Fast separations of chiral β-blockers on a cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia monolithic column by capillary electrochromatography

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) is an excellent chiral selector for enantioseparation of a wide variety of chiral compounds. The monolithic chiral columns are becoming popular in liquid chromatography and capillary electrochromatography. In this work, we present the fast separation of chiral β-blockers on a CDMPC-modified zirconia monolithic column by capillary electrochromatography (CEC). The porous zirconia monolithic capillary column was prepared by using the sol-gel technology and then zirconia surface modified with CDMPC. The enantioseparations were performed in reversed-phase (RP) eluents of a phosphate solution (pH 4.4) modified with acetonitrile or alcohol. The enantioseparations of a set of eight chiral β-blockers were achieved in less than one minute. Influences of the applied voltage, column temperature, concentration of acetonitrile and the type of alcohol as the organic modifier in the mobile phase, and sample injection time on enantioseparation were investigated. CEC separations at the applied voltage of 10 kV and 15 °C in the ACN-modified mobile phase provided the best resolutions for the analytes studied. Run-to-run and day-to-day repeatabilities of the column in the RP-CEC separation were less than 1 and 2%, respectively.     

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.     

Chemically modified chiral monolithic silica column prepared by a sol-gel process for enantiomeric separation by micro high-performance liquid chromatography

In this work a new type of chiral monolith silica column was developed for the chiral separation by micro high-performance liquid chromatography (micro-HPLC). The chiral monolith column with a continuous skeleton and a large through-pore structure was prepared inside a capillary of 100 microm I.D. by a sol-gel process, and chemically modified with chiral selectors, such as L-phenylalaninamide, L-alaninamide and L-prolinamide, on the surface of the monolithic silica column. Based on the principle of ligand exchange, these chiral monolithic columns were successfully used for the separation of dansyl amino acid enantiomers, as well as hydroxy acid enantiomers by micro-HPLC. The chromatographic conditions, the enantioselectivity and the performance of columns are discussed.     

Chiral covalent organic framework incorporated organic polymer monolithic capillary column for enantioseparations

A chiral covalent organic framework was synthesized, characterized, and incorporated into organic polymer monolithic capillary columns to provide chiral stationary phases for enantioseparations. The prepared monolithic capillary columns were characterized by scanning electron microscopy and elemental analysis. To obtain better enantioseparations, the columns’ preparation conditions, and enantioseparation conditions were optimized. Baseline resolutions of several chiral compounds were obtained with good reproducibility and stability. Furthermore, the mechanism of chiral recognition was investigated using molecular docking with AutoDock. Docking results showed that the enantioselectivity factor rather than resolution is correlated with the binding free energy difference between enantiomers with the chiral covalent organic framework. And abundant acetoxy and nitrile groups as well as benzene rings in the chiral covalent organic framework are responsible for the enantioseparation ability of the chiral monolithic capillary columns.     

含多面体低聚倍半硅氧烷杂化毛细管整体柱的制备及修饰

本文采用自由基聚合法原位制备了两种杂化毛细管整体柱。首先以含有一个甲基丙烯酸基团的多面体低聚倍半硅氧烷(POSS)试剂(Bu-POSS)为单体、以含有多个甲基丙烯酸基团的POSS试剂(POSS-MA)为交联剂在二元致孔剂(正丙醇/聚乙二醇400)和引发剂(偶氮二异丁腈)存在下发生热引发聚合,在毛细管中形成聚(Bu-POSS-co-POSS-MA)杂化整体柱;另外仅以POSS-MA为单体在相同条件下制备聚(POSS-MA)杂化整体柱,并将这两种杂化整体柱应用于小分子的毛细管液相色谱(cLC)分析。结果表明,含POSS杂化整体柱具有制备简单、重现性好以及稳定性高的特点。此外,利用聚(POSS-MA)杂化整体柱表面剩余的甲基丙烯酸基团,可以将功能单体(甲基丙烯酸硬脂酸酯等)化学键合到整体柱上,不但可以提高色谱柱效,而且使其具有不同的选择性。本文所发展的以POSS试剂为原料采用自由基聚合法制备杂化整体柱的方法为新型杂化整体柱的制备提供了一种新思路。     

Photografted methacrylate‐based monolithic columns coated with cellulose tris(3,5‐dimethylphenylcarbamate) for chiral separation in CEC

A chiral capillary monolithic column for enantiomer separation in capillary electrochromatography was prepared by coating cellulose tris(3,5‐dimethylphenylcarbamate) on porous glycidyl methacrylate‐co‐ethylene dimethacrylate monolith in capillary format grafted with chains of [2(methacryloyloxy)ethyl] trimethylammonium chloride. The surface modification of the monolith by the photografting of [2(methacryloyloxy)ethyl] trimethylammonium chloride monomer as well as the coating conditions of cellulose tris(3,5‐dimethylphenylcarbamate) onto the grafted monolithic scaffold were optimized to obtain a stable and reproducible chiral stationary phase for capillary electrochromatography. The effect of organic modifier (acetonitrile) in aqueous mobile phase for the enantiomer separation by capillary electrochromatography was also investigated. Several pairs of enantiomers including acidic, neutral, and basic analytes were tested and most of them were partially or completely resolved under aqueous mobile phases. The prepared monolithic chiral stationary phases exhibited a good stability, repeatability, and column‐to‐column reproducibility, with relative standard deviations below 11% in the studied electrochromatographic parameters.     

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.     

纤维素键合型手性整体柱的制备及其在快速手性分离中的应用

采用N-丙烯酰琥珀酰亚胺(NAS)为基质单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,原位聚合制备聚(NAS-co-EDMA)毛细管整体柱,并通过化学键合法将自合成的纤维素-三(4-甲基苯甲酸酯)(CTMB)共价键合到整体柱上,制备用于快速手性分离的纤维素键合型手性整体柱.优化了整体柱制备和衍生化条件;通过对固定相红...     

Repeatability in column preparation of a reversed-phase C18 monolith and its application to separation of tocopherol homologues

This work investigated the repeatability of column preparation for a reversed-phase C18 monolith, namely stearyl methacrylate-co-ethylene glycol dimethacrylate (SMA-EDMA). The columns were thermally polymerised using three commonly available heating devices (GC oven, hot air oven and water bath) and their chromatographic performance evaluated using micro-liquid chromatography for separation of five test compounds. Precision in terms of %RSD of retention times were 9.0, 6.5, and 12.5 using GC oven, hot air oven and water bath, respectively. Between-batch precision for the hot air oven (n = 3 days) was less than 10.4% for retention time. The SMA-EDMA monolith was applied to the separation of tocopherol homologues by capillary electrochromatography. Usually tocopherol homologues cannot be completely separated by conventional reversed-phase C8- or C18-packed bed or C18-silica based monolithic columns. Polymer monolith has been shown to give remarkable selectivity towards the tocopherols compared to the conventional microparticulate phase and silica based monolith. Successful separation of the tocopherol isomers was achieved on the SMA-EDMA monolith without any column modification.     

胃蛋白酶亲和有机聚合物毛细管整体柱的制备及性能考察

以热引发原位聚合方法制备了聚（甲基丙烯酸缩水甘油酯（glycidyl methacrylate,GMA）-乙二醇二甲基丙烯酸酯（ethyleneglycol dimethacrylate,EDMA））毛细管整体柱,对整体柱的性能进行了表征。结果表明,柱内部结构均匀、渗透性好;整体柱能够实现苯等中性小分子化合物的分离,具有反相色谱特征,重现性和稳定性良好。利用整体柱环氧基团的活性,采用间接法,以戊二醛为连接臂制备胃蛋白酶亲和手性整体柱。在毛细管电色谱模式下进行了柱分离性能研究,并对缓冲液pH值和运行电压等分离条件进行了考察。结果表明,亲和整体柱对4种碱性手性药物（奈福泮、氨氯地平、西酞普兰、扑尔敏）有拆分效果,奈福泮、氨氯地平、西酞普兰能达到基线分离。本文为蛋白质亲和毛细管电色谱整体柱的制备和应用提供了新的思路和方法。     

Evaluation of a methacrylate-bonded cyclodextrins as a monolithic chiral stationary phase for capillary electrochromatography (CEC)-UV and CEC coupled to mass spectrometry

Glycidyl methacrylate-bonded β-cyclodextrin (GMA-β-CD) is synthesized as a new chiral monomer by direct chemical bonding with GMA using a fast and simple alternative procedure. Next, rigid and homogenous monolithic columns were prepared by polymerization of GMA-β-CD monomer with ethylene dimethacrylate (EDMA), in the presence of commonly used porogens and a charged achiral monomer to form a versatile chiral monolith. This is the first report in which a preparation procedure for a methacrylate-bonded CD is introduced for chiral separations in CEC. The degree of substitution of GMA-β-CD monomer and mobile-phase parameters were optimized to achieve the highest enantioselectivity and plate number. To evaluate the GMA-β-CD monolithic column, different classes of chiral compounds were screened. Under the optimized β-CD monolith phase and the optimum mobile-phase conditions, 30 neutral and basic chiral compounds and two acidic compounds could be separated. The high chemical and mechanical stability, homogenous microflow and no loss of material at the interface allows for the first time the feasibility of applying this polymer-based monolithic column for CEC coupled to ESI-MS. Compared with CEC-UV, CEC-ESI-MS showed higher sensitivity and lower resolution. However, resolution greater than 1.0 can still be obtained for majority of the select tested compound in CEC-ESI-MS with at least three out of seven compound providing Rs≥1.5. The results reinforce the potential of GMA-β-CD monolithic columns for chiral separations with high sensitivity in CEC-ESI-MS. Finally, using hexobarbital as the model chiral analyte, the monolithic column demonstrated excellent stability and reproducibility of retention time and enantioselectivity.     

Pepsin‐modified chiral monolithic column for affinity capillary electrochromatography

Pepsin‐modified affinity monolithic capillary electrochromatography, a novel microanalysis system, was developed by the covalent bonding of pepsin on silica monolith. The column was successfully applied in the chiral separation of (±)‐nefopam. Furthermore, the electrochromatographic performance of the pepsin‐functionalized monolith for enantiomeric analysis was evaluated in terms of protein content, pH of running buffer, sample volume, buffer concentration, applied voltage, and capillary temperature. The relative standard deviation (%RSD) values of retention time (intraday <0.53, n = 10; interday <0.53, n = 10; column‐to‐column <0.70, n = 20; and batch‐to‐batch <0.80, n = 20) indicated satisfactory stability of these columns. No appreciable change was observed in retention and resolution for chiral recognition of (±)‐nefopam in 50 days with 100 injections. The proteolytic activity of this stationary phase was further characterized with bovine serum albumin as substrate for online protein digestion. As for monolithic immobilized enzyme reactor, successive protein injections confirmed both the operational stability and ability to reuse the bioreactor for at least 20 digestions. It implied that the affinity monolith used in this research opens a new path of exploring particularly versatile class of enzymes to develop enzyme‐modified affinity capillary monolith for enantioseparation.     

Effect of fabrication strategy on the enantioseparation performance of β‐cyclodextrin‐functionalized polymethacrylate monoliths: A comparative evaluation

To evaluate the effect of the preparation strategy on the enantioseparation performance of β‐cyclodextrin‐functionalized monoliths, a series of β‐cyclodextrin‐functionalized organic polymeric monolithic columns were prepared through two‐step, single‐step, and one‐pot approaches, using the same cyclodextrin, linker–spacer, and crosslinker. Physicochemical characterization of the columns was carried out by determining the morphology, β‐cyclodextrin density, permeability, and chromatographic efficiency. For each type of monolithic column, the enantioresolution of 22 chiral compounds, including mandelic acid derivatives, nonsteroidal anti‐inflammatory drugs, N‐derivatized amino acids, and herbicides, was comparatively studied under optimum chromatographic conditions. The β‐cyclodextrin‐functionalized monolithic columns prepared through the one‐pot approach exhibited higher enantioresolution for most chiral compounds, and they have the advantage of good controllability and simple preparation. On the other hand, the enantioresolution obtained on columns prepared through the single‐step approach was quite unsatisfactory, and therefore the effect of using different linking spacers and crosslinkers was studied. A significant improvement of enantioresolution for 2‐chloro‐mandelic acid was obtained by using N ,N‐methylenebisacrylamide instead of ethylene dimethacrylate as the crosslinker in the single‐step preparation.     

Enantiomer separation by capillary electrochromatography on a cyclodextrin-modified monolith

A chiral monolithic stationary phase was prepared by packing a capillary with bare porous silica and sintering the silica bed at high temperature. The resulting silica monolith was polymer-coated with Chirasil-Dex, a permethylated beta-cyclodextrin covalently linked via an octamethylene spacer to dimethylpolysiloxane. Subsequently, Chirasil-Dex was thermally immobilized on the silica support and a chiral monolith of very high stability (30 kV, more than 400 bar pressure) was obtained. The enantiomer separation of various chiral compounds by monolithic (rod) capillary electrochromatography (rod-CEC) was feasible. This method was compared with capillary liquid chromatography (LC) in a single-column mode using unified equipment. About two to three times higher efficiency was found in the rod-CEC mode as compared to rod-LC. The influence of pressure-driven flow support on efficiency, resolution, elution time and baseline stability was investigated. The amount and nature of organic modifier strongly influences efficiency and resolution.     

Preparation of cyclodextrin‐modified monolithic hybrid columns for the fast enantioseparation of hydroxy acids in capillary liquid chromatography

Cyclodextrins and their derivatives are one of the most common and successful chiral selectors. However, there have been few publications about the use of cyclodextrin‐modified monoliths. In this study, organic hybrid monoliths were prepared by the immobilization of derivatized β‐cyclodextrin alone or with l‐ 2‐allylglycine hydrochloride to the polyhedral oligomeric silsesquioxane methacryl substituted monolith. The main topic of this study is a combined system with dual chiral selectors (l‐ 2‐allylglycine hydrochloride and β‐cyclodextrin) as monolithic chiral stationary phase. The effect of l‐ 2‐allylglycine hydrochloride concentration on enantioseparation was investigated. The enantioseparation of the four acidic compounds with resolutions up to 2.87 was achieved within 2.5 min on the prepared chiral monolithic column in capillary liquid chromatography. Moreover, the possible mechanism of enantioseparation was discussed.     

A silica monolithic column prepared by the sol-gel process for enantiomeric separation by capillary electrochromatography

A method for the preparation of a silica monolithic capillary electrochromatography (CEC) column for the separation of enantiomers has been developed. The porous silica monolith was fabricated inside a fused-silica capillary column by using the sol-gel process. After gelation for 24 h, hydrothermal treatment at 100 degrees C for 24 h was performed to prevent the sol-gel matrix from cracking. The prepared monolith was then coated with Chirasil-beta-Dex which represents a chiral polymer prepared by grafting permethyl-beta-cyclodextrin to polymethylsiloxane with an octamethylene spacer. Immobilization of Chirasil-beta-Dex was performed by heat treatment at 120 degrees C for 48 h to give a nonextractable coating. The column performance was evaluated by using racemic hexobarbital as a model compound. The efficiency of 9.2 x 10(4) theoretical plates/m for the first eluted enantiomer of hexobarbital was obtained at an optimal flow rate of the mobile phase. The effect of mobile phase composition on enantiomeric separation of hexobarbital was also investigated. The column proved to be stable for more than one hundreds of runs during a two-months period. The enantiomers of several neutral and negatively charged chiral compounds were baseline separated on this column.     

β-环糊精衍生物/Cu_2O毛细管电色谱整体柱的制备及其应用

采用一步键合法制备了烯丙胺-β-环糊精/Cu_2O(Ally-β-CD/Cu_2O)毛细管电色谱整体柱,并考察了制备整体柱的主要影响因素。通过扫描电子显微镜(SEM)、X-射线光电子能谱(XPS)、X-射线衍射(XRD)对整体柱柱内固定相进行表征。以硫脲为中性标记物测定了柱效,柱效达到47 658 N/m。对D,L-组氨酸对映体的分离度RS达到3.82,整体柱在连续使用72h或间歇使用2个月后仍具有良好的分离能力,表明整体柱具有良好的重现性和稳定性,同时具备较好的手性拆分能力。运用该整体柱对盐酸克伦特罗对映体进行了拆分,达到基线分离,分离度达到2.89。     

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.     

Methacrylate monolithic columns of 320 microm I.D. for capillary liquid chromatography

Monolithic capillary columns (320 microm I.D.) were prepared for capillary liquid chromatography (CLC) by radical polymerization of butylmethacrylate (BMA) and ethylenedimethacrylate (EDMA) in the presence of a porogen solvent containing propan-1-ol, butane-1,4-diol and water. The influence of the contents of the porogen solvent and EDMA in the polymerization mixture on the monolith porosity and column efficiency was investigated. The composition of the polymerization mixture was optimized to attain a minimum HETP of the order of tens of microm for test compounds with various polarities. The separation performance and selectivity of the most efficient monolithic column prepared was characterized by van Deemter curves, peak asymmetry factors and Walters hydrophobicity and silanol indices. It was demonstrated that the 320-microm I.D. monolithic column exhibited CLC separation performance similar to that observed for 100- and 150-microm I.D. monolithic columns reported in the literature; moreover, the 320-microm I.D. column was easier to operate in CLC and exhibited a higher sample loadability.     

Synthesis of zirconia monoliths for chromatographic separations

The aim of this work is to join the advantages of two different kinds of stationary phases: monolithic columns and zirconia-based supports. On the one hand, silica monolithic columns allow a higher efficiency with a lower back-pressure than traditional packed columns. On the other hand, chromatographic stationary phases based on zirconia have a higher thermal and chemical stability and specific surface properties. Combining these advantages, a zirconia monolith with a macroporous framework could be a real improvement in separation sciences. Two main strategies can be used in order to obtain a zirconia surface on a monolithic skeleton: coating or direct synthesis. The coverage by a zirconia layer of the surface of a silica-based monolith can be performed using the chemical properties of the silanol surface groups. We realized this coverage using zirconium alkoxide and we further grafted n-dodecyl groups using phosphate derivatives. Any loss of efficiency was observed and fast separations have been achieved. The main advance reported in this paper is related to the preparation of zirconia monoliths by a sol-gel process starting from zirconium alkoxide. The synthesis parameters (hydrolysis ratio, porogen type, precursor concentration, drying step, etc.) were defined in order to produce a macroporous zirconia monoliths usable in separation techniques. We produced various homogeneous structures: zirconia rod 2 cm long with a diameter of 2.3 mm, and zirconia monolith inside fused silica capillaries with a 75 microm I.D. These monoliths have a skeleton size of 2 microm and have an average through pore size of 6 microm. Several separations have been reported.