Silica sol-gel/organic hybrid material for protein encapsulated column of capillary electrochromatography

A new-type of sol-gel/organic hybrid composite material using gelatin or chitosan with tetramethoxysilane was developed for the bovine serum albumin (BSA)-encapsulated monolithic column for capillary electrochromatography (CEC). The composite monolith was used to immobilize BSA in a fused-silica capillary. The addition of gelatin and chitosan to the alkoxysilane enabled the enantioseparation of Trp. A very small amount of these polymers were effective for the enantioseparation. Especially, the monolithic column prepared from chitosan with tetramethoxysilane showed a high enantioselectivity for Trp enantiomers and the value (alpha' = t2/t1, t1: fast eluted enantiomer, t2: second eluted enantiomer) reached 1.15 on CEC mode. Furthermore, the composite materials exhibited a higher stability compared to the silica sol-gel column. These results showed that the sol-gel/organic hybrid composite was useful as a monolithic matrix for the BSA-encapsulated column for CEC.     

Preparation and characterization of a polystyrene/bovine serum albumin nanoparticle‐coated capillary for chiral separation using open‐tubular capillary electrochromatography

Polystyrene (PS) nanoparticles coated by BSA, hereafter denoted as PS/BSA, were prepared and chemically immobilized for the first time onto a capillary inner wall for open‐tubular CEC (OTCEC). EOF and scanning electron micrography were used to characterize the prepared nanoparticle‐coated capillaries. To investigate the performance of the prepared columns in OTCEC, chiral separation of d ,l ‐tryptophan (dl ‐Trp) was performed in monolayer BSA‐modified capillary and PS/BSA nanoparticle‐coated columns. The results indicated that the nanoparticle‐modified column afforded a higher resolution compared with the monolayer type. Rapid enantioseparation of dl ‐Trp (within 3 min) was achieved with the PS/BSA‐immobilized column using an electroosmotic pump‐assisted CEC. Enantiomer separations of other compounds like dl ‐tyrosine and warfarin were also achieved with the column. Besides, run‐to‐run and column‐to‐column repeatabilities of the PS/BSA‐coated column in the chiral separation were systematically introduced.     

A mesoporous silica nanoparticles immobilized open-tubular capillary column with a coating of cellulose tris(3,5-dimethylphenyl-carbamate) for enantioseparation in CEC

An approach of immobilizing mobile crystalline material (MCM)-41 mesoporous silica nanoparticles on the inner wall of an open-tubular (OT) capillary as the support for coating chiral selector of cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) was carried out. By taking advantage of the improved phase ratio of OT capillary with the immobilization of MCM-41 mesoporous material, the cellulose derivative of CDMPC as the chiral selector was simply coated on the MCM-41 nanoparticle layer via the hydrogen-bonding interaction, and the enantioseparation was successively carried out. Eight pairs of acidic, neutral and basic enantiomers were resolved in capillary electrochromatography or capillary liquid chromatography mode. The concentration of CDMPC for coating was systematically investigated to obtain the optimized chromatographic properties on enantioseparation by controlling the supposed film thickness of CDMPC on MCM-41 nanoparticle layer. Comparing with a bare fused silica capillary column coated with CDMPC under the same coating procedure as MCM-41-modified capillary did, the MCM-41-modified capillary column offered much higher enantioselectivity. This result indicated the significance of using the mesoporous nanoparticles as the electrochromatographic support to enhance the phase ratio of OT capillary column in capillary electrochromatography and capillary liquid chromatography. For investigating the effect of experimental conditions on the enantioseparation with this prepared OT capillary column, the content of organic modifier acetonitrile in the mobile phase was thus extensively evaluated to achieve a better chiral separation.     

金纳米粒子修饰的手性毛细管电色谱固定相的制备及性能表征

制备了粒径为15 nm的金纳米粒子(GNPs)并将其修饰到氨基衍生化的硅胶整体柱内,通过化学键合法将牛血清白蛋白(BSA)固载到GNPs的表面作为手性固定相。通过透射电子显微镜、扫描电子显微镜等方法进行表征,结果表明,GNPs分散性良好,并被成功地修饰到毛细管柱内,含量高达17.18%。优化了BSA手性柱的制备条件,最终确定了体积分数为10%的3-氨丙基三乙氧基硅烷(APTES)和15 g/L BSA为最佳反应条件。在毛细管电色谱分离模式下,对缓冲液pH值、电压等分离条件进行了考察,最终选择了10 mmol/L pH 7.4的磷酸缓冲液和15 kV运行电压作为最佳分离条件。手性柱对3种手性化合物(色氨酸、阿替洛尔和麻黄碱)有拆分效果,对色氨酸能实现基线分离。与物理吸附法相比,化学键合法制备的手性柱拆分效果好,分析物无需柱前衍生化,且色谱柱稳定性良好。该文的制备方法也为其他类型手性选择剂的引入提供了良好的思路。     

Enantioselective open-tubular capillary electrochromatography using cyclodextrin-modified gold nanoparticles as stationary phase

Enantioselective open-tubular CEC (OTCEC) with thiolated β-CD modified gold nanoparticles (CD-GNPs) as stationary phase was developed. The enantioselective OT capillary column was fabricated by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by self-adsorption of negatively charged CD-GNPs. The enantioselective capillary column has a steady EOF mobility over a wide pH range of 3.0 to 9.2 (RSD 4.8%), and is quite stable over 240 min with very good column to column reproducibility. Efficient enantioseparation of the presented method was demonstrated by analyzing three drug enantiomers. Our results show that the column exhibits good run-to-run repeatability for enantioseparations and can maintain the enantioselectivity for more than 1 month if the column was stored in CD-GNPs solution at 4 °C.     

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。     

Affinity capillary electrophoresis coupling with partial filling technique and field‐amplified sample injection for enantioseparation and determination of DL‐tetrahydropalmatine

A novel, simple and sensitive method for the enantioseparation and determination of DL ‐tetrahydropalmatine (DL ‐THP) was developed using ACE in combination with partial filling technique and field‐amplified sample injection. A chiral selector, i.e. BSA, was used for the enantioseparation of DL ‐THP in ACE. Effects of BSA concentration, pH and separation voltage on the effectiveness of the enantiomer separation were evaluated. In an optimal condition, D ‐ and L ‐THP were completely enantio‐separated in less than 9 min by partially filling an electrophoretic capillary with 50 μmol/L BSA (50 mbar, 100 s) and carrying out an electrophoresis with 20 mmol/L phosphate buffer (pH 7.4) at 15 kV. The sensitivity was further improved by making use of field‐amplified sample injection to lower the LOD (defined as S/N=3) down to 6 ng/mL. Real samples were also tested and promising results for the determination of DL ‐THP enantiomers were obtained.     

Preparation of a thiols β‐cyclodextrin/gold nanoparticles‐coated open tubular column for capillary electrochromatography enantioseparations

Inspired by the distinct chemical and physical properties of nanoparticles, here a novel open‐tubular capillary electrochromatography column was prepared by electrostatic assembly of poly(diallydimethylammonium chloride) onto the inner surface of a fused‐silica capillary, followed by self‐adsorption of negatively charged SH‐β‐cyclodextrin/gold nanoparticles. The formation of the SH‐β‐cyclodextrin/gold nanoparticles coated capillary was confirmed and characterized by scanning electron microscopy and energy dispersive spectrometry. The results of scanning electron microscopy and energy dispersive spectrometry studies indicated that SH‐β‐cyclodextrin/gold nanoparticles were successfully coated on the inner wall of the capillary column. The performance of the SH‐β‐cyclodextrin/gold nanoparticles coated capillary was validated by the analysis of six pairs of chiral drugs, namely zopiclone, carvedilol, salbutamol, terbutaline sulfate, phenoxybenzamine hydrochloride, and ibuprofen. Satisfactory enantioseparation results were achieved, confirming the use of gold nanoparticles as the support could enhance the phase ratio of the open‐tubular capillary column. Additionally, the stability and reproducibility of the SH‐β‐cyclodextrin/gold nanoparticles coated capillary column were also investigated. Then, this proposed method was well validated with good linearity (≥0.999), recovery (90.0–93.5%) and repeatability, and was successfully used for enantioseparation of ibuprofen in spiked plasma samples, which indicated the new column's potential usage in biological analysis.     

High-performance liquid chromatographic enantioseparations on capillary columns containing monolithic silica modified with cellulose tris(3,5-dimethylphenylcarbamate)

Monolithic capillary columns containing native silica gel were modified with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) and used for enantioseparations in capillary liquid chromatography. The method adopted for in situ enantioselective modification of monolithic fused silica capillary columns by coating with CDPMC appears to be fairly simple and fast. High efficiency enantioseparations of test racemic compounds and s(everal chiral drugs were achieved in a short time. It was possible to increase the amount of chiral selector present by multiple coating of monoliths with CDMPC. The baseline enantioseparation of 2,2,2-trifluoro-1-(9-anthryl)ethanol was achieved in an analysis time less than 30 s with this capillary column. In addition, reproducible enantioseparations were obtained when the chiral selector was removed from the monolithic column by flushing it with appropriate solvent and the column recoated.     

Enantioseparation on cellulose dimethylphenylcarbamate-modified zirconia monolithic columns by reversed-phase capillary electrochromatography

This work reports the preparation of monolithic zirconia chiral columns for separation of enantiomeric compounds by capillary electrochromatography (CEC). Using sol–gel technology, a porous monolith having interconnected globular-like structure with through-pores is synthesized in the capillary column as a first step in the synthesis of monolithic zirconia chiral capillary columns. In the second step, the surface of the monolith is modified by coating with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) as the chiral stationary phase to obtain a chiral column (CDMPCZM). The process of the preparation of the zirconia monolithic capillary column was investigated by varying the concentrations of the components of the sol solution including polyethylene glycol, water and acetic acid. CDMPCZM is mechanically stable and no bubble formation was detected with the applied current of up to 30 μA. The enantioseparation behavior of the CDMPCZM columns was investigated by separating a set of 10 representative chiral compounds by varying the applied voltage and pH and organic composition of the aqueous organic mobile phases.     

Cyclodextrin-NH-MIL-53 open tubular stationary phase for capillary electrochromatography enantioseparation

A novel chiral group functionalized metal-organic framework, Cyclodextrin-NH-MIL-53, was synthesized and modified on the inner wall of a capillary column via a post-synthetic process. The prepared chiral metal-organic framework was utilized as a chiral capillary stationary phase and used in an open-tubular capillary electrochromatography method to enantioseparate several racemic amino acids. Excellent enantioseparation of five pairs of enantiomers was obtained in this chiral separation system (Resolutions of D/L-Alanine = 16.844, D/L-Cysteine = 3.617, D/L-Histidine = 9.513, D/L-Phenylalanine = 8.133, and D/L-Tryptophan = 2.778). The prepared Cyclodextrin-NH-MIL-53 and the Cyclodextrin-NH-MIL-53-based capillary columns were characterized by scanning electron microscopy, X-ray diffraction, Fourie-transform infrared spectroscopy, and circular dichroism. The chiral capillary electrochromatography conditions, such as separation conditions, amount of Cyclodextrin-NH-MIL-53, and electroosmotic flow, were optimized. This research is estimated to present a novel insight and method for the design and use of metal-organic framework-based capillaries for enantioseparation.     

一种简单的毛细管色谱柱电加热装置的制作方法及其在液相色谱-质谱联用系统中的应用

为了实现蛋白质组的深度覆盖,特别是低丰度蛋白质的定性鉴定和定量分析,目前常用的方法是采用更长或装填更小粒径填料的毛细管色谱柱,但因此带来的问题是色谱柱反向柱压显著升高。针对以上问题发展了一种简单的毛细管色谱柱电加热装置制作方法,并将该装置安装于液相色谱-质谱联用系统,分别以牛血清白蛋白(BSA)酶切肽段混合物和酵母蛋白(yeast)酶切肽段混合物为样品,从柱压和柱效两方面对该装置的性能进行了评价。实验结果表明,所制作的毛细管柱电加热装置安装在装填粒径为3 μm反相色谱填料的毛细管柱上,在最佳电流(100 mA)下对BSA及yeast酶切肽段混合物进行分离时的柱压比不加电流时的柱压降低至少50%,柱效略有升高。这说明所制作的毛细管色谱柱电加热装置能显著降低柱压,为在较低的柱压条件下选择更小粒径色谱颗粒填料的毛细管色谱柱提供了一种有效的方法。     

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...     

The application of functional silica nanoparticles to fulfill the rapid and improved enantioselective capillary electrophoresis separation of amino acid derivatives

In this study, diamino moiety functionalized silica nanoparticles with the size of 118 ± 12 nm were successfully synthesized and directly introduced into a chiral capillary electrophoresis system to improve the enantioseparation of 9‐fluorenyl methoxycarbonyl derivatized amino acids using norvancomycin as chiral selector. Under acidic background electrolyte conditions, functional silica nanoparticles can be readily adsorbed onto the inner surface of bare silica capillary column through electrostatic interaction to form a dynamic coating, resulting in a reversed anodic electro‐osmotic flow (i.e. from cathode to anode). As expected, chiral amino acid derivatives (usually negatively charged) can be rapidly separated under co‐electro‐osmotic flow conditions in the current separation system. Furthermore, the column performance and detection sensitivity for the enantioseparation were also obviously improved because the adsorption of chiral selector of norvancomycin to the capillary wall was greatly suppressed. Some important factors influencing the separation, such as the coating thickness, background electrolyte concentration, functional silica nanoparticles concentration, and the organic modifier were also investigated and the optimized separation conditions were obtained.     

新型毛细管内固相萃取-气相色谱法检测纺织品中烷基酚类物质

建立了毛细管内固相萃取(SPE)-气相色谱(GC)检测纺织品中壬基酚和辛基酚含量的分析方法。通过比较4种性质不同固相萃取剂的萃取效果,筛选出对烷基酚(APs)类物质萃取效果最佳的固相萃取剂,将其作为填充物质制作毛细管内固相萃取柱,将毛细管内固相萃取法与气相色谱联用进行分析检测。最佳固相萃取剂为Abselut NEXUS,毛细管内固相萃取最佳条件为:1.2 μL甲醇和1.2 μL超纯水活化,1.2 μL甲醇洗脱,上样速率是0.4 μL/min。该法在较低浓度范围内呈现良好的线性相关性,对烷基酚的富集倍数约为100倍,对辛基酚和壬基酚的检出限分别为3.7 μg/L和4.5 μg/L,加标回收率分别为85.6%~98.2%和83.8%~95.7%,结果表明,此法能够简捷、迅速、有效地检测出纺织品中残留的烷基酚类物质。     

Specific capture of phosphopeptides by Zr4+-modified monolithic capillary column

A method to prepare zirconium phosphate (ZrP)-modified monolithic capillary column for highly specific capture of phosphopeptides is presented. In this method, the phosphate monolithic capillary column was prepared by direct copolymerization of the functional monomer containing phosphate group (ethylene glycol methacrylate phosphate) and cross-linker (bis-acrylamide) in a ternary porogenic solvent. Copolymerization of cross-linker and functional monomer simplifies the procedure for the preparation of phosphate chromatographic media. After Zr4+ was immobilized, the ZrP-modified monolithic capillary column was evaluated by the analysis of standard phosphoproteins and the excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and BSA with molar ratio of 1:200.     

Sol‐gel technique for the preparation of β‐cyclodextrin gold nanoparticles as chiral stationary phase in open‐tubular capillary electrochromatography

A novel open‐tubular capillary electrochromatography column coated with β‐cyclodextrin was prepared using the sol‐gel technique. In the sol‐gel approach, owing to the three‐dimensional network of sol‐gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating enantiomers were shown. The influences of capillary inner diameter, coating time, organic modifier, buffer pH, and buffer concentration on separation were investigated. The sol‐gel‐coated β‐cyclodextrin column has shown improved enantioseparation efficiency of chlorphenamine, brompheniramine, pheniramine, zopiclone in comparison with the sol‐gel matrix capillary column. The migration time relative standard deviation of the separation of the enantiomers was less than 0.89% over five runs and 2.9% from column to column. This work confirmed that gold nanoparticles are promising electrochromatographic support to enhance the phase ratio of open‐tubular capillary electrochromatography column in capillary electrochromatography.     

Enantioseparation of cetirizine by sulfated-beta-cyclodextrin-mediated capillary electrophoresis

A sulfated beta-cyclodextrin (sulfated beta-CD)-mediated capillary electrophoresis method is described for the enantioseparation of cetirizine using achiral cefazolin as an internal standard. The enantioseparation of the drug was performed in a borate buffer (5 mM, pH 8.7) with 1% sulfated beta-CD (w/v) as chiral selector at 10 kV. Several parameters affecting the separation were studied, including the pH and the concentration of borate buffer and chiral selector. Under optimized conditions, a baseline separation of two enantiomers was achieved in less than 7 min. Using cefazolin as an internal standard (IS), the linear range of the method for the determination of levocetirizine was over 1.0 to 50.0 microg/mL; the detection limit (signal-to-noise ratio = 3) of levocetirizine was 0.5 microg/mL. The method allowed the enantioseparation of cetirizine in bulk samples and enantiomeric purity evaluation of levocetirizine (R-enantiomer) in pharmaceutical tablets (Xyzal), and it was also found to be suitable for enantioseparation in human plasma.     

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.     

一种多通道匀浆装填毛细管色谱柱的新装置及应用

针对目前毛细管色谱柱装柱效率低、不同批次装填的毛细管色谱柱之间性能差异大的问题,我们发展了一种多通道匀浆装填毛细管色谱柱的新装置。该装置以液相色谱泵提供压力、采用磁力搅拌保持匀浆液均匀分散,一次可装填多达6根毛细管色谱柱。以牛血清白蛋白(BSA)的胰蛋白酶酶切肽段混合物为样本,选择峰容量、蛋白覆盖率、3个特定离子的保留时间以及毛细管色谱柱柱压为指标,在毛细管液相色谱-质谱联用系统上对装填的反相毛细管色谱柱的性能进行了评价。分别考察了一次装填的6根毛细管色谱柱、两次装填的12根毛细管色谱柱以及一次装填1根与一次装填6根毛细管色谱柱的性能及稳定性。实验结果表明:同一批次装填的6根毛细管色谱柱的性能相近;不同批次装填的12根毛细管色谱柱的峰容量和覆盖率没有明显的区别,但保留时间和毛细管色谱柱柱压的稳定性较差;一次装填1根和一次装填6根毛细管色谱柱柱性能的稳定性与两次分别装填6根毛细管色谱柱的稳定性相近,即采用本装置可显著提高毛细管色谱柱的装填效率且每次装填毛细管色谱柱的数量不会对柱性能产生影响。