Hybrid silica monolithic column for capillary electrochromatography with enhanced cathodic electroosmotic flow

A hybrid silica monolithic stationary phase for RP CEC was prepared by in situ co-condensation of (3-mercaptopropyl)-trimethoxysilane (MPTMS), phenyltriethoxysilane (PTES), and tetraethoxysilane (TEOS) via a sol-gel process. The thiol groups on the surface of the stationary phase were oxidized to sulfonic acids by peroxytrifluoroacetic acid. The introduced sulfonic acid moieties on the monoliths were characterized by a strong and relatively stable EOF in a broad pH range from 2.35 to 7.0 in CEC. Aromatic acids and neutral compounds can be simultaneously separated in this column under cathodic EOF. The CEC column exhibited a typical RP chromatographic mechanism for neutral compounds due to the introduced phenyl groups.     

Preparation and characterization of mixed-mode monolithic silica column for capillary electrochromatography

A silica-based monolithic stationary phase with mixed-mode of reversed phase (RP) and weak anion-exchange (WAX) for capillary electrochromatography (CEC) has been prepared. The mixed-mode monolithic silica column was prepared using the sol–gel technique and followed by a post-modification with hexadecyltrimethoxysilane (HDTMS) and aminopropyltrimethoxysilane (APTMS). The amino groups on the surface of the stationary phase were used to generate a substantial anodic EOF as well as to provide electrostatic interaction sites for charged compounds at low pH. A cathodic EOF was observed at pH above 7.3 due to the full ionization of residual silanol groups and the suppression in the ionization of amino groups. A variety of analytes were used to evaluate the electrochromatographic characterization and column performance. The monolithic stationary phase exhibited RP chromatographic behavior toward neutral solutes. The model anionic solutes were separated by the mixed-mode mechanism, which comprised RP interaction, WAX, and electrophoresis. Symmetrical peaks can be obtained for basic solutes because positively charged amino groups can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

有机-无机杂化硅胶基质整体柱的制备及其电色谱性能

采用溶胶-凝胶法,以四乙氧基硅烷和苯基三乙氧基硅烷作为反应单体,通过酸碱两步催化在毛细管中进行原位缩聚反应,制备了新型有机-无机杂化硅胶基质毛细管整体柱,制备过程简单。整体柱基质中均匀分布的苯基基团可直接用于反相毛细管电色谱的分离,因而不需要对基质再进行衍生化。优化了整体柱的制备条件,采用扫描电镜和压汞法对整体柱的微观结构和孔径分布进行了表征。分别考察了溶胶-凝胶初始反应液中水的用量对柱床结构的影响和两种单体的配比对材料孔径分布的影响。研究了稠环芳烃类化合物在整体柱上的保留行为,用所制备的整体柱分离了7种苯酚类化合物,平均柱效达100000塔板/m。     

Reversed-phase and weak anion-exchange mixed-mode silica-based monolithic column for capillary electrochromatography

A silica-based CEC monolithic column with mixed modes of RP and weak anion-exchange (WAX) was successfully prepared by using the sol-gel technique at mild temperature. The synthesizing procedure was optimized by changing the ratios of tetraethoxysilane (TEOS), aminopropyltriethoxysilane (APTES), and octyltriethoxysilane (C(8)-TEOS) in the mixture. While serving as WAX group, the amino group dominated the charge on the surface of the capillary column and generated an EOF from cathode to anode at low pH. At pH above 7.5, a cathodic EOF was observed due to the full ionization of silanol group and the suppression in the ionization of amino group. The morphology of monolithic columns was examined by SEM, and the performance of column was evaluated in detail by separating different kinds of compounds. As expected, the monolithic column exhibited RP chromatographic behavior for neutral solutes. Fast and efficient separation of six aromatic acids was obtained using acidic mobile phase with column efficiency up to 160,000 plates/m. Symmetrical peaks can be obtained for aromatic amines because positively charged amino groups on the surface can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

Preparation and evaluation of rigid porous polyacrylamide-based strong cation-exchange monolithic columns for capillary electrochromatography

A CEC monolithic column with strong cation-exchange (SCX) stationary phase based on hydrophilic monomers was prepared by in situ polymerization of acrylamide, methylenebisacrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a complete organic binary porogenic solvent consisting of DMSO and dodecanol. The sulfonic groups provided by the monomer AMPS on the surface of the stationary phase generate an EOF from anode to cathode, and serve as an SCX stationary phase at the same time. The monolithic stationary phase exhibited normal-phase chromatographic behavior for neutral analytes. For charged analytes, electrostatic interaction/repulsion with the monolith was observed. The strong SCX monolithic column has been successfully employed in the electrochromatographic separation of basic drugs, peptides, and alkaloids extracted from natural products.     

Electrochromatographic performance of conventional and polar-embedded C16 silica monolithic stationary phases

A novel monolithic silica column that has a polar‐embedded amide‐secondary amine group linking with C16 functionality for RP‐CEC is described. The amide‐secondary aminealkyloxysilane was synthesized by the reaction of 3‐(2‐aminoethylamino) propyltrimethoxysilane with hexadecanoyl chloride. Then, the silylant agent was bonded to the silica monolith matrix to produce hexadecanamide‐secondary amine bonded silica (HDAIS) monolithic column. The electrochromatographic performance of HDAIS monolithic column for the separation of neutral, basic and polar solutes was studied, which was compared to that using the hexadecyl bonded silica monolithic column. The HDAIS monolithic column displayed reduced hydrophobic retention characteristics in the separation of five hydrophobic n‐alkylbenzenes and four polar phenols when compared to the hexadecyl bonded silica monolithic column. A very much reduced silanol activity of HDAIS monolithic column was observed in the separation of test basic mixture including four aromatic amines, atenolol and metoprolol with 10 mM borate buffer (pH 7.5) containing 30% v/v ACN as the mobile phase. The comparison results indicate good performance for both polar and basic mixtures on HDAIS monolithic column in RP‐CEC, and also show promising results for further applications.     

Single-step preparation and characterization of polymeric monolith for pressurized capillary electrochromatography of typical homologs

A monolithic stationary phase was prepared in a single step by in situ copolymerization of iso-butyl methacrylate (IBMA), ethylene dimethacrylate (EDMA), and N,N-dimethylallylamine (DMAA) in a binary porogenic solvent consisting of N,N-dimethylformamide (DMF) and 1,4-butanediol. As the frame structures of monoliths, the amino groups are linked to support the EOF necessary for driving the mobile phase through the monolithic capillary, while the hydrophobic groups are introduced to provide the nonpolar sites for the chromatographic retention. To evaluate the column performance, separations of typical kinds of neutral or charged homologs, such as alkylbenzenes, phenols (including isomeric compounds of hydroquinone, resorcin, and catechol), and anilines (including isomeric compounds of o-phenylenediamine and 1,4-phenylenediamine), were performed, respectively on the prepared column under the mode of pressurized pCEC. Effects of the buffer pH and the mobile phase composition on the linear velocity of mobile phase and the retention factors of these compounds were investigated. It was found that the retention mechanism of charged solutes could be attributed to a mixed mode of hydrophobic interaction and electrophoresis, while an RP chromatographic behavior on the monolithic stationary phases was exhibited for neutral solutes. Especially, basic compounds such as anilines were well separated on the monolithic columns in the "counterdirectional mode," which effectively eliminated the electrostatic adsorption of basic analytes on the charged surface of the stationary phases.     

Polyacrylamide-based monolithic capillary column with coating of cellulose tris(3,5-dimethylphenyl-carbamate) for enantiomer separation in capillary electrochromatography

A hydrophilic chiral capillary monolithic column for enantiomer separation in CEC was prepared by coating cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) on porous hydrophilic poly(acrylamide-co-N,N'-methylene-bisacrylamide) (poly(AA-co-MBA)) monolithic matrix with confine of a fused-silica capillary. The coating conditions were optimized to obtain a stable and reproducible chiral stationary phase for CEC. The effect of organic modifier of ACN in aqueous mobile phase for the enantiomer separation by CEC was investigated, and the significant influence of ACN on the enantioresolution and electrochromatographic retention was observed. Twelve pairs of enantiomers including acidic, neutral, and basic analytes were tested and nine pairs of them were baseline-enantioresolved with acidic and basic aqueous mobile phases. A good within-column repeatability in retention time (RSD = 2.4%) and resolution (RSD = 3.2%) was obtained by consecutive injections of a neutral compound, benzoin, on a prepared chiral monolithic column, while the between-column repeatability in retention time (RSD = 6.4%) and resolution (RSD = 9.6%) was observed by column-to-column examination. The prepared monolithic stationary phase showed good stability in either acidic or basic mobile phase.     

Pressurized CEC of neutral and charged solutes using silica monolithic stationary phases functionalized with 3-(2-aminoethylamino)propyl ligands

A novel silica monolithic stationary phase functionalized with 3-(2-aminoethylamino)propyl ligands for pressurized CEC has been presented. The monolithic capillary columns were prepared by a sol-gel process in 75 microm id fused-silica capillaries and followed by a chemical modification. The diamino groups on the surface of the stationary phase are meant to generate the chromatographic surface and a substantial anodic EOF as well as to provide electrostatic interaction sites for charged solutes. The electrochromatographic characterization and column performance were evaluated by a variety of neutral and charged solutes. It was observed that the anodic EOF for the diamine-bonded monolith was greatly affected by the reaction time with 3-(2-aminoethylamino)propyltrimethoxysilane and the PEG amount in the sol-gel reaction mixture in addition to the mobile phase conditions. The monolithic stationary phase exhibited hydrophilic interaction chromatographic behavior toward neutral solutes. Good separations of various solutes including phenols, nucleic acid bases, nucleosides and nucleotides were achieved under different experimental conditions. Fast and efficient separations were obtained with high plate counts reaching more than 130,000 plates/m.     

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.     

Separation of Polar and Basic Compounds in Hydrophilic Interaction Pressurized CEC Using Diethylenetriaminopropyl Silica Monolithic Columns

A novel silica-based monolithic column possessing diethylenetriaminopropyl ligands for hydrophilic interaction pressurized capillary electrochromatography is described. The preparation of monolithic stationary phase was based on the individual silica matrix forming and subsequent chemical bonding. The triamino groups on the surface of the novel stationary phase generated a sustainable anodic electroosmotic flow under acidic conditions. A variety of neutral and basic analytes were used to evaluate the column performance. The monolithic silica stationary phase exhibited hydrophilic interaction chromatographic behavior toward neutral solutes. For basic tetracycline antibiotics, hydrophilic interaction as well as electrophoretic migration process with the monoliths was observed and peak tailing was avoided.     

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.     

Preparation and evaluation of the highly cross‐linked poly(1‐hexadecane‐co‐trimethylolpropane trimethacrylate) monolithic column for capillary electrochromatography

In this paper, a novel highly cross‐linked porous monolithic stationary phase having a long alkyl chain ligand (C16) was introduced and evaluated in CEC. The monolithic stationary phase was prepared by in situ copolymerization of 1‐hexadecene, trimethylolpropane trimethacrylate, and 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid (AMPS) in the presence of ternary porogenic solvent (cyclohexanol/1,4‐butanediol/water). In preparing monoliths, the ternary cross‐linker trimethylolpropane trimethacrylate was usually applied to preparing molecularly imprinted polymers or molecularly imprinted solid‐phase extraction, instead of binary cross‐linker ethylene dimethacrylate. 1‐Hexadecene was introduced to provide the non‐polar sites (C16) for chromatographic retention, while AMPS was used to generate the EOF for transporting the mobile phase through the monolithic capillary. Monolithic columns were prepared by optimizing proportion of porogenic solvent and AMPS content in the polymerization solution as well as the cross‐linkers. The monolithic stationary phases could generate a strong and stable EOF in various pH values and exhibit an RP‐chromatographic behavior for neutral compounds. For charged compounds, the separation was mainly based on the association of hydrophobic, electrostatic and electrophoretic interaction.     

Preparation and evaluation of a hydrophilic poly(2‐hydroxyethyl methacrylate‐co‐N,N′‐methylene bisacrylamide) monolithic column for pressurized capillary electrochromatography

A polar polymethacrylate‐based monolithic column was introduced and evaluated as a hydrophilic interaction CEC stationary phase. The monolithic stationary phase was prepared by in situ copolymerization of a neutral monomer 2‐hydroxyethyl methacrylate and a polar cross‐linker N,N′‐methylene bisacrylamide in a binary porogenic solvent consisting of dodecyl alcohol and toluene. The hydroxyl and amino groups at the surface of the monolithic stationary phase provided polar sites which were responsible for hydrophilic interactions. The composition and proportion of the polymerization mixture was investigated in detail. The mechanical stability and reproducibility of the obtained monolithic column preformed was satisfied. The effects of pH and organic solvent content on the EOF and the separation of amines, nucleosides, and narcotics on the optimized monolithic column were investigated. A typical hydrophilic interaction CEC was observed on the neutral polar stationary phase. The optimized monolithic column can obtain high‐column efficiencies with 62 000–126 000 theoretical plates/m and the RSDs of column‐to‐column (n = 9), run‐to‐run (n = 5), and day‐to‐day (n = 3) reproducibility were less than 6.3%. The calibration curves of these five narcotics exhibited good linearity with R in the range of 0.9959–0.9970 and linear ranges of 1.0–200.0 μg/mL. The detection limits at S/N = 3 were between 0.2 and 1.2 μg/mL. The recoveries of the separation of narcotics on the column were in the range of 84.0–108.6%. The good mechanical stability, reproducibility, and quantitation capacity was suitable for pressure‐assisted CEC applications.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Capillary electrochromatography with zwitterionic stationary phase on the lysine-bonded poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column

A polymer-based neutral monolithic capillary column was prepared by radical polymerization of glycidyl methacrylate and ethylene dimethacrylate in a 100 mum id fused-silica capillary, and the prepared monolithic column was subsequently modified based on a ring opening reaction of epoxide groups with 1 M lysine in solution (pH 8.0) at 75 degrees C for 10 h to produce a lysine chemically bonded stationary phases in capillary column. The ring opening reaction conditions were optimized so that the column could generate substantial EOF. Due to the zwitterionic functional groups of the lysine covalently bonded on the polymer monolithic rod, the prepared column can generate cathodic and anodic EOF by varying the pH values of running buffer during CEC separation. EOF reached the maximum of -2.0 x 10(-8) m2v(-1)s(-1) and 2.6 x 10(-8) m2v(-1)s(-1) with pH of the running buffer of 2.25 and 10, respectively. As a consequence, neutral compounds, ionic solutes such as phenols, aromatic acids, anilines, and basic pharmaceuticals were all successfully separated on the column by CEC. Hydrophobic interaction is responsible for separation of neutral analytes. In addition, the electrostatic and hydrophobic interaction and the electrophoretic migration play a significant role in separation of the ionic or ionizable analytes.     

Monolithic column with double mixed-modes of hydrophilic interaction/cation-exchange and reverse-phase/cation-exchange stationary phase for pressurized capillary electrochromatography

A monolithic capillary column with double mixed-modes of hydrophilic interaction/cation-exchange and RP/cation-exchange stationary phase was prepared by in situ thermal polymerization and then hydrolyzed with hydrochloric acid. The polymerization solution containing glycidyl methacrylate (GMA), 3-sulfopropyl methacrylate potassium salt (SPMA), and ethylene dimethacrylate (EDMA) in a binary porogenic solvent consisting of dimethylformamide (DMF) and 1,4-butanediol was polymerized in a fused-silica capillary pretreated with 3-(trimetoxysilyl) propyl methacrylate. The epoxy groups on the surface were hydrolyzed to diol groups with hydrochloric acid to enhance the polarity of the stationary phase. By simply altering the ACN content in the mobile phase, two mixed-mode mechanisms could be achieved on the same monolithic column in different mobile phase condition. Hydrophilic interaction (or hydrophilic interaction/cation-exchange) was observed at high ACN content, as well as RP (or RP/cation-exchange) was observed at low ACN content. The monolithic column provided good selectivity and high efficiency for separation of neutral polar analytes and basic compounds. Phenols, anilines, alkaloids, nucleic acid bases, and narcotic pharmaceuticals have been successfully separated. Effects of salt concentration and ACN content on the separation have also been investigated. High column efficiencies of up to 352 000 plates/meter were obtained by the separation of narcotic pharmaceuticals.     

Chiral separation of basic compounds on a cellulose 3,5-dimethylphenylcarbamate-coated zirconia monolithin basic eluents by capillary electrochromatography

Porous zirconia monolith (ZM) modified with cellulose 3,5-dimethylphenylcarbamate (CDMPC) was used as chiral stationary phase to separate basic chiral compounds in capillary electrochromatography. The electroosmotic flow behavior of bare and CDMPC-modified zirconia monolithic (CDMPC-ZM) column was studied in ACN/phosphate buffer eluents of pH ranging from 2 to 12. The CDMPC-ZM column was evaluated by investigating the influences of pH, the type and composition of organic modifier of the eluent on enantioseparation. CEC separations at pH 9 provided the best resolutions for the analytes studied, which are better than those observed on CDMPC-modified silica monolithic columns under similar chromatographic conditions. No appreciable decline in retention and resolution factors after over 200 injections, and run-to-run and day-to-day repeatabilities of the column of less than 3% indicate the stability of the zirconia monolithic column in basic media.