A new zwitterionic electrochromatographic stationary phase based on poly(3‐chloro‐2‐hydroxypropyl methacrylate‐co‐ethylene dimethacrylate) reactive monolith

A new reactive monolith, poly(3‐chloro‐2‐hydroxypropyl methacrylate‐co‐ethylene dimethacrylate), poly(HPMA‐Cl‐co‐EDMA) was synthesized and post‐functionalized by taurine (2‐aminoethane sulfonic acid) to obtain a zwitterionic stationary phase for capillary electrochromatography. The new stationary phase contained charged groups such as secondary amine providing anodic electroosmotic flow and sulfonic acid groups providing cathodic electroosmotic flow. Hence, the capillary electrochromatography separations with the new zwitterionic monolith were performed with either anodic or cathodic electroosmotic flow. The electrochromatographic separation of alkylbenzenes and phenols was successfully performed. The zwitterionic monolith also allowed the separation of nucleosides using only electrokinetic mode. Theoretical plate numbers up to ~10⁵ plates/m were achieved. Our study is the first report based on poly(HPMA‐Cl‐co‐EDMA) reactive monolith post‐functionalized with a zwitterionic ligand allowing to operate in both anodic and cathodic electroosmotic flow modes. Copyright © 2014 John Wiley & Sons, Ltd.     

Dipyridyl‐immobilized ionic liquid type hybrid silica monolith for hydrophilic interaction electrochromatography

A pyridinium‐based immobilized ionic liquid type multifunctional hybrid silica monolith was prepared by the in situ polymerization of 3‐chloropropyl‐silica matrix and 4,4′‐dipyridyl for hydrophilic interaction CEC. The obtained hybrid monolith possessed of high stable skeletal microstructures with obviously hydrophilic retention mechanism under ACN content >50% in the mobile phase. Strong and stable anodic EOF could be observed under a broad pH range from pH 3.0 to 9.0. Due to the immobilized dipyridyl groups bonded to the silica matrix surface, the resulting hydrophilic hybrid monolith possessed multiple separation interactions including hydrogen bond, π–π, and anion exchange. Excellent separations of various polar analytes including electroneutral phenols, charged acid nucleotides, and basic analytes were successfully achieved. The highest column efficiencies up to 120 000, 164 000, and 106 000 plates/m were obtained for nucleotides, nucleic acid bases, and nucleosides and nicotines, respectively. These results demonstrated that the dipyridyl‐immobilized ionic liquid functionalized hybrid monolith possessed highly mechanical stability and good chromatographic performance for hydrophilic interaction electrochromatography.     

One‐step strategy for the synthesis of a derivatized cyclodextrin‐based monolithic column

Derivatized β‐cyclodextrin (β‐CD) functionalized monolithic columns were prepared by a “one‐step” strategy using click chemistry. First, the intended derivatized β‐CD monomers were synthesized by a click reaction between propargyl methacrylate and mono‐6‐azido‐β‐CD and then sulfonation or methylation was carried out. Finally, monolithic columns were prepared through a one‐step in situ copolymerization of the derivatized β‐CD monomer and ethylene glycol dimethacrylate. The sulfated β‐CD‐based monolith was successfully applied to the hydrophilic interaction liquid chromatography separation of nucleosides and small peptides, while the methylated β‐CD‐functionalized monolith was useful for the separation of nonpolar compounds and drug enantiomers in capillary reversed‐phase liquid chromatography. The structures of the monomers were characterized by Fourier transform infrared spectroscopy and mass spectrometry. The physicochemical properties and column performance of monoliths were evaluated by scanning electron microscopy and micro high performance liquid chromatography. This strategy has considerable prospects for the preparation of other derivatized CD‐functionalized methacrylate monoliths.     

Polymer monolithic capillary column fabricated by using monodisperse iron oxide nanocrystal template to enhance the electrochromatographic separation of small molecules

Monodisperse iron oxide nanocrystals and organic solvents were utilized as coporogens in monolithic poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) capillary columns to afford stationary phases with enhanced electrochromatographic performance of small molecules. While the conventional monoliths using organic solvents only as a porogen exhibited poor resolution (Rs) <1.0 and low efficiency of 40 000–60 000 plates/m, addition of a small amount of nanocrystals to the polymerization mixture provided increased resolution (Rs > 3.0) and high efficiency ranged from 60 000 to 100 000 plates/m at the same linear velocity of 0.856 mm/s. It was considered that the mesopores introduced by the nanocrystals played an important role in the improvement of the monolith performance. This new strategy expanded the application range of the hydrophobic monoliths in the separation of polar alkaloids and narcotics. The successful applications demonstrated that the glycidyl methacrylate based monoliths prepared by using nanocrystal template are a good alternative for enhanced separation efficiency of small molecules.     

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.     

Polymer monolith microextraction online coupled to hydrophilic interaction chromatography/mass spectrometry for analysis of β2‐agonist in human urine

An analytical method based on online combination of polymer monolith microextraction (PMME) technique with hydrophilic interaction LC (HILIC)/MS is presented. The extraction was performed with a poly(methacrylic acid‐co‐ethylene glycol dimethacrylate) monolithic column while the subsequent separation was carried out on a Luna silica column by HILIC. After 1:1 v/v dilution with 20 mM phosphate solution at pH 7.0 and centrifugation, urine sample was directly used for extraction. After optimization, 85% ACN (containing 0.3% formic acid v/v) was used for rapid online elution, which was also the mobile phase in HILIC to avoid band broadening during separation or carry‐over that was usually observed in PMME‐RP LC system. Online automation of extraction and separation procedures was realized under the control of a program in this study. The developed method was applied to rapid and sensitive monitoring of three β₂‐agonist traces in human urine. The LODs (S/N = 3) of the method were found to be 0.05–0.09 ng/mL of β₂‐agonists in urine. The recoveries of three β₂‐agonists spiked in five different urine samples ranged from 79.8 to 119.8%, with RSDs less than 18.0%.     

Thiol‐yne Click Adamantane Monolithic Stationary Phase for Capillary Electrochromatography

A porous crosslinked organic polymer based on N‐acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) was prepared inside 75 µm i.d. fused silica capillary as functionalizable monolithic stationary phase for electrochromatographic applications. Succinimide groups on the monolith surface provide reactive sites able to react readily through standard electrophile‐nucleophile chemistry. Propargylamine was used to prepare alkyne functionalized poly(NAS‐co‐EDMA). Onto this thiol‐reactive polymer surface was grafted adamantane units via a photochemically‐driven addition reaction. Chemical characterization was performed in situ after each synthetic step by means of Raman spectroscopy and grafting kinetics was investigated to ensure quantitative grafting of 1‐adamantanethiol. The as‐designed monolithic stationary phase exhibited typical reversed‐phase separation mechanism as evidenced by the linear increase of the logarithm of retention factor of neutral aromatic solutes with the increase of the aqueous buffer content in the mobile phase.     

New vinylester‐based monoliths as a new stationary phase for capillary electrochromatography

Vinyl ester‐based monoliths are proposed as a new group of stationary phase for CEC. The capillary monolithic columns were prepared by using two vinyl ester monomers, vinyl pivalate (VPV), and vinyl decanoate (VDC) by using ethylene dimethacrylate (EDMA) as the cross‐linking agent, and 2‐acrylamido‐2‐methylpropane sulfonic acid as the charge‐bearing monomer. The monoliths with different pore structures and permeabilities were obtained by varying the type and composition of the porogen mixture containing isoamyl alcohol and 1,4‐butanediol. The electrochromatographic separation of alkylbenzenes was successfully performed by using an acetonitrile/aqueous buffer system as the mobile phase in a CEC system. Vinyl ester monoliths with short alkyl chain length (i.e. poly(VPV‐co‐EDMA) exhibited better separation performance compared with the monolith with long alkyl chain length (i.e. poly(VDC‐co‐EDMA). In the case of VPV‐based monoliths, the theoretical plate numbers higher than 250 000 plates/m were achieved by using a porogen mixture containing 33% v/v of isoamyl alcohol. For both VDC and VPV‐based monoliths, the column efficiency was almost independent of the superficial velocity in the range of 2–12 cm/min.     

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.     

Use of the Synthesized Titania Monolith to Determine Benzoic Acid and Vanillin in Foodstuffs by HPLC

A simple and reliable HPLC method for the determination of benzoic acid and vanillin in food samples has been developed, in which a pure titania monolithic column synthesized through a template-free sol-gel synthesis route was used as chromatography column. To fully understand the retention mechanism of benzoic acid and vanillin on titania, acetonitrile (ACN) percentage, buffer concentration, and buffer pH of the mobile phase were investigated. The retention mechanism of benzoic acid and vanillin on the titania monolith column belongs to hydrophilic interaction and ligand exchange. When the high %ACN and appropriate acetate existed in eluent, the hydrophilic interaction was the dominant retention mode. Benzoic acid and vanillin in preserved fruit and jelly samples were successfully determined and quantitative analysis was carried out by external standard method with correlation coefficient (R ² ) of 0.9994 for benzoic acid and 0.9989 for vanillin. The relative standard deviations (RSDs) of benzoic acid and vanillin were 0.94% and 1.50%, respectively. The developed titania-based HPLC method is simple, rapid, accurate, and competent for the separation of polar and hydrophilic compounds, and this work has also promoted the application of titania monolith in chromatographic separation.     

Preparation of chiral polystyrene monoliths by utilizing W/O emulsion polymerization and their optical resolution ability

We synthesized two kinds of chiral polystyrene‐based monoliths, which are macroporous gel with continuous open‐celled monolith structure. Thus, two chiral styrene monomers, (–)‐p‐[dimethyl(10‐pinanyl)silyl]styrene ((–)‐PSSt) and (–)‐p‐(menthoxycarbonyl)styrene ((–)‐MtSt]), were prepared and subjected to water‐in‐oil emulsion polymerization in the presence of divinylbenzene and AIBN. The macroporous structure of the obtained monoliths was directly confirmed by SEM observation. The obtained monoliths showed an optical resolution ability. That is, in the enantioselective adsorption using trans‐stilbene oxide, poly[(–)‐PSSt] monolith and poly[(–)‐MtSt] monoliths preferentially adsorbed (S,S)‐isomer [poly[(–)‐PSSt] monolith: α^(S,S) = 1.49 (0.25 wt % acetone solution); poly[(–)‐MtSt] monolith: α^(S,S) = 1.39 (0.25 wt % toluene solution)]. Depinanylsilylation of the poly[(?)‐PSSt] monolith and removal of menthyl groups from the poly[(–)‐MtSt] monolith were achieved by acid‐catalyzed scission of the Si? C bond and base‐catalyzed hydrolysis, respectively. In addition, de‐poly[(–)‐PSSt] and de‐poly[(–)‐MtSt] showed enantioselectivity ((S,S)‐isomer preferentially absorbed) in adsorption using trans‐stilbene oxide in spite of the absence of chiral substituents in the monoliths. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2348–2357, 2005     

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.     

Incorporation of graphene oxide nanosheets into boronate‐functionalized polymeric monolith to enhance the electrochromatographic separation of small molecules

Graphene oxide (GO) nanosheets were incorporated into an organic polymer monolith containing 3‐acrylamidophenylboronic acid (AAPBA) and pentaerythritol triacrylate (PETA) to form a novel monolithic stationary phase for CEC. The effects of the mass ratio of AAPBA/PETA, the amount of GO, and the volume of porogen on the morphology, permeability and pore properties of the prepared poly(AAPBA‐GO‐PETA) monoliths were investigated. A series of test compounds including amides, alkylbenzenes, polycyclic aromatics, phenols, and anilines were used to evaluate and compare the separation performances of the poly(AAPBA‐GO‐PETA) and the parent poly(AAPBA‐co‐PETA) monoliths. The results indicated that incorporation of GO into monolithic column exhibited much higher resolutions (>1.5) and column efficiency (62 000 ～ 110 000 plates/m for toluene, DMF, formamide, and thiourea) than the poly(AAPBA‐co‐PETA). The successful application in isocratic separation of peptides suggests the potential of the GO incorporated monolithic column in complex sample analysis. In addition, the reproducibility and stability of the prepared poly(AAPBA‐GO‐PETA) monolith was assessed. The run‐to‐run, column‐to‐column and batch‐to‐batch reproducibilities of this monolith for alkylbenzenes’ retention were satisfactory with the RSDs less than 1.8% (n = 5), 3.7% and 5.6% (n = 3), respectively, indicating the effectiveness and practicability of the proposed method.     

Proline‐based polymeric monoliths: Synthesis,characterization, and applications as organocatalysts in aldol reaction

Proline‐based polymer monoliths were synthesized via green protocol using lipase‐catalyzed esterification of methacrylic acid and 4‐hydroxyproline. Prolinyl methacrylate thus prepared was polymerized in situ as crosslinked monolith. The monolith was characterized by various techniques such as Fourier transform infrared, ¹H‐NMR, ¹³C‐NMR, scanning electron microscopy (SEM), X‐ray diffraction (XRD), and nitrogen analysis and used as catalyst in aldol reactions. The swelling behavior of the monolith was also studied as function of various external parameters like pH and temperature. The monoliths synthesized with 1% crosslinker was selected as candidate monolith for use as catalyst in aldol reaction, which was studied as a function of time, temperature, substrate structure, and amount of water:EtOH. The catalysts exhibited high efficiency in the cross aldol reaction, especially with the aromatic substrates having electron withdrawing substituent, and also good activity retention was observed when recycleability was studied up to five cycles. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1007–1015, 2010     

Epinephrine‐modified methacrylate monolithic column with fumed silica nanoparticle for pressurized capillary electrochromatography

Via the ring‐opening reaction of epoxy groups with epinephrine, a novel epinephrine functionalized polymethacrylate monolith with fumed silica nanoparticles has been fabricated for pressurized capillary electrochromatography. The preparation of epinephrine‐modified monoliths has been optimized. In addition, morphology, electroosmotic flow, separation mechanism and column performance have been studied. The internal structure of the monolithic stationary phase was more uniform and the column efficiency increased after the incorporation of nanoparticles. With this column, satisfactory separation capability of aromatic compounds and alkaloids has been achieved and the column efficiency for naphthalene reached 138 696 plates/m. As for the real sample, 3 alkaloids were separated in Huanglian Shangqing capsules, a Chinese traditional medicine.     

Preparation of an Electrochromatographic Stationary Phase Using a New Polymethacrylate Monolith with Chloropropyl Functionality

A new reactive capillary monolith as an alternative to the commonly employed glycidyl methacrylate-based stationary phases in capillary electrochromatography (CEC) applications was synthesized and post-functionalized with charge-bearing groups. For this purpose, a hydrophilic capillary monolith with reactive 3-chloro-2-hydroxypropyl moiety was first obtained by the copolymerization of a new methacrylate-based monomer, 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl), with a methacrylic crosslinking agent, ethylene glycol dimethacrylate (EDMA). The presence of 3-chloro-2-hydroxypropyl functionality in poly(HPMA-Cl-co-EDMA) monolith allowed the synthesis of monoliths carrying strongly ionizable anionic, cationic or neutral groups such as sulfonic acid, quaternary ammonium or octadecyl, respectively, via simple and single-stage reactions. In the present study, a reactive poly(HPMA-Cl-co-EDMA) capillary monolith was functionalized with sodium bisulfite (NaHSO₃) to have a strongly ionizable sulfonic acid group on the monolith. The resulting monolith providing cathodic electroosmotic flow in CEC was successfully used for the separation of phenol derivatives. The theoretical plate numbers up to 63,000 plates/column were achieved. The results showed that a new promising, reactive support that could be functionalized with different chromatographic ligands for different chromatographic applications was obtained in the study.     

One‐pot preparation of an organic polymer monolith by thiol‐ene click chemistry for capillary electrochromatography

A novel organic monolith was successfully fabricated by a one‐pot thiol‐ene click reaction of triallyl isocyanurate with pentaerythritol tetrakis‐(2‐mercaptoacetate) and mercaptopropionic acid in the presence of porogens. We investigated the effects of the ratio of monomer and cross‐linking agent, the type and ratio of porogen, and click reaction temperature on the permeability and morphology of the prepared poly triallyl isocyanurate‐co‐pentaerythritol tetrakis (2‐mercaptoacetate) monoliths. The monolith was also characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. The results indicated that the monoliths had continuous porous framework, good permeability, and high mechanical stability. A series of analytes with different properties such as alkylbenzenes, polycyclic aromatic hydrocarbons, anilines, and phenols were used to evaluate the electrochromatographic performance of the prepared monoliths in pressurized capillary electrochromatography. The prepared polymer monolith showed typical reversed‐phase electrochromatographic behavior for hydrophobic substances. Moreover, the prepared monolith showed a mix of reversed‐phase and cation exchange interaction modes for basic aniline compounds. The minimum plate height of the monolith was 8.76 μm (132 100 plates/m) for propylbenzene. These results demonstrated that one‐pot thiol‐ene click chemistry can provide a simple and reliable method for the preparation of organic monoliths.     

Separation of peptide diastereomers using CEC and a hydrophobic monolithic column

A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has been evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. Using an acidic mobile phase, the peptides migrated due to their own electrophoretic mobility. Hydrophobic interactions with the stationary phase contributed to the separation. Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of the mobile phase. Peak resolution increased with buffer pH and concentration. Di‐ and tripeptides composed only of L ‐configured amino acids migrated faster than peptides containing D ‐amino acids. A mixture of isomeric Asp tripeptides that could not be completely resolved by either CZE or HPLC as well as the 24mer peptides tetracosactide and ¹⁶[D ‐Lys]‐tetracosactide could also be separated by CEC on the hydrophobic monolith.     

Biocompatible polymeric monoliths for protein and peptide separations

The concept of biocompatibility with reference to chromatographic stationary phases for separation of biomolecules (including proteins and peptides) is introduced. Biocompatible is a characteristic that indicates resistance to nonspecific adsorption of biomolecules and preservation of their structures and biochemical functions. Two types of biocompatible polymeric monoliths [i. e., polyacrylamide‐ and poly(meth)acrylate‐based monoliths] used for protein and peptide separations are reviewed in detail, with emphasis on size exclusion, ion exchange, and hydrophobic interaction chromatographic modes. Biocompatible monoliths for enzyme reactors are also included. The two main synthetic approaches to produce biocompatible monoliths are summarized, i. e., surface modification of a monolith that is not inherently biocompatible and direct copolymerization of hydrophilic monomers to form a biocompatible monolith directly. Integration of polyethylene glycol into the poly(meth)acrylate monolith network is becoming popular for reduction of non‐specific protein interactions.     

Phenylalanine functionalized zwitterionic monolith for hydrophobic interaction electrochromatography

A novel phenylalanine (Phe) functionalized zwitterionic monolith for hydrophobic electrochromatography was prepared by a two‐step procedure involving the synthesis of glycidyl methacrylate based polymer monolith and subsequent on‐column chemical modification with Phe via ring‐opening reaction of epoxides. Benefitting from the hydrophobicity of both methacrylate‐based matrix and aromatic group of Phe, this monolith could exhibit good hydrophobic interaction for the separation. Typical RP chromatographic behavior was observed toward various solutes. The well‐controlled cathodic or anodic EOF of the prepared column could be facilely switched by altering the pH values of running buffers. The separation mechanism of this Phe functionalized zwitterionic monolith is discussed in detail. Two mixed‐mode mechanisms of RP/cation exchange and RP/anion exchange could be further realized on the same monolith in different pH condition of the mobile phase. Versatile separation capabilities of neutral, basic, and acidic analytes have been successfully achieved in this zwitterionic monolith by CEC method.