Fabrication and characterization of open-tubular CEC modified with tentacle-type metal-chelating polymer chains

A novel stationary phase with tentacle-type metal-chelating polymer chains was fabricated for open-tubular CEC. The preparation procedure of the stationary phase included the synthesis of monomer, silanization of capillary inner wall, in situ polymerization, and metal complexation. The effects of initiator concentration and reaction time on the column capacity were investigated. To compare with the tentacle-type metal-chelating capillary column, a monolayer ligand-modified capillary was also prepared. Immobilized copper(II) capacity of the tentacle-type polymer stationary phase was nearly 900 times higher than that of the monolayer one. The electroosmotic mobility was examined for its dependence on pH as well as phosphate and ACN concentrations. The tentacle-type metal-chelating capillary with high ligand capacity has proven to afford better retention and resolution for the separation of phenylalanine, tryptophan, and tyrosine mixtures and three purine derivatives. The separation was considered to be effected by a combination of ligand exchange and electrophoretic mobility.     

Branched polyethyleneimine-bonded tentacle-type polymer stationary phase for peptides and proteins separations by open-tubular capillary electrochromatography

A novel tentacle-type polymer stationary phase covalently modified with branched polyethyleneimine (PEI) was developed for peptides and proteins separations by open-tubular CEC (OT-CEC). The preparation procedure included the silanization of capillary inner wall, in situ graft polymerization and PEI functionalization. A wrinkly polymer surface of multitudinous steric amine groups was evenly formed on the capillary inner wall, and anodic EOF could be gained within a wide pH range of 2.5-7.5. The electroosmotic mobility was examined for its dependence on pH as well as PEI concentrations. Good repeatability was gained with RSD for the migration time of EOF marker within 4.8% and satisfactory chemical stability was validated. Due to the existence of amine groups on the surface of tentacle-type polymer stationary phase, the silanol effect that occurs between the positively charged biomolecules and the silanols of the capillary column was greatly suppressed. Compared with a monolayer-coating capillary, seven enkephalin-related peptides were well resolved on the PEI-bonded column with high efficiencies. Favorable separations of peptides and proteins with high column efficiencies were obtained in 144,000-189,000 and 97,000-170,000 plates/m. Branched PEI-bonded tentacle-type polymer stationary phase has been proven to afford satisfactory retention and resolution of peptides and proteins.     

C18衍生的微米金颗粒的制备及其作为毛细管液相色谱和加压电色谱固定相的评价

制备了用于色谱的微米纯金颗粒并键合上十八烷基(C18)官能团;对其进行了扫描电镜、红外光谱、元素分析、氮气吸附分析等表征。测得衍生的金颗粒的粒径、孔径以及比表面积分别为3.5 μm、5.0 nm、49.0 m2/g;红外光谱表明C18官能团已键合在金颗粒表面上;衍生后的金颗粒的含碳量为0.56%。通过电填充法得到长度为36 cm(固定相填充长度为19 cm)、内径为100 μm的毛细管色谱柱。利用极端pH的流动相(80%甲醇,pH 1.0以及pH 12.0)冲洗该色谱柱140 h,比较冲洗前后分析物的保留因子,以考察色谱柱的耐酸耐碱性能。结果表明,冲洗前后分析物的保留因子没有明显的变化,说明该色谱柱有良好的耐酸耐碱性。在毛细管液相色谱模式下,用该柱分离尿嘧啶、苯、萘、2-甲基萘、苊以评价色谱柱的一般性能;在碱性条件下分离咖啡因、茶碱、洛贝林以测定色谱柱分离碱性物质的能力。其分离结果表明,该色谱柱的柱效超过了50000理论塔板/m,且色谱峰形较好。在毛细管加压电色谱模式下,施加+5 kV和~5 kV的电压均可以使苯甲酸和苯胺分离,但电场方向不同时,二者的出峰顺序不同。     

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.     

Preparation of stationary phases for open-tubular capillary electrochromatography using the sol-gel method

Capillary electrochromatography requires the deposition of a stationary phase inside the capillary. In this paper the sol-gel method is proposed for this purpose. The gels were prepared externally and injected into a fused-silica capillary, where anchorage to the capillary wall was possible through condensation reactions between the silanol groups of the capillary wall and the residual silanol groups the gel. Contrary to a commonly used practice, alkaline pretreatment of the inner capillary wall prior to the introduction and anchoring of the gel was found to be only marginally effective in improving the mechanical stability of the column. The influence of various parameters, such as the pH, the water content, the presence of alcohol (ethanol) on the formation of tetraethoxysilane (TEOS)-n-octyltriethoxysilane (C8-TEOS) hybrid gels of varied composition is discussed. The pH and the amount of water present were found to be the determining factors in the preparation of a stable gel with the desired mechanical and chromatographic properties. By carrying out the gel formation at 80 degrees C, capillary columns could be produced in 2.5 h. While an acidic pH was required during (external) gel formation, subsequent treatment of the gel inside the capillary with an alkaline solution ('aging') was found to improve separation and stationary phase capacity significantly. The capillary columns were subsequently used to separate a mixture of polycyclic aromatic hydrocarbons in less than 3 min.     

Aptamer stationary phase for protein capture in affinity capillary chromatography

The thrombin-binding DNA aptamer was used with thrombin as a model system to investigate protein capture using aptamer stationary phases in affinity capillary chromatography. The aptamer was covalently attached to the inner surface of a bare fused-silica glass capillary to serve as the stationary phase. Proteins were loaded onto the capillary via an applied pressure. The capillary was then washed to remove unbound and non-specifically associated proteins. Finally, the bound protein was released and eluted using 20 mM Tris buffer containing 8 M urea, pH 7.3, at 50 degrees C. Eluate was collected after each step (load, wash and elute) and relative amounts of protein each were compared using fluorescence spectroscopy. The identity of the protein in the collections was confirmed using matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The experiment was repeated for thrombin on a bare (unmodified) capillary and a capillary coated with a scrambled-sequence, non-G-quartet forming oligonucleotide that does not bind with thrombin. The results show that the aptamer stationary phase captures approximately three times as much thrombin as the control columns. The experiment was also repeated using human serum albumin (HSA) alone and in an equimolar mixture with thrombin. HSA was not retained on the aptamer capillary, nor did it affect the capture of thrombin from the mixture.     

1 μm硅胶颗粒固定相的加压电色谱分离性能

制备了1 μm无孔硅胶颗粒。通过电动填充法得到总长度为45 cm(固定相填充长度为20 cm)、内径为100 μm的毛细管色谱柱。以乙腈-水体系作为流动相,详细考察了碱性化合物在该色谱柱上的加压电色谱(pCEC)分离性能,讨论了流动相比例、缓冲液浓度、pH值及操作电压等因素对分离的影响。实验结果表明,裸硅胶柱在乙腈-水体系分离碱性样品中表现出典型的反相色谱分离性能;缓冲液浓度的改变则对分离影响不大。当pH值改变时,碱性化合物的解离程度发生变化,它们与固定相之间的作用力发生变化,使得分离度发生相应的变化。分离柱效随施加电压的增加而增加,在1 kV电压下,裸硅胶柱对邻甲苯胺的柱效为35000理论塔板/m。     

Enantiomeric separation by capillary electrochromatography. I. Chiral separation of dansyl amino acids and organochlorine pesticides on a diol-silica dynamically coated with hydroxypropyl-beta-cyclodextrin

In this work, a commercially available diol-silica stationary phase was converted in situ to a chiral stationary phase by dynamically coating it with hydroxypropyl-beta-cyclodextrin (HP-beta-CD). This stationary phase was shown useful for the capillary electrochromatography (CEC) separation of neutral and anionic enantiomers such as some organochlorine pesticides and dansyl amino acids, respectively. The inclusion of HP-beta-CD in the mobile phase to produce the in situ chiral stationary phase allowed the rapid separation of the anionic dansyl amino acid enantiomers at relatively low electroosmotic flow (EOF). The formation of host-guest complexes between the dansyl amino acids and the neutral HP-beta-CD in the mobile phase lowered the actual charge-to-mass ratios of the anionic solutes, thus speeding up their transport by the EOF across the packed capillary column. Several parameters affecting enantioseparation were investigated, including the concentration of HP-beta-CD, ionic strength, pH, and organic modifier content of the mobile phase.     

Chromatographic behavior of packing materials for capillary electrochromatography with a coating of different immobilized polysiloxanes

Octadecyl silyl silica (ODS) phase coated with immobilized polysiloxanes (OV1701, SE-54, SE-30) were synthesized, their characteristics as capillary electrochromatography (CEC) column packing materials were studied. It was found that, although the polysiloxane coatings were different in polarity, the resulting packing materials showed the highest efficiencies when the respective coating ratios (polysiloxane:ODS, w/w) were all 20-30%. As expected, packing materials coated with different polysiloxanes resulted in different selectivity on solute pairs. Separations on these stationary phases were studied with different factors such as pH values and acetonitrile contents of the mobile phases. It was found that all these kind of stationary phases could resist basic mobile phase with a pH value as high as 11.6. Tests were made to analyze polar, basic drugs with CEC using the stationary phases.     

Preparation and characterization of mixed-mode monolithic silica column for capillary electrochromatography

A silica-based monolithic stationary phase with mixed-mode of reversed phase (RP) and weak anion-exchange (WAX) for capillary electrochromatography (CEC) has been prepared. The mixed-mode monolithic silica column was prepared using the sol–gel technique and followed by a post-modification with hexadecyltrimethoxysilane (HDTMS) and aminopropyltrimethoxysilane (APTMS). The amino groups on the surface of the stationary phase were used to generate a substantial anodic EOF as well as to provide electrostatic interaction sites for charged compounds at low pH. A cathodic EOF was observed at pH above 7.3 due to the full ionization of residual silanol groups and the suppression in the ionization of amino groups. A variety of analytes were used to evaluate the electrochromatographic characterization and column performance. The monolithic stationary phase exhibited RP chromatographic behavior toward neutral solutes. The model anionic solutes were separated by the mixed-mode mechanism, which comprised RP interaction, WAX, and electrophoresis. Symmetrical peaks can be obtained for basic solutes because positively charged amino groups can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

丁基胺丙基硅胶毛细管整体柱的制备及其电色谱性能研究

采用溶胶-凝胶技术制备了丁基胺丙基硅胶毛细管整体柱,此整体固定相表面同时含有能产生阳极的电渗流的仲胺官能团和产生疏水作用的正丁基和丙基官能团。对所制备的整体柱电色谱性能进行了详细的表征和分析。考察了流动相pH值对电渗流的影响;对烷基苯同系物、有机酸酸性化合物和苯胺类碱性化合物保留行为进行了研究,并对其可能的保留机理进行了探讨。实验结果表明,对于中性化合物的保留机理主要基于反相作用;而对于酸性化合物的保留行为则是基于混合模式作用机理,即除了电泳作用外,还包括阴离子交换和疏水作用。碱性化合物在丁基胺丙基硅胶毛细管整体柱上的峰形较好,没有明显的峰拖尾现象。     

混合固定相电中溶质的输运特征

 以离子交换和反相固定相构成的混合固定相电中 ,溶质迁移在受到疏水、离子交换作用的同时 ,对于带电溶质而言 ,还受到电泳迁移的影响。根据离子独立迁移原理 ,结合过程中的多种相互作用 ,得到了描述溶质表观迁移速率与其各形态迁移速率、各种相互作用之间相互关系的理论表达式 ;讨论了混合模式电中流动相的 pH及其中的有机调节剂浓度、混合固定相配比等对电渗流的影响及不同形态溶质在柱内的输运特征。结果表明 ,在电中采用混合固定相可以在较大的 pH和有机调节剂浓度范围内得到较强且稳定的电渗流。     

Protein separation by open tubular capillary electrochromatography employing a capillary coated with phenylalanine functionalized tentacle-type polymer under both cathodic and anodic electroosmotic flows

The use of a phenylalanine (Phe) functionalized tentacle-type polymer coated capillary column for protein separation by open tubular capillary electrochromatography (OTCEC) was demonstrated in this work. The tentacle-type stationary phase was prepared from silanized fused-silica capillaries of 50 microm I.D. by glycidyl methacrylate graft polymerization and subsequent Phe functionalization. Due to the amphoteric functional groups of the Phe bonded on the tentacle-type polymer stationary phase, protein separation in the prepared column can be performed under both cathodic and anodic electroosmotic flow (EOF) by varying the pH values of the mobile phase. Model proteins including ribonuclease A (RNase A), myoglobin, transferrin, insulin were baseline separated under cathodic EOF with a mobile phase of pH 8.8. Comparison between the separation result of the four proteins under conditions of OTCEC and capillary zone electrophoresis indicates that the migration behavior of the four proteins in the prepared column was the result of the interplay of chromatographic retention and electrophoretic migration. Besides, three basic proteins including RNase A, cytochrome c (Cyt-c) and lysozyme (Lys) were fully resolved under anodic EOF with an acidic running buffer (pH 2.5). The elution order was the same as the isoelectric point values of the proteins (RNase A相似文献   

Enantiomeric separation by capillary electrochromatography. II. Chiral separation of dansyl amino acids and phenoxy acid herbicides on sulfonated silica having surface-bound hydroxypropyl-beta-cyclodextrin

A chiral silica-based stationary phase having surface-bound hydroxypropyl-beta-cyclodextrin (HP-beta-CD) with a relatively strong electroosmotic flow (EOF) was introduced for enantioseparation by capillary electrochromatography (CEC). The stationary phase contained a hydrophilic sulfonated sublayer to which a chiral top layer of HP-beta-CD was immobilized. While the sulfonated sublayer was to provide a relatively strong EOF, the top HP-beta-CD was to confer the desired chiral recognition towards enantiomeric solutes. This HP-beta-CD sulfonated silica (CDSS) stationary phase proved useful for the rapid separation of anionic enantiomers such as dansyl amino acids and phenoxy acid herbicides. The effects of the organic modifier content, pH, and ionic strength of the mobile phase on enantioseparation were investigated. Under the optimized separation conditions, ten dansyl amino acids and six phenoxy acid herbicides were enantioseparated with a resolution greater than unity.     

Brush-type chiral stationary phase for enantioseparation of acidic compounds. Optimization of chiral capillary electrochromatographic parameters

The capillary electrochromatographic separations of three acidic enantiomers (carprofen, coumachlor and warfarin) were studied on a capillary column packed with 5 microm (3R,4S)-Whelk-O 1 chiral stationary phase. The influence of several experimental parameters (mobile phase pH, type of background electrolyte, acetonitrile ratio, temperature, applied voltage and ionic strength) on electroosmotic flow velocity, retention factor, selectivity factor, efficiency, resolution and effectiveness of chiral separation was evaluated. It was notable that the optimum resolution of the acidic enantiomers was achieved at pH 3.0 phosphate buffer, suggesting that capillary electrochromatography in the ion-suppressed mode can be applied for chiral separations of a range of acidic compounds.     

Novel open tubular CEC with tentacle-type polymer stationary phase functionalized by phenylalanine

A novel stationary phase with tentacle-type polymer chains with epoxy groups was fabricated for open tubular CEC. The fabrication procedure of the stationary phase included pretreatment of capillary inner wall, silanization, and glycidyl methacrylate (GMA)-grafted polymerization. The influence of initiator concentration and polymerization time on the resulting polymer stationary phase was studied by measuring EOF mobility and hydrophobicity of the prepared capillary column. SEM showed that the capillary inner wall was evenly coated with a polymer film showing a wrinkly surface. The prepared GMA-grafted stationary phase could be easily modified with different chromatographic ligands by the ring-opening reaction of the epoxy groups. In this study, phenylalanine (Phe) was selected as the ligand. Benzene derivatives and three amino acids were then separated, respectively, using the Phe modified tentacle-type capillary column. As a control, separations were also performed on a monolayer Phe-modified capillary. The tentacle-type polymer stationary phase was found affording stronger retention and better resolution in the separation of benzene derivatives and tryptophan, tyrosine, and Phe mixtures compared with the monolayer stationary phase.     

Open-tubular capillary electrochromatography using a capillary coated with octadecylamine-capped gold nanoparticles

Octadecylamine-capped gold nanoparticles (ODA-Au-NPs) were prepared and characterized by using UV-Vis adsorption spectrum, transmission electron chromatography (TEM), SEM, and FT-IR. A simple but robust hydrophobic coating was easily developed by flushing a capillary with a solution of ODA-Au-NPs, because the positive charges were carried by the nanoparticles which strongly adsorb to the negatively charged inner surface of a fused-silica capillary via electrostatic and hydrophobic interactions. The chromatographic characteristics of the coated capillary was investigated by varying the experimental parameters such as buffer pH, buffer concentration, and percentage of organic modifier in the mobile phase. The results show that (i) resolution between thiourea and naphthalene is almost the same when comparing the electrochromatograms obtained using pH 7 buffer as mobile phase after and before the capillary column was operated using pH 11 and 3 mobile phase; (ii) no significant changes in retention time and deterioration in peak efficiency were found after 60 runs of test aromatic mixtures; and (iii) column efficiency up to 189 000 theoretical plates/meter for testosterone was obtained. All of the results indicated that the coating could act as a stable stationary phase for open tubular CEC as well as for bioanalysis.     

Open tubular columns with mixed‐mode reversed‐phase and weak anion‐exchange stationary phase for capillary electrochromatography

Columns for open tubular capillary electrochromatography, coated with a mixed‐mode (RP/ion‐exchange) stationary phase, were prepared by using the sol–gel method. The synthetic procedure was optimized by changing the ratios of tetraethoxysilane, octyltriethoxysilane, and 3‐aminopropyltriethoxysilane in the initial sol. SEM studies reveal that a coating with about 400 nm thickness can be obtained. The inner surface properties of these capillaries were probed by measuring the EOF as a function of pH. The surface of this stationary phase contains octyl, amine, and residual silanol moieties; the amine and silanol groups determine the net charge on the inner surface of the capillary and can produce a switchable EOF (anodal/cathodal). The performances of the columns were evaluated by open tubular capillary electrochromatography using a wide range of compounds (polycyclic aromatic hydrocarbons, aromatic acids, and aromatic amines).     

十二烷基键合氧化锆填充电色谱柱的研究

以十二烷基键合氧化锆(C12-ZrO2)作为固定相,制备了填充毛细管电色谱(CEC)柱,较为系统地研究了流动相条件对电渗流的影响、填充CEC柱的稳定性、碱性与中性化合物的保留与流动相pH值和有机溶剂含量的关系。C12-ZrO2固定相填充CEC柱在pH3～11．7范围内具有极好的稳定性;利用磷酸盐与氧化锆表面之间较强的相互作用,能够有效解决传统硅胶键合烷基固定相在有机溶剂含量低的流动相条件下不稳定的问题;同时吸附磷酸盐的固定相表面使得在更宽的流动相pH值范围内CEC柱有足够的电渗流,进一步拓宽CEC的应用领域。     

Open-tubular capillary electrochromatography with bare gold nanoparticles-based stationary phase applied to separation of trypsin digested native and glycated proteins

In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins.