed the packed column in terms of system robustness under capillary electrochromatography conditions and showed excellent column longevity. The enantioselective strong cation-exchange-type monolithic silica column performed also well in comparison to the organic polymer monolith.     

非电荷型毛细管电色谱原位柱的色谱行为研究──阴离子表面活性剂的动态改性

 采用原位聚合的方法在毛细管中合成了非电荷型连续床电色谱原位柱 ,通过在电色谱流动相中加入阴离子表面活性剂十二烷基硫酸钠 (SDS)进行动态改性使其产生电渗流 ,考察了SDS浓度及有机改性剂浓度等因素对电渗流的影响。此类连续床柱制备容易 ,柱效可达 14万理论塔板 /m ,在不同的操作条件下有良好的稳定性 ,连续 10次运行 ,其死时间t0 与保留时间的精密度分别为 0 .2 2 %和 <0 .5 6 %。     

Development of a new C14 monolithic silica column containing embedded polar groups for pressurized capillary electrochromatography

Monolithic columns have been prepared with a novel bonded silica stationary phase, tetradecylamine bonded silica (TDAS), and used in pressurized capillary electrochromatography (pCEC). The monolithic silica column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and tetradecylamine. The introduced embedded polar amine groups dominated the charge on the surface of the monolithic stationary phase and generated an EOF from cathode to anode under acidic conditions. The tetradecyl hydrophobic chains in TDAS provide chromatographic interactions. The chromatographic characteristics of the prepared monolithic column were studied. Some aromatic compounds including alkylbenzenes, aromatic hydrocarbons, phenols, and anilines were successfully separated on the TDAS monolithic column in pCEC mode. As expected, the TDAS monolithic stationary phases exhibit typical reversed-phase electrochromatographic behavior toward neutral solutes due to the introduced tetradecyl groups. Hydrophobic as well as electrophoretic migration processes within the monoliths were observed in the separation of basic anilines. Symmetrical peaks can be obtained for anilines because the embedded polar amine groups on the surface can effectively shield the adsorption of positively charged analytes onto the stationary phase.     

Capillary liquid chromatography using a hydrophilic/cation-exchange monolithic column with a dynamically modified cationic surfactant

A novel form of reversed-phase liquid chromatography (RPLC) by the dynamically modified hydrophilic interaction monolithic column has been described in this paper. A porous poly(SPMA-co-PETA) monolith with strong cation-exchange (SCX) was prepared and the resulting monolith showed a typical hydrophilic interaction chromatography (HILIC) mechanism at higher organic solvent content (ACN% > 50%). The good selectivity for neutral, basic and acidic polar analytes was observed in the HILIC mode. In order to increase the hydrophobic interaction, the monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX monolithic material, and the resulting hydrophobic layer was used as the stationary phase. Using the dynamically modified SCX monolithic column, neutral, basic and acidic hydrophobic analytes were well separated with the RPLC mode.     

反相毛细管电色谱中无盐体系的优势及分离研究

本文将电中性溶质在缓冲盐体系和无盐体系下的电渗流、分离选择性等进行了对比,从理论和实验两方面讨论了电中性溶质在无盐体系下的分离,提出了采用无盐流动相既可以增加电渗流也可以减小焦耳热的优势。反相毛细管电色谱的实验结果也充分证明了这一点。