Novel preparation of monolithic imprinted columns for electrochromatographic separation by photopolymerization

A monolithic molecularly imprinted polymer with specific recognition ability for 4-hydroxybenzoic acid (4-HBA) was prepared by in situ photopolymerization, using methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EDMA) as a cross-linking agent, toluene and isooctane as porogenic solvents and Irgacure 1800 as an initiator. Baseline separation of isomers of hydroxybenzoic acid was achieved in less than 8 min on this monolithic column using 4-HBA as template, but not on the blank polymer. Furthermore, some neutral compounds could also be baseline-separated on the imprinted polymer column in the mode of pressure-driven capillary electrochromatography.     

Mechanism of molecular recognition on molecular imprinted monolith by capillary electrochromatography

The recognition mechanism of molecularly imprinted polymer (MIP) in capillary electrochromatography (CEC) is complicated since it possesses a hybrid process, which comprises the features of chromatographic retention, electrophoretic migration and molecular imprinting. For an understanding of the molecular recognition of MIP in CEC, a monolithic MIP in a capillary with 1,1'-binaphthyl-2,2'-diamine (BNA) imprinting was prepared by in situ copolymerization of imprinted molecule, methacrylic acid and ethylene glycol dimethacrylate in porogenic solvent, a mixture of toluene-isooctane. Strong recognition ability and high column performance (theory plates was 43,000 plates/m) of BNA were achieved on this monolithic MIP in CEC mode. In addition, BNA and its structural analogue, 1,1'-bi-2, 2'-naphthol, differing in functional groups, were used as model compounds to study imprinting effect on the resultant BNA-imprinted monolithic column, a reference column without imprinting of BNA and a open capillary. The effects of organic modifier concentration, pH value of buffer, salt concentration of buffer and column temperature on the retention and recognition of two compounds were investigated. The results showed that the molecular recognition on MIP monolith in CEC mode mainly derived from imprinting cavities on BNA-imprinted polymer other than chromatographic retention and electrophoretic migration.     

Dual‐templates molecularly imprinted monolithic columns for the evaluation of serotonin and histamine in CEC

To extend the application of molecularly imprinted polymers, the dual‐templates molecularly imprinted monolithic columns were developed in a capillary format. Two templates serotonin and histamine were simultaneously imprinted using two different functional monomers such as methacrylic acid (MAA) and methylenesuccinic acid (MSA) in a mixture of ethylene glycol dimethacrylate (EDMA) as a cross‐linker and AIBN as polymerization initiator dissolved in DMF as porogen. The resulting molecular imprinted polymers (MIPs) were characterized based on their performance in the CEC separation of two imprinted templates. The optimization parameters such as pH, ACN composition, and concentration of the eluent were varied to achieve best resolution and efficiency for CEC separation of templates with each MIP column. It was found that the MIP monolith column fabricated using MSA offered better resolution and separation efficiency compared to column fabricated with MAA. This work utilized the dual‐templates imprinting approach successfully and broadens the scope of multi‐templates imprinting capabilities in capillary format in CEC application.     

Recognition of oxytocin by capillary electrochromatography with monolithic tetrapeptide-imprinted polymer used as the stationary phase

Using YPLG (Tyr-Pro-Leu-Gly), a tetrapeptide, as the template, an imprinted monolithic column was prepared and applied to the selective recognition of oxytocin based on the epitope approach and capillary electrochromatography (CEC). By optimizing the polymerization solution in terms of functional monomer, cross-linking reagent, porogen, and imprinted template via CEC evaluations of synthesized columns, an imprinted monolith with good recognition capacity (the imprinting factors for YPLG and oxytocin were 4.499 and 4.013, respectively) and high column efficiency (theoretical plates for YPLG and oxytocin were 22,995 plates/m and 16,952 plates/m, respectively) was achieved. In addition, the effects of various experimental parameters on the recognition of oxytocin, including the organic modifier content, the buffer concentration, and the pH value, were studied systematically. Furthermore, a mixture of oxytocin and other proteins was analyzed using this monolithic CEC column, and oxytocin was eluted much more slowly than other large biomolecules, which demonstrated the high selective recognition ability of such an imprinted monolith for oxytocin with PLG (Pro-Leu-Gly) as the epitope. Figure Separation of a mixture of oxytocin, BSA, bovine hemoglobin, ovalbumin, and lysozyme on the open column, the blank monolithic column, and the monolithic YPLG-imprinted column     

4-氨基吡啶分子印迹聚合物毛细管整体柱的制备及电色谱性能研究

以4-氨基吡啶(4-AP)为印迹分子,热引发原位合成了分子印迹聚合物毛细管整体柱,聚合物通过共价键和石英毛细管内壁相连,制备方法简单、快捷.在最佳电色谱条件下,4-AP和2-AP之间的分离度在印迹聚合物柱上高达2.5,而在不含印迹的对照柱上仅为0.35.通过研究流动相条件对4-AP,2-AP和硫脲迁移的影响,对4-AP印迹聚合物的电色谱识别机理进行了探讨.印迹识别能力随缓冲溶液离子强度的减小或流动相中乙腈比例的增大而增大.上述两种情形下,流动相中阳离子浓度均减少,使得聚合物孔穴中可与4-AP发生静电作用的有效羧基作用位点增加,从而显现出孔穴对印迹分子的专一亲和作用(形状、大小和作用力).缓冲溶液的种类和pH值对该印迹聚合物识别能力的影响较为复杂,在磷酸盐缓冲溶液体系中,pH值对识别能力的影响呈抛物线形,pH=5时识别能力最强;在醋酸盐缓冲溶液体系中,高pH值有利于分离.     

Preparation and characterization of a molecularly imprinted monolithic column for pressure‐assisted CEC separation of nitroimidazole drugs

A polymethacrylate‐based molecularly imprinted monolithic column bearing mixed functional monomers, using non‐covalent imprinting approach, was designed for the rapid separation of nitroimidazole compounds. The new monolithic column has been prepared via simple in situ polymerization of 2‐hydroxyethyl methacrylate, dimethylaminoethyl methacrylate and ethylene dimethacrylate, using (S)‐ornidazole ((S)‐ONZ) as template in a binary porogenic mixture consisting of toluene and dodecanol. The composition of the polymerization mixture was systematically altered and optimized by altering the amount of monomers as well as the composition of the porogenic solvent. The column performance was evaluated in pressure‐assisted CEC mode. Separation conditions such as pH, voltage, amount of organic modifier and salt concentration were studied. The optimized monolithic column resulted in excellent separation of a group of structurally related nitroimidazole drugs within 10 min in isocratic elution condition. Column efficiencies of 99 000, 80 000, 103 000, 60 000 and 99 000 plates/m were obtained for metronidazole, secnidazole, ronidazole, tinidazole and dimetridazole, respectively. Parallel experiments were carried out using molecularly imprinted and non‐imprinted capillary columns. The separation might be the result of combined effects including hydrophobic, hydrogen bonding and the imprinting cavities on the (S)‐ONZ‐imprinted monolithic column.     

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.     

分子印迹整体柱在高效液相色谱和电色谱手性分离中的应用

在常规不锈钢色谱管中以甲基丙烯酸为功能单体,采用原位聚合法制备了(5S,11S)-特罗格尔碱(S-TB)的印迹整体柱。考察了流动相中添加不同量的醋酸和水对分离的影响,结合台阶梯度洗脱模式在S-TB整体柱上实现了对TB消旋体的快速分离。另外,以碱性单体2-二甲基乙基胺甲基丙烯酸酯(DAMA)为功能单体,在毛细管中采用原位聚合法制备了毛细管分子印迹整体柱,用于在毛细管电色谱（CEC）中对消旋体1,1′-联-2-萘酚(BNL)进行手性分离。结果表明,以AMA为功能单体可以制备其他酸性模板的分子印迹聚合物,从而扩大了分子印迹聚合物MIP）在CEC分离中的应用范围。     

分子印迹毛细管电色谱整体柱研究进展

从分子印迹毛细管电色谱整体柱的基本原理入手,介绍了毛细管的预处理方法;讨论了色谱柱制备过程中,印迹分子、功能单体、交联剂、引发剂、溶剂的选择以及比例的影响;比较了光引发及热引发两种聚合方式的特点;阐述了聚合时间、聚合温度的控制;并探讨了色谱分析过程中检测电压、检测温度、流动相组成等操作条件的影响。最后介绍了分子印迹毛细管电色谱整体柱领域一些新的研究方向,并对其今后的发展进行了展望。     

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.     

S-citalopram imprinted monolithic columns for capillary electrochromatography enantioseparations

In this study, the molecular imprinting method was used to separate enantiomeric forms of chiral antidepressant drug, R,S-citalopram (R,S-CIT) in aqueous solution by CEC system combining the advantages of capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). For that, an amino acid-based molecularly imprinted monolithic capillary column was designed and used as a stationary phase for selective separation of S-citalopram (S-CIT) for the first time. S-CIT was selectively separated from the aqueous solution containing the other enantiomeric form of R-CIT, which is the same in size and shape as the template molecule. Morphology of the molecularly imprinted (MIP S-CIT) and non-imprinted (NIP S-CIT) monolithic capillary columns was observed by scanning electron microscopy. Imprinting efficiency of MIP S-CIT monolithic capillary column used for selective S-CIT separation was verified by comparing with NIP S-CIT and calculated imprinting factor (I.F:1.81) proved the high selectivity of the MIP S-CIT for S-CIT. Cavities formed for S-CIT form enabled selective (α = 2.08) separation of the target molecule from the other enantiomeric R-CIT form. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 7.68 × 10⁻⁶ m²/Vs for R,S-CIT at pH 7.0 10 mM PB and 50% ACN ratio. The performance of both MIP S-CIT and NIP S-CIT columns was estimated by repeating the R,S-CIT separations with intra-batch and inter-batch studies for reproducibility of retention times of R,S-CITs. Estimated RSD values that are lower than 2% suggest that the monolithic columns separate R,S-CIT enantiomers without losing separation efficiency.     

丙基脲硅胶毛细管整体柱的制备及性能研究

采用溶胶-凝胶技术在毛细管中原位合成硅胶整体柱,通过表面化学修饰技术制备了极性的丙基脲硅胶整体柱.对所制备的整体柱柱性能进行了评价,考察了极性物质在该整体柱上的保留行为,并对其可能的保留机理进行了探讨.研究表明,该柱在亲水作用电色谱模式下能有效分离苯酚类极性小分子化合物.     

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.     

Chiral separation of binaphthol enantiomers on molecularly imprinted polymer monolith by capillary electrochromatography.

A novel enantioseparational monolithic stationary phase for binaphthol based on a molecular imprinting method was introduced and evaluated in capillary electrochromatography (CEC). The monolithic stationary was prepared by the in situ copolymerization of methacrylic acid and ethylene glycol dimethacrylate in a porogenic solvent (toluene or toluene-isooctane) in the presence of an imprinting molecule, (R)-1,1'-bi-2,2'-naphthol. Such stationary phases could separate the enantiomers of binaphthol. The influence of several parameters on the column permeability was investigated. These parameters included the polymerization time, the molar ratio of the functional monomer to the imprinting molecule and the content of porogen. The influence of the polymerization condition and the electrochromatographic parameters on the enantiomer separation was also studied. Initial studies showed that a higher molecular ratio of the imprinted molecule to the functional monomer, a higher content of porogen, a higher content of acetonitrile, a higher pH, as well as the addition of Tween 20, gave a higher enantiomer selectivity.     

Open tubular layer of S‐ofloxacin imprinted polymer fabricated in silica capillary for chiral CEC separation

An open tubular molecule imprinted polymer (OT‐MIP) capillary column has been prepared for chiral separation of ofloxacin enantiomers in CEC. The S‐ofloxacin imprinted OT column was fabricated by thermally initiated non‐covalent polymerization procedure inside a pretreated and silanized fused silica capillary. The template molecule was incorporated with methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA) and 4‐styrenesulfonic acid (4‐SSA) and dissolved in a porogen mixture of ACN/2‐propanol (9:1). The separation efficiency of the 4‐SSA MIP column was found quite better than that of the MIP column without 4‐SSA. It has been demonstrated that our OT‐MIP column can separate ofloxacin enantiomers with excellent chiral separation efficiency after tuning the various chromatographic conditions. The optimized chromatographic eluent was 85:15, v/v%, ACN/60 mM sodium acetate at pH 7. The separation efficiency and selectivity of chiral separation of this study were far better than those obtained by previous methods for chiral separation of R‐ and S‐ofloxacin.     

Preparation of an open-tubular capillary column with a monolithic layer of S-ketoprofen imprinted and 4-styrenesulfonic acid incorporated polymer and its enhanced chiral separation performance in capillary electrochromatography

Enhanced chiral separation performance has been observed for ketoprofen enantiomers in capillary electrochromatography (CEC) with an open-tubular (OT) column prepared with a specific molecule imprinted polymer (MIP) on the innerwall of 50mum ID capillary. The column was prepared by in situ thermal polymerization inside the pretreated and silanized fused silica capillary. A specific diluted monomer mixture composed of S-ketoprofen, methacrylic acid (MAA, functional monomer), ethylene glycol dimethacrylate (EDMA, cross-linker), and 4-styrenesulfonic acid (4-SSA) dissolved in 9/1 (v/v) acetonitrile/2-propanol was used to fabricate the OT-MIP layer. 4-SSA was added to form a MIP layer capable of stable and strong electro-osmotic flow (EOF) over the pH range of this study securing CEC elution of ketoprofen having partial negative charge near the optimized pH. Various parameters such as buffer pH, organic modifier composition, salt concentration, and applied potential have been optimized for CEC chiral separation of ketoprofen enantiomers. Very good separation selectivity and efficiency were observed, thus the chromatographic resolution of ketoprofen enantiomers was as high as 10.5, and the number of theoretical plates of R-ketoprofen, 156,000/m (40,000/m for S-ketoprofen), which proves that the OT-MIP-CEC type approach is a promising strategy in MIP study.     

电色谱模式下四肽印迹整体柱分子识别机理的研究

本文采用原位聚合法制备了以四肽YPLG为模板的毛细管分子印迹整体柱,在毛细管电色谱模式下以模板分子和它的结构类似物YPGL为样品,对分子印迹聚合物的识别机理进行了研究。这两种四肽由于化学结构相似且等电点非常相近,普通的电色谱和毛细管电泳方法分离非常困难。但我们的实验表明,印迹整体柱对模板分子具有特异性识别能力,因此YPLG与YPGL之间的分离因子为1.73,分离度达3.72。实验中系统地研究了流动相中有机溶剂的含量、缓冲溶液的pH值、缓冲溶液的盐浓度以及柱温对四肽识别的影响。实验中我们观察到模板在印迹柱上具有非线性的Van’t Hoff行为,揭示可能存在多重保留机理。本研究结果表明,在毛细管电色谱模式下,分子印迹整体柱的分子识别主要决定于样品与印迹聚合物之间的氢键作用以及印迹孔穴的三维结构。     

Molecularly Imprinted Polymer Monolithic Column Separation of Isomers and Analogues of Vanillin by Capillary Electrochromatography

A vanillin imprinted capillary monolithic column was synthesized by in situ polymerization reaction using ethylene-glycol dimethacrylate as cross-linking monomer and methacrylic acid as functional monomer. Under the optimum conditions of capillary electrochromatography, this molecularly imprinted polymer （MIP）-based column showed high selectivity and could recognize not only template molecule vanillin but also positional isomer o-vanillin from their structural analogues.     

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.     

Growth of two-layer copolymer as the stationary phase with very high separation efficiency for separating peptides in capillary electrochromatography

Open tubular CEC (OT-CEC) column with a very high separation efficiency was prepared for peptides separation. A pretreated silica-fused capillary was reacted with 3-(methacryloxy) propyltrimethoxysilane followed by vinylbenzyl chloride and divinylbenzene to produce first thin monolithic monolayer. The second copolymer layer was formed on thin monolithic monolayer of the capillary by reversible addition-fragmentation transfer polymerization of N-phenylacrylamide and styrene. The key parameters including buffer pH value and organic modifier were systematically evaluated to provide the optimal chromatographic condition. The resultant OT-CEC columns were validated by separating a synthetic mixture of peptides and cytochrome C tryptic digest in capillary electrochromatography. The number of theoretical plates as high as 2.4 million per column was achieved for synthetic mixture peptides. In addition, the fabricated OT-CEC column also resolved more than 18 high-efficiency digestion peptides from a mixture containing tryptic digest of cytochrome C. The column to column and inter- to intraday repeatabilities of OT-CEC column through RSD% were found better than 3.0%, exhibiting satisfactory stability and repeatability of the two-layer deposited OT-CEC column. The results reveal that the open tubular capillary column modified with two-layer copolymer shows the great prospect for the separation of proteins in capillary electrochromatography.