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。     

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.     

Methacrylate-based monolithic column with mixed-mode hydrophilic interaction/strong cation-exchange stationary phase for capillary liquid chromatography and pressure-assisted CEC

A novel porous polymethacrylate-based monolithic column by in situ copolymerization of 3-sulfopropyl methacrylate (SPMA) and pentaerythritol triacrylate in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was prepared. The monolith possessed in their structures bonded sulfonate groups and hydroxyl groups and was evaluated as a hydrophilic interaction and strong cation-exchange stationary phases in capillary liquid chromatography (cLC) and pressure-assisted CEC using small polar neutral and charged solutes. While the SPMA was introduced as multifunctional monomer, the pentaerythritol triacrylate was used to replace ethylene glycol dimethacrylate as cross-linker with much more hydrophilicity due to a hydroxyl sub-layer. The different characterization of monolithic stationary phases were specially designed and easily prepared by altering the amount of SPMA in the polymerization solution as well as the composition of the porogenic solvent for cLC and pressure-assisted CEC. The resulting monolith showed the different trends about the effect of the permeabilities on efficiency in the pressure-assisted CEC and cLC modes. A typical hydrophilic interaction chromatography mechanism was observed at higher organic solvent content (ACN%>70%) for polar neutral analytes. For polar charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Therefore, for charged analytes, selectivity can be readily manipulated by changing the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). With the optimized monolithic column, high plate counts reaching greater than 170 000 plates/m for pressure-assisted CEC and 105 000 plates/m for cLC were easily obtained, respectively.     

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.     

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.     

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.     

Preparation and evaluation of a sulfoalkylbetaine‐based zwitterionic monolithic column for CEC of polar analytes

A novel polymethacrylate‐based monolithic column with covalently bonded zwitterionic functional groups was prepared by in situ copolymerization of N,N‐dimethyl‐N‐methacryloxyethyl N‐(3‐sulfopropyl) ammonium betaine (SPE), pentaerythritol triacrylate (PETA), and vinylsulfonic acid (VS) in a binary porogenic solvent consisting of cyclohexanol and ethylene glycol. This monolith was developed as a separation column for CEC. While SPE functioned as both an electrostatic interaction stationary phase and the polar ligand provider, VS was employed to generate EOF. PETA, which has much more hydrophilicity due to a hydroxyl sub‐layer, was used to replace ethylene dimethacrylate as a cross‐linker. The monolith provided an adequate EOF when VS level was maintained at 0.6% w/w. Different monolithic stationary phases were easily prepared by adjusting the ratio of PETA/SPE in the polymerization solution as well as the composition of the porogenic solvent. The observed RSD were ≤3.6, ≤4.3 and ≤5.6% for the EOF velocity, retention time, and column efficiency, respectively. The column efficiencies greater than 145 000 theoretical plates/m for thiourea and 132 000 theoretical plates/m for charged cytidine were obtained. The poly(SPE‐co‐PETA‐co‐VS) monolith showed good selectivity for neutral and charged polar analytes. It was found that the separation mechanism of charged polar solutes was attributed to a mixed mode of hydrophilic interaction and electrostatic interaction, as well as electrophoresis. No peak tailing was observed for the separation of basic compounds, such as basic nucleic acid bases and nucleoside on the monolith.     

Capillary electrochromatography with monolithic stationary phases: 1. Preparation of sulfonated stearyl acrylate monoliths and their electrochromatographic characterization with neutral and charged solutes

A novel monolithic stationary phase having long alkyl chain ligands (C17) was introduced and evaluated in capillary electrochromatography (CEC) of small neutral and charged species. The monolithic stationary phase was prepared by the in situ copolymerization of pentaerythritol diacrylate monostearate (PEDAS) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent consisting of cyclohexanol/ethylene-glycol/water. While AMPS was meant to support the electroosmotic flow (EOF) necessary for transporting the mobile phase through the monolithic capillary, the PEDAS was introduced to provide the nonpolar sites for chromatographic retention. Monolithic columns at various EOF velocities were readily prepared by conveniently adjusting the amount of AMPS in the polymerization solution as well as the composition of the porogenic solvent. The monolithic stationary phases thus obtained exhibited reversed-phase chromatography behavior toward neutral solutes and yielded a relatively strong EOF. For charged solutes (e.g., dansyl amino acids), nonpolar as well as electrostatic interaction/repulsion with the monoliths were observed in addition to electrophoretic migration. Therefore, for charged solutes, selectivity and migration can be readily manipulated by changing various parameters including the nature of the monolith and the composition of the mobile phase (e.g., pH, ionic strength and organic modifier). Ultrafast separation on the time scale of seconds of 17 different charged and neutral pesticides and metabolites were performed using short capillary columns of 8.5 cm x 100 microm ID.     

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.     

Monolithic silica columns with mixed mode of hydrophilic interaction and weak anion-exchange stationary phase for pressurized capillary electrochromatography

A silica-based monolithic column as polar stationary phase is proposed for pressurized CEC (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by 3-aminopropyltrimethoxysilane to produce a column for hydrophilic interaction applications. The amino groups on the surface of the polar stationary phase generated anodic EOF under acidic conditions and served at the same time as a weak anion-exchanger. The anion solutes such as nucleotides were separated by the mixed mode mechanism, which comprised hydrophilic interaction, weak anion-exchange, and electrophoresis. The influences of buffer concentration and organic modifier content on the separation of nucleotides by pCEC have been investigated. In addition, the monolithic silica columns were also able to separate various polar compounds such as phenols, nucleic acid bases, and nucleosides in the hydrophilic interaction CEC mode.     

Development of acrylate-based monolithic stationary phases for electrochromatographic separations

Organic monolithic stationary phases were synthesized in fused-silica capillaries. They were prepared by in situ polymerization under UV irradiation of various alkyl acrylates, 1,3-butanediol diacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid in a ternary porogenic solvent. The resulting stationary phases were tested in CEC. The influence of UV irradiation energy on the resulting separative performances of the monoliths was studied. It was thus demonstrated that the use of hexyl acrylate rather than butyl acrylate and lauryl methacrylate gives highly efficient monoliths (more than 300 000 plates per meter) with optimized EOF. It was also confirmed that the mobile phase ionic strength may affect significantly the separation efficiency. The influence of the nature of the mobile phase organic modifier (ACN or methanol) on EOF, retention, efficiency, and selectivity was studied and differences were observed. Finally, the performances of monolithic stationary phases developed and optimized for CEC separations were evaluated in nanoLC.     

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.     

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.     

A novel, neutral hydroxylated octadecyl acrylate monolith with fast electroosmotic flow velocity and its application to the separation of various solutes including peptides and proteins in the absence of electrostatic interactions

A neutral hydroxylated octadecyl monolith (ODM-OH) for reversed-phase capillary electrochromatography has been developed. The ODM-OH was prepared by the in situ polymerization of octadecyl acrylate and pentaerythritol triacrylate (PETA) in a ternary porogenic solvent. Pentaerythritol triacrylate possesses a hydroxyl functional group, which imparts the monolith with a hydrophilic group, thus the acronym ODM-OH. The ODM-OH 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. This ODM-OH monolith exhibited stronger EOF than its counterpart the ODM made from the in situ polymerization of octadecyl acrylate and trimethylolpropane trimethacrylate. Similar to ODM, it is believed that the EOF was due to the adsorption of ions from the mobile phase onto the surface of the monolith thus imparting the neutral monolithic column the zeta potential necessary to support the EOF. The higher EOF exhibited by ODM-OH was due to the presence of polar OH groups on its surface, which would favor stronger adsorption of ions from the mobile phase. The wide applications of the neutral ODM-OH column were demonstrated in the separation of a wide range of small and large solutes. As a typical result, the ODM-OH was able to separate proteins quite rapidly yielding 200,000 plates/m.     

Preparation of a mixed-mode hydrophilic interaction/anion-exchange polymeric monolithic stationary phase for capillary liquid chromatography of polar analytes

A novel cationic hydrophilic interaction monolithic stationary phase based on the copolymerization of 2-(methacryloyloxy)ethyltrimethylammonium methyl sulfate (META) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was designed for performing capillary liquid chromatography. While META functioned as both the ion-exchange sites and polar ligand provider, the PETA, a trivinyl monomer, was introduced as cross-linker. The monolithic stationary phases with different properties were easily prepared by adjusting the amount of META in the polymerization solution as well as the composition of the porogenic solvent. The hydrophilicity of the monolith increased with increasing content of META in the polymerization mixture. A typical hydrophilic interaction chromatography mechanism was observed when the content of acetonitrile in the mobile phase was higher than 20%. The poly(META-co-PETA) monolith showed very good selectivity for neutral, basic and acidic polar analytes. For polar-charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Peak tailing of basic compounds was avoided and the efficient separation of benzoic acid derivatives was obtained.     

Preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate) monolithic column for hydrophilic interaction chromatography

A method for the preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate copolymerization)-based monolithic capillary column was reported for the separation of polar small molecular weight compounds with nano-liquid chromatography in hydrophilic interaction chromatography mode. The monolithic columns were prepared by in situ copolymerization of N-vinylpyrrolidone and a cross-linker pentaerythritol triacrylate in a binary porogenic agent consisting of methanol and water. The composition of the polymerization solution was systematically optimized in terms of column permeability, theoretical plate number, asymmetric factor, and retention factor. A typical hydrophilic chromatography retention mechanism was observed with a mobile phase composed of a high content of organic solvent. The preparation method is simple and robust, the precursor N-vinylpyrrolidone is chemically stable, cheap, and easily available. The N-vinylpyrrolidone-based hydrophilic interaction chromatography stationary phase displays satisfactory separation selectivity for a range of polar test analytes, including benzoic acid derivatives, nucleosides, and phenols.     

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.     

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.     

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.     

Capillary electrochromatography with monolithic stationary phases. 4. Preparation of neutral stearyl-acrylate monoliths and their evaluation in capillary electrochromatography of neutral and charged small species as well as peptides and proteins

A neutral, nonpolar monolithic capillary column having a relatively strong electroosmotic flow (EOF) yet free of electrostatic interactions with charged solutes was developed for the reversed-phase capillary electrochromatography (RP-CEC) of neutral and charged species including peptides and proteins. The neutral nonpolar monolith is based on the in situ polymerization of pentaerythritol diacrylate monostearate (PEDAS) in a ternary porogenic solvent composed of cyclohexanol, ethylene glycol, and water. PEDAS plays the role of both the cross-linker and the ligand provider, generating a macroporous nonpolar monolith having C17 chains as the chromatographic ligands. Despite the fact that the neutral PEDAS monolith is devoid of fixed charges, the monolithic capillary columns exhibited a relatively strong EOF due to the ability of PEDAS to adsorb sufficient amounts of electrolyte ions from the mobile phase. The adsorbed ions imparted the neutral PEDAS monolith the zeta potential necessary to support the EOF required for mass transport across the monolithic column. The absence of fixed charges on the surface of the neutral PEDAS monolith and in turn the adsorption sites for electrostatic attraction of charged solutes allowed the rapid and efficient separations of proteins and peptides at pH 7.0, with an average plate number of 255,000 and 121,000 plates/m, respectively. To the best of our knowledge, this constitutes the first report on the separation of proteins at neutral pH by RP-CEC using a neutral monolithic column.