Selectivity of quadruplex DNA stationary phases toward amino acids in homodipeptides and alanyl dipeptides

Series of dipeptides, including homodipeptides and alanyl dipeptides, were separated using quadruplex (G-quartet) DNA stationary phases in open-tubular capillary electrochromatography (OTCEC). The stationary phases were constructed by covalently attaching the DNA oligonucleotides to the inner capillary surface. Three different G-quartet forming oligonucleotides were investigated: the two-plane G-quartet forming thrombin-binding aptamer, the four-plane analogue of the thrombin-binding aptamer, and a two-plane oligonucleotide identical to the thrombin-binding aptamer except for the replacement of the guanine by thymine in the central bridging loop of the G-quartet structure. Results were compared with results obtained using capillary electrophoresis on a bare capillary and OTCEC using an oligonucleotide with the same base composition as the thrombin-binding aptamer but in a different sequence that does not allow G-quartet formation as the stationary phase.     

Effects of G-quartet DNA stationary phase destabilization on fibrinogen peptide resolution in capillary electrochromatography

The migration of fibrinogen peptides in capillaries coated with G-quartet-forming DNA oligonucleotides in open-tubular CEC (OTCEC) was studied, in order to investigate factors affecting the retention of peptides on G-quartet DNA stationary phases. At 25 degrees C, the peptides eluted in the same order in OTCEC using a two-plane G-quartet DNA stationary phase as in CZE, including two peptides that were completely overlapped. It was found that baseline resolution of the coeluting peptides could be achieved in the OTCEC experiment, but not in CZE, at run temperatures of 35-40 degrees C. A stationary phase formed by a scrambled-sequence oligonucleotide that does not form a G-quartet did not provide any resolution of the two coeluting peptides, even at the higher temperatures, indicating that some destabilization of the G-quartet enhances resolution but that some degree of G-quartet structure is necessary. The effects of destabilization were further explored through variation of the cations (sodium or potassium) used in attachment of the G-quartet oligonucleotide to the capillary surface and in the mobile-phase buffer. Resolution was lower when a more stable, four-plane G-quartet stationary phase was used, supporting the conclusion that some flexibility in the G-quartet structure facilitates differential interactions that resolve otherwise coeluting peptides. The increase in peptide resolution upon destabilization of the G-quartet structure could prove to be an important factor in the application of G-quartet DNA stationary phases for nonaffinity-based separation of native proteins and peptides.     

Graphene oxide and reduced graphene oxide as novel stationary phases via electrostatic assembly for open‐tubular capillary electrochromatography

We present here the application of graphene oxide (GO) and reduced graphene oxide (GOOH) sheet as novel stationary phases for open‐tubular CEC (OTCEC) separation based on electrostatic assembly. The inner walls of a bare capillary column was first modified by ionic assembly of poly (diallyldimethylammonium chloride) (PDDA), and then negatively charged GO or GOOH was easily assembled on a positively charged interior walls of the capillary by electrostatic force. Scanning Electron Microscope images showed that GO and GOOH can still maintain sheet‐layer‐like structure when coated onto the capillary via electrostatic assembly. The chromatographic properties of the GO and GOOH coated columns were evaluated via OTCEC separations of various kinds of analytes, including three acid nitrophenol isomers, three basic nitroaniline isomers, and four neutral PAHs. Efficient separations of all the analytes were achieved with optimized buffer pH and organic additive. The reproducibility and stability of the GO or GOOH coated columns were investigated. Our results indicate the capability of application GO or GOOH sheet in OTCEC separation, which can be coated on the inner wall of fused‐silica capillary via electrostatic assembly.     

Alkylthiol gold nanoparticles in sol-gel-based open tubular capillary electrochromatography

A novel open-tubular capillary electrochromatography (OTCEC) column was prepared by immobilizing dodecanethiol gold nanoparticles on prederivatised fused-silica capillary columns with sol-gel technology. 3-Mercaptopropyl-trimethoxysilane (MPTMS) was selected as sol-gel precursor to develop a sol-gel layer on the inner wall of the capillary, prior to assembly of dodecanethiol gold nanoparticles onto the generated sol-gel layer through specific interaction between the gold nanoparticles and surface terminating thiol groups. The electrochromatographic behaviour of the sol-gel gold nanoparticle capillary was compared with a gold nanoparticle capillary prepared via MPTMS surface functionalisation, through variation of the percentage of the organic modifier, pH, and separation voltage. Efficient separation for a "reversed-phase" test mixture of thiourea, naphthalene, and biphenyl and for selected polycyclic aromatic hydrocarbons (PAHs) was obtained on the sol-gel based gold nanoparticle capillaries. OTCEC separations of three selected drug substances (propiophenone, benzoin, and warfarin) were also demonstrated. Scanning electron microscopy was used for the characterization of the sol-gel gold nanoparticle capillary surface. The results confirm that dodecanethiol gold nanoparticles, bound on the sol-gel-based inner layer of fused-silica capillary, can provide sufficient solute-bonded phase interactions for OTCEC with reproducible retention as well as characteristic reversed-phase behaviour.     

开管毛细管电色谱进展

开管毛细管电色谱是近年发展起来的一种高效、快速的新型微柱分离方法。它是在毛细管管壁涂布或键合固定相,以电渗流驱动流动相的一种色谱分离模式。该文对开管毛细管电色谱的发展、柱制备、理论进行了较为详细的综述,引用文献４７篇     

Interaction between netropsin and double-stranded DNA in capillary zone electrophoresis and affinity capillary electrophoresis

Poor sensitivity and low phase ratio are the main drawbacks of open tubular capillary electrochromatography (OTCEC). The poor sensitivity results from the use of narrow bore size capillary, whereas the low phase ratio, which limits the separation capability, is caused by the limited surface area of conventional capillary. Two strategies may be useful to overcome these disadvantages. First, an extended light path (ELP) capillary, which has a bubble cell at the detection point, is used to improve the sensitivity. Secondly, an etched capillary of a 1,000-fold increased surface area is used to enhance the phase ratio. In this work, use of an ELP capillary and an etched capillary in OTCEC was evaluated with a chiral stationary phase of avidin prepared with the physical adsorption method. With a 20 microm I.D. ELP capillary with a 150 microm bubble cell, the peak height was enhanced by 4-10-fold and the corrected peak area was increased by 12-fold relative to a 20 microm I.D. conventional capillary. However, the peak efficiency and resolution decreased noticeably. The phase ratio on the etched capillary was slightly enhanced, by a factor of 1.64 relative to an unetched capillary. Consequently, the separation capability was slightly improved. The increase in the phase ratio was much lower than that expected from the increase in surface area, the reason for which is probably the reduced density of surface silanol group and the generation of nitrogen-containing groups due to the etching process.     

溶胶-凝胶法制备丙二酰胺型二氧大环多胺用作开管柱电色谱固定相的研究

开管柱毛细管电色谱（OTCEC）兼有HPLC和CE的优点^[1] 。柱内径相同时,柱效是OTLC的2倍^[2]。现在常用的直接键合法的制备步骤多,周期长,柱容量小。溶胶－凝胶（sol-gel)能在很温和的条件下使有机物附着在无机介质的表面上,经化学键合作用使涂层对基质有强烈的粘附性。与通常方法相比,sol-gel法制得的涂层有高的相比和抗水解能力。Guo等^[3]用sol-gel技术制备了高相比、高样品容量的OTCEC柱,叶明亮等6[4]用sol-gel法制备了C8开管柱电色谱柱并进行了评价。我们^[5]用sol-gel法将含羟基的冠醚涂渍固化在毛细管内,用于GC分析取得满意结果。丙二酰胺型二氧大环多胺具有大环多胺和寡肽的双重性质,用作OTCEC的固定相更有助于提高分离物的选择性。本文采用sol-gel技术制备含有丙二酰胺型二氧大环多胺的OTCEC柱,可将大环化合物键合在多孔的玻璃状基体上,使毛经表面粗糙化和固定相键合两步合二为一。用制得的OTCEC柱成功地分离了苯二酚、硝基酚、氨基酚和苯二胺的位置异构体及邻卤代苯胺和生物单胺神经递质。与键合法制得的二氧大环多胺柱子^[6,7]比较,用该法制得的柱子有较高的柱效,重现性好,迁移时间短,可进行快速分析。     

Multimodal open-tubular capillary electrochromatographic analysis of amines and peptides

This study describes a comparison of different modes of open-tubular electrochromatography (OTCEC) in bare and etched capillaries. To carry out the investigation, the separation of impurities of two synthetic peptides and the separation of a mixture of five heterocyclic aromatic amines were studied. Three different types of stationary phase were evaluated: (i) fluorosurfactants (anionic and zwitterionic) adsorbed in the inner wall of the capillary (electrochromatography with dynamically modified stationary phases (DMS)CEC); (ii) physically adsorbed polymers (DMA-SO(3-) and DMA-N(+)(CH(3))(3)) and (iii) chemically modified capillaries (C(18), cholesteryl 10-undecanoate and diol). The results confirm that electrochromatography can be a viable alternative to capillary electrophoresis (CE) and liquid chromatography, more established separation techniques. It is possible to differentiate some minor species for the synthetic peptides that cannot be resolved by CE or high-performance liquid chromatography (HPLC). Moreover the separation of the amine mixture depends strongly on the stationary phase used.     

混合烷基开管电色谱柱的制备和评价

利用溶胶－凝胶（Sol-Gel）技术制备了混合烷基开管毛细管电色谱柱（C8－C13OT－CEC），并考察了其电渗流行为和电色谱性能。研究了流动相中甲醇含量对芳香族中性化合物保留的影响。发现C8－C18OT－CEC柱体现反相分配机理。5种芳香族化合物和4种苯同系物在C8－C13OT－CEC柱上分离良好，同时还考察了分离电压和柱内径对柱效的影响，结果表明高的电压和较小的柱内径能提高柱效。     

Study of physically absorbed stationary phases for open tubullar capillary electrochromatography.

A novel method based on the adsorption of positively charged compounds on the wall of a fused-silica capillary was applied to prepare stationary phases for open tubular capillary electrochromatography (OTCEC). The positively charged substances including cationic surfactant such as cetyltrimethylammonium bromide (CTAB) and basic chiral selectors such as protein, peptide and amino acid were physically adsorbed onto the capillary wall under specially selected conditions. The adsorbed stationary phase of CTAB was used to separate neutral compounds, while the others were used for chiral separations. The run-to-run reproducibility of retention time was rather good with relative standard deviation (RSD) values of less than 2.3%. The separation efficiency was excellent with the highest theoretical plate number of up to 590000/m and the average one above 250000/m. Stored at 2-8 degrees C in the refrigerator, the adsorbed stationary phase can last at least one month. It was observed that the UV spectra for the enantiomers are significantly different due to the diastereomeric interactions of enantiomers with the chiral stationary phase in the detection window. With the use of the same capillary, the same instrument, and the same mobile phase, the superiority of OTCEC over open tubular liquid chromatography (OTLC) and capillary zone electrophoresis (CZE) was illustrated.     

丙二酰胺型大环多胺键合固定相在毛细管开管电色谱中的应用

毛细管电色谱 ( Capillary electrochromatography,简称 CEC)是近年发展起来的一种高效、快速的新型微柱分离方法 [1～ 4] .由于它在毛细管柱内填充液相色谱固定相或者在毛细管内壁键合固定相 ,且采用电渗流作为驱动力 ,因而兼有高效液相色谱和毛细管电泳的分离特点 ,已应用于复杂的药物分析 [2 ] .填充柱毛细管柱具有工艺要求高、容易产生气泡、焦耳热和价格昂贵等缺点 .开管柱电色谱( Open- tubular CEC,简称 OTCEC)是将固定相键合或涂覆在毛细管的内壁 ,避免了上述缺陷 .因此已引起高度重视 [3,4] .大环多胺的结构与冠醚类似 ,是一…     

Separation of Trp-Arg and Arg-Trp using G-quartet-forming DNA oligonucleotides in open-tubular capillary electrochromatography

DNA oligonucleotides that form intramolecular G-quartet structures were investigated as stationary phase reagents for separation of mixtures of the isomeric dipeptides Trp-Arg and Arg-Trp in open-tubular capillary electrochromatography (OTCEC). The oligonucleotides included a thrombin-binding aptamer that forms a biplanar G-quartet structure and an oligonucleotide that forms a 4-plane G-quartet structure. Fluorescence, circular dichroism and UV-visible absorbance spectroscopies were used in batch solution studies to indicate interactions between the dipeptides and the biplanar G-quartet structure. Results for OTCEC separations were compared with results obtained for capillary zone electrophoresis separations on a bare capillary. Temperature studies suggest that resolution is improved when the G-quartet structure is partially destabilized, but control experiments in which potassium chloride was not included in the mobile phase indicate that the G-quartet structure nevertheless plays a role in the separations.     

Open tubular capillary electrochromatography using capillaries coated with films of alkanethiol-self-assembled gold nanoparticle layers

We have used alkanethiol self-assembly and dithiol layer-by-layer (LBL) self-assembly processes to prepare an Au nanoparticle (NP)-coated open tubular capillary electrochromatography (OTCEC) column for the separation of three neutral steroid drugs (testosterone, progesterone, and testosterone propionate). The CEC column was fabricated through LBL self-assembly of Au NPs on a 3-aminopropyltrimethoxysilane (APTMS)-modified fused-silica capillary and subsequent surface functionalization of the Au NPs through self-assembly of alkanethiols. We investigated the electrochromatographic properties of the resulting Au NP-coated CEC column using a "reversed phase" test mixture of three steroid drugs. We found that the key factors affecting the separation performance were the number of Au NP layers, the length of the carbon-atom chain of the alkanethiol self-assembled on the Au NPs, the percentage of organic modifier, and the pH of the running electrolyte. This study reveals that the self-assembly of alkanethiols and dithiols onto Au NPs provides stationary phases for CEC separation that are easy to prepare and whose retention behavior is highly controllable and reproducible. We believe that our findings will contribute to further studies of the application of nanotechnology to separation science.     

吸附固定相开管毛细管电色谱方法的建立（英文）

 首次将管壁吸附作用作为开管毛细管电色谱固定相制备的推动力,成功地建立了称为“吸附固定相开管毛细管电色谱”的一种新方法。原理是：选择合适的条件,让荷正电的化合物在毛细管管壁上充分吸附,直接用吸附层作为固定相。目前,已有数类化合物被用作固定相物质,其中包括阳离子表面活性剂如十六烷基三甲基溴化铵（CTAB）、碱性蛋白质如溶菌酶和细胞色素C、碱性小肽如赖氨酸-酪氨酸和赖氨酸-丝氨酸-酪氨酸、以及碱性氨基酸如L-赖氨酸。CTAB吸附固定相用于分离电中性化合物,其它吸附固定相用于手性分离。     

吸附固定相开管毛细管电色谱方法的建立（英文）

首次将管壁吸附作用作为开管毛细管电色谱固定相制备的推动力,成功地建立了称为“吸附固定相开管毛细管电色谱”的一种新方法。原理是：选择合适的条件,让荷正电的化合物在毛细管管壁上充分吸附,直接用吸附层作为固定相。目前,已有数类化合物被用作固定相物质,其中包括阳离子表面活性剂如十六烷基三甲基溴化铵（CTAB）、碱性蛋白质如溶菌酶和细胞色素C、碱性小肽如赖氨酸-酪氨酸和赖氨酸-丝氨酸-酪氨酸、以及碱性氨基酸如L-赖氨酸。CTAB吸附固定相用于分离电中性化合物,其它吸附固定相用于手性分离。所建立的方法重现性好（迁移时间次间RSD值小于2．3％）,柱效高（最高柱效可达590000／m,平均柱效在250000／m以上）,寿命较长（2～8℃下保存,至少30d）。与已有的固定相制备方法比较,所建立的方法具有简便、快速和稳定等优点。     

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   

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.     

Advances in sol-gel based columns for capillary electrochromatography: sol-gel open-tubular columns

The development of sol-gel open-tubular column technology in capillary electrochromatography (CEC) is reviewed. Sol-gel column technology offers a versatile means of creating organic-inorganic hybrid stationary phases. Sol-gel column technology provides a general approach to column fabrication for microseparation techniques including CEC, and is amenable to both open-tubular and monolithic columns. Direct chemical bonding of the stationary phase to the capillary inner walls provides enhanced thermal and solvent stability to sol-gel columns. Sol-gel stationary phases inherently possess higher surface area, and thus provide an effective one-step alternative to conventional open-tubular column technology. Sol-gel column technology is applicable to both silica-based and transition metal oxide-based hybrid stationary phases, and thus, provides a great opportunity to utilize advanced material properties of a wide range of nontraditional stationary phases to achieve enhanced selectivity in analytical microseparations. A wide variety of stationary phase ligands can be chemically immobilized on the capillary inner surface using a single-step sol-gel procedure. Sol-gel chemistry can be applied to design stationary phases with desired chromatographic characteristics, including the possibility of creating columns with either a positive or a negative charge on the stationary phase surface. This provides a new tool to control electroosmotic flow (EOF) in the column. Column efficiencies on the order of half a million theoretical plates per meter have been reported for sol-gel open-tubular CEC columns. The selectivity of sol-gel stationary phases can be easily fine-tuned by adjusting the composition of the coating sol solution. Open-tubular columns have significant advantages over their packed counterparts because of the simplicity in column making and hassle-free fritless operation. Open-tubular CEC columns possess low sample capacity and low detection sensitivity. Full utilization of the analytical potential of sol-gel open-tubular columns will require a concomitant development in the area of high-sensitivity detection technology.     

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).     

Characterization and applications of etched chemically modified capillaries for open-tubular capillary electrochromatography

In this article, the effects of the stationary phase, buffer pH, organic modifier type, organic modifier composition, applied voltage, and temperature on the migration of several synthetic peptides in etched chemically modified open-tubular capillaries are discussed. With these solutes, migration is due to two effects: electrophoretic mobility and solute/bonded phase interactions. In addition, relative migration rates are evaluated for the peptide samples as a function of these experimental variables in order to determine which parameters might be useful for optimizing separations in open-tubular capillary electrochromatography (OTCEC). Some examples of synthetic peptide separations are presented where the sample contains a major component and several minor species, demonstrating how the resolution of these mixtures can be affected by the appropriate choice of experimental variables.