Mechanism of formation of monodisperse polystyrene hollow particles prepared by membrane emulsification technique. Effect of hexadecane amount on the formation of hollow particles

In a previous study, it was found that monodisperse polystyrene (PSt) hollow particles can be prepared under special conditions by combining a Shirasu Porous Glass (SPG) emulsification technique and subsequent suspension polymerization process. The dispersed phase mainly containing St, hexadecane (HD), and initiator, was pressed through the uniform pores of a SPG membrane into the continuous phase to form uniform droplets. Then, the droplets were polymerized at 70°C. It was proposed that rapid phase separation between PSt and HD was a main reason responsible for the formation of hollow particle. Rapid phase separation confined the HD inside the droplets, it belonged to a non-equilibrium morphology. In this study, HD/St ratio was increased to a high value to confirm the above proposition by promoting rapid phase separation further between HD and PSt, to prevent monomer diffusion into aqueous phase, and to obtain hollow particle with a large hole.     

Evaluating the utility of ion mobility separation in combination with high-pressure liquid chromatography/mass spectrometry to facilitate detection of trace impurities in formulated drug products

Many formulated products contain complex polymeric excipients such as polyethylene glycols (PEGs). Such excipients can be readily ionized by electrospray and may be present at very high concentrations, thus making it very difficult to identify trace level impurities such as degradants in samples, even if hyphenated techniques such as liquid chromatography/mass spectrometry (LC/MS) are used. Ion mobility (IM) spectrometry is a very rapid gas-phase separation technique and offers additional separation capability within the LC timeframe. This work investigates the use of an IM separator in combination with high-pressure liquid chromatography (HPLC) and MS, to improve the separation of drug-related materials from excipients, thus aiding the identification of trace-level impurities in an anti-HIV medication, Combivir.     

Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry

In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back-exchange level of H for D after the exchange has been quenched, to accurately assign protein conformation and protein-protein or protein-ligand interactions. Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back exchange averages roughly 30% because of the long exposure to H₂O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the dramatically reduced retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated. The rapid separation coupled with high-resolution FT-ICR MS at 14.5 T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability.     

Miniaturization in carbohydrate analysis

Recent progress of microchip electrophoresis (ME) of carbohydrates is overviewed. Carbohydrate analysis by ME encounters difficulties such as lack of electric charge and deficiency of a chromophore/fluorophore in analyte molecules, however, it benefits from the accumulated knowledge of capillary electrophoresis (CE) and rapid separation of simple sugars also by ME, with high column efficiency comparable to CE, has become possible. Analysis at high pH, with electrochemical detection, is a promising approach because carbohydrates can be ionized by weak dissociation of the hydroxyl groups and the in situ formed ionic species can be effectively separated by the zone electrophoresis mode. The separated species can be sensitively monitored by electrochemical detection on a gold or copper electrode. Ionization as borate complexes and refractometric detection is also possible, though sensitivity is lower. Introduction of UV-absorbing or fluorescent tags is potentially useful but the time-consuming derivatization processes sacrifice the rapidity of ME. Examples of ME of carbohydrates as 1-phenyl-3-methyl-5-pyrazolone (PMP; for simple mono- and oligosaccharides with UV detection), 8-aminopyrene-1,3,6-trisulfonate (APTS; for oligosaccharides ladders with LIF detection), and 4-nitro-2,1,3-benzoxadiazole (NBD-F; for amino sugars and aminoalditols with LIF detection) derivatives are presented, with details of the analytical conditions. Since ME in a short separation channel enables rapid analysis within 1 min, it presents an ideal tool for clinical analysis, as shown in a few papers reporting protocols for specific blood glucose assay. Finally, the usefulness of microfluidic reactors and microarrays for enzyme-assisted carbohydrate analysis as well as glycan profiling is pointed out.     

Very fast electrophoretic separation on commercial instruments using a combination of two capillaries with different internal diameters

A capillary formed by connecting a 9.7 cm‐long separation capillary with id 25 μm with an auxiliary 22.9 cm‐long capillary with id 100 μm (coupled capillary) was tested for electrophoretic separation at high electric field intensities. The coupled capillary was placed in the cassette of a standard electrophoresis apparatus. It was used in the short‐end injection mode for separation of a mixture of dopamine, noradrenaline, and adrenaline in a BGE of 20 mM citric acid/NaOH, pH 3.2. An intensity of 2.7 kV/cm was attained in the separation part of the capillary at a separation voltage of 30 kV, which is 2.9 times more than maximum intensity value attainable in a capillary with the same length with uniform id. At these high electric field intensities, the migration times of the tested neurotransmitters had values of 12.3–13.3 s and the attained separation efficiency was between 2350 and 2760 plates/s. It is thus demonstrated that an effective separation instrument ‐ a coupled capillary ‐ can be used for very rapid separation in combination with standard, commercially available instrumentation.     

用短毛细管柱分离一、二、三硝基甲苯的异构体

一、二、三硝基甲苯(简称MNT、DNT和TNT)共有15个异构体.分离这些异构体文献中虽有一些报道^[1-9],但均不太完善.我们曾用液晶填充柱分离和测定MNT的三个异构体[10];用OV-225填充柱分离DNT的六个异构体^[11];用OV-25和OV-210混合固定液填充柱分离TNT六个异构体^[12].     

Two-dimensional capillary electrophoresis using tangentially connected capillaries

A novel type of fused silica capillary system is described where channels with circular cross-sections are tangentially in contact with each other and connected through a small opening at the contact area. Since the channels are not crossing each other in the same plane, the capillaries can easily be filled with different solutions, i.e. different solutions will be in contact with each other at the contact point. The system has been used to perform different types of two-dimensional separations and the complete system is fully automated where a high voltage switch is used to control the location of the high voltage in the system. Using two model compounds it is demonstrated that a type of two-dimensional separation can be performed using capillary zone electrophoresis at two different pH values. It is also shown that a compound with acid/base properties can be concentrated using a dynamic pH junction mechanism when transferred from the first separation to the second separation. In addition, the system has been used to perform a comprehensive two-dimensional capillary electrophoresis separation of tryptic digest of bovine serum albumin using capillary zone electrophoresis followed by micellar electrokinetic chromatography.     

Ash removal from viscous media by supercritical gas extraction

The separation of solid μ-sized particles from viscous media sometimes raises difficulties because sedimentation is too slow, so that use of a centrifuge is required. In the centrifuge, however, the particles may act as an abrasive material. Examples are finely dispersed solid ingredients in shale oil, in oils from tar sand, in hydrogenation process (slurries) suspensions etc.

The media containing the finely dispersed particles are processed with a compressed gas at high density using an entrainer. The conditions are chosen so that the ternary (or pseudoternary) system composed of supercritical gas, entrainer and viscous substance (or mixture) is supercritical. In this way, high concentration of the viscous medium are obtained in the gas phase. As the viscosity of the supercritical gas phase is relatively low, the density of the gas phase can be adjusted by variation of pressure and temperature so that the difference between the solid and the gas phase is sufficiently high for rapid separation of the solid particles. The velocity of sedimentation at constant temperature and pressure depends on the concentration of the viscous substance in the gaseous phase. Even at concentrations of the viscous medium in the gaseous phase, of 30 to 35 % in weight the velocity of sedimentation is sufficiently rapid for the process to be of economical interest.     

Rapid determination of 210Po in water samples

A new rapid method for the determination of ²¹⁰Po in water samples has been developed at the Savannah River National Laboratory (SRNL) that can be used for emergency response or routine water analyses. If a radiological dispersive device event or a radiological attack associated with drinking water supplies occurs, there will be an urgent need for rapid analyses of water samples, including drinking water, ground water and other water effluents. Current analytical methods for the assay of ²¹⁰Po in water samples have typically involved spontaneous auto-deposition of ²¹⁰Po onto silver or other metal disks followed by counting by alpha spectrometry. The auto-deposition times range from 90 min to 24 h or more, at times with yields that may be less than desirable. If sample interferences are present, decreased yields and degraded alpha spectrums can occur due to unpredictable thickening in the deposited layer. Separation methods have focused on the use of Sr Resin?, often in combination with ²¹⁰Pb analysis. A new rapid method for ²¹⁰Po in water samples has been developed at the SRNL that utilizes a rapid calcium phosphate co-precipitation method, separation using DGA Resin^® (N,N,N′,N′ tetraoctyldiglycolamide extractant-coated resin, Eichrom Technologies or Triskem-International), followed by rapid microprecipitation of ²¹⁰Po using bismuth phosphate for counting by alpha spectrometry. This new method can be performed quickly with excellent removal of interferences, high chemical yields and very good alpha peak resolution, eliminating any potential problems with the alpha source preparation for emergency or routine samples. A rapid sequential separation method to separate ²¹⁰Po and actinide isotopes was also developed. This new approach, rapid separation with DGA resin plus microprecipitation for alpha source preparation, is a significant advance in radiochemistry for the rapid determination of ²¹⁰Po.     

Pressure-assisted capillary electrochromatography with electrospray ionization-mass spectrometry based on silica-based monolithic column for rapid analysis of narcotics

A pressure-assisted CEC (pCEC) with ESI-MS based on silica-based monolithic column was developed for rapid analysis of narcotics. Combining the extremely high permeability and separation efficiency of silica-based monolithic column with the high selectivity and sensitivity of pCEC-ESI-MS, the developed system exhibited its prominent advantages in separation and detection. A systematic investigation of the pCEC separation and ESI-MS detection parameters was performed. Experiment results showed that the optimized separation efficiency could be obtained at 8 bar assisted pressure with 25 kV separation voltage, using the solution containing 65% ACN v/v and 20 mmol/L ammonium acetate with pH 6.0 as running buffer. 3 microL/min of sheath liquid was considered as the optimized flow rate since it could provide the maximum signal intensity. Under the optimum conditions, the tested five narcotics could be completely separated within 10 min with the detection limit in the range of 2.0-80 nmol/L. The proposed method has been successfully used for detection of narcotics in real urine samples.     

High-performance capillary electrophoresis using open tubes and gels

Summary This paper overviews several aspects of high performance capillary electrophoresis (HPCE), a promising new method of analytical and micropreparative separation of biochemically important samples. The basic migration equations of electrophoresis are first presented and the benefit of high fields for rapid analysis and high performance emphasized. Since power is generated with high voltages, Joule heating results and this heat must be dissipated. The use of capillary columns is shown to be important in efficient heat removal and in minimizing the temperature differences within the column. The various factors influencing band broadening are next described, and it is shown how plate counts close to 10⁶ can be achieved. Various results from our laboratory on open tube and gel columns are then presented to illustrate the potential of this method. Chiral resolution of dansylated amino acids using a chiral metal chelate micelle in open tube HPCE is shown. With the gel columns, the baseline separation of a 2-chain variant from methionine growth hormone (met-hGH) under non-denaturing conditions at fields close to 1000 V/cm is presented. Finally, the micropreparative purification of a 20-mer oligonucleotide using the gel column is described.     

Detecting deviations from pure EOF during CE separations

CE separations are known for their high separation efficiencies. In systems with EOF, the high efficiencies benefit from the flat, plug profile that is characteristic of EOF. When a velocity gradient is present, such as in separations which have nonuniform buffer ionic strength, surface adsorption or differences in the height of the ends of the capillary, a parabolic flow component is introduced. This deviation from purely EOF yields increased peak dispersion and a subsequent decrease in separation performance. This work details a rapid method for detecting deviations from ideal plug flow during the course of a separation using the radially averaged flow profile of a photobleached fluorophore added to the BGE. By comparing the ratio of two different data analysis procedures, deviations from ideal plug flow can be detected. This method allows rapid measurement of flow character and does not interfere with the concurrent separation. We demonstrate easy detection of the onset of hydrodynamic flow induced by both gravity siphoning and an ionic strength buffer discontinuity. A brief analysis of the radially averaged peak shapes is also presented.     

Sorbent materials for separation and preconcentration of gold in environmental and geological samples – A review

Determination of gold in environmental and geological samples requires very often preconcentration and separation due to the high concentration of interfering matrix components and the low content of this metal. Solid phase extraction technique with different kind of solid sorbents offers for this purpose high enrichment factor, rapid phase separation and the ability of combination with different detection techniques. It can be easily implemented and controlled in flow systems, The recent developments in this area are presented and discussed.     

Overview of software options for processing,analysis and interpretation of mass spectrometric proteomic data

Recently, the interests in proteomics have been intensively increased, and the proteomic methods have been widely applied to many problems in cell biology. If the age of 1990s is considered to be a decade of genomics, we can claim that the following years of the new century is a decade of proteomics. The rapid evolution of proteomics has continued through these years, with a series of innovations in separation techniques and the core technologies of two‐dimensional gel electrophoresis and MS. Both technologies are fueled by automation and high throughput computation for profiling of proteins from biological systems. As Patterson ever mentioned, ‘data analysis is the Achilles heel of proteomics and our ability to generate data now outstrips our ability to analyze it’. The development of automatic and high throughput technologies for rapid identification of proteins is essential for large‐scale proteome projects and automatic protein identification and characterization is essential for high throughput proteomics. This review provides a snap shot of the tools and applications that are available for mass spectrometric high throughput biocomputation. The review starts with a brief introduction of proteomics and MS. Computational tools that can be employed at various stages of analysis are presented, including that for data processing, identification, quantification, and the understanding of the biological functions of individual proteins and their dynamic interactions. The challenges of computation software development and its future trends in MS‐based proteomics have also been speculated. Copyright © 2014 John Wiley & Sons, Ltd.     

A flow detector based on square-wave polarography at the dropping mercury electrode

Square-wave polarography (s.w.p.) at the DME has been adapted for detection in various analytical flow systems. The combination of high sensitivity with rapid potential scan rates results in detector characteristics significantly superior to those previously reported for techniques involving constant applied potential. In an automated flow system for the analysis of discrete samples, s.w.p. allows sensitive and reproducible multi-component sample analysis, at a sampling rate of 22.5 samples per hour (at a 1:1 sample/wash ratio and with relatively low sample volumes). The electrochemical selectivity of the detector may be exploited for monitoring Chromatographic column effluents, in cases where the chromatographic separation is incomplete: species eluted simultaneously but having different reduction potentials can be determined with satisfactory sensitivity. The in-situ monitoring system based on s.w.p. allows rapid simultaneous determinations (~ 300 per hour) of a number of contaminants present at the sub-ppm level; because of its long-term stability and reproducibility, it seems well suited for continuous contaminant control.     

Electrokinetic chromatography for drug analysis. Separation and determination of cefpiramide in human plasma

Electrokinetic chromatography using a fused silica capillary and sodium dodecyl sulfate (SDS) solution has been applied to the separation and determination of cefpiramide (CPM) in human plasma with the use of antipyrine (AP) as an internal standard. A plasma sample was introduced into the capillary by siphoning. The calibration plot for CPM in plasma sample showed good linearity in the concentration range over 10 to 300 micrograms/ml. This method has advantages over usual high performance liquid chromatography (HPLC) in that it needs only a very small volume (less than 10 nl) of plasma without pretreatment, and an extremely high separation efficiency (10 times or much higher plate number than usual HPLC) is obtained. The addition of SDS to the supporting electrolyte solution enabled (1) rapid release of protein-bound drug which allowed the total concentration to be determined, (2) reproducible results to be obtained by suppressing adsorption of protein onto the fused silica capillary and (3) rapid separation of drug from proteins by selective retardation of protein peaks.     

外加场分离技术与微流控技术联用在微纳尺度物质分离中的研究进展

微纳尺度物质的分离和分选在精准医学、材料科学和单细胞分析等研究中至关重要。精准、高效和快速的分离微纳尺度物质能够为癌症的早期诊断、生物样品检测和细胞筛选提供重要帮助,其中基于外加场分离技术的分离微纳尺度物质因可以对微纳尺度物质高效在线分离和分选,被广泛应用于微纳米颗粒、外泌体以及生物细胞的分离工作中,而目前多数外加场分离技术存在装备繁琐和样品消耗大等问题。微流控技术是一种通过制作微通道和微流控芯片操纵微小流体对微纳尺度样品组分进行分离的技术,因具有快速检测、高通量、在线分离、集成性高、成本低等优势现被应用于微纳尺度物质分离分析中,是一种微纳尺度物质分离的有效方法,通过在微流控芯片上设计不同的通道及外部配件提高主动场对微纳尺度物质分离效率。外加场分离技术与微流控技术联用可以实现微纳尺度物质的无损、高效、在线分离。该综述主要概述了近年来在微流控芯片上依托流动场、电场、磁场及声场等外加场分离技术来提高对微纳尺度物质分离效率的研究现状,并将各个外力场对单细胞、微颗粒等微纳尺度物质的分离进行分类介绍,总结各自的优缺点及发展应用,最后展望了外加场分离技术与微流控技术联用在应用于癌细胞的早期筛查、精确分离微尺度物质领域的未来发展前景,并提出联用技术的优势和未来应用等。     

A microchip device to enhance free flow electrophoresis using controllable pinched sample injections

Micro free flow electrophoresis (µFFE) is a valuable technique capable of high throughput rapid microscale electrophoretic separation along with mild operating conditions. However, the stream flow separation nature of free flow electrophoresis affects its separation performance with additional stream broadening due to sample stream deflection. To reduce stream broadening and enhance separation performance of µFFE, we presented a simple microfluidic device that enables injection bandwidth control. A pinched injection was formed in the reported µFFE system using operating buffer at sample flow rate ratio (r) setting. Initial bandwidth at the entrance of separation chamber can be shrunk from 800 to 30 µm when r increased from 1 to 256. Stream broadening at the exit of separation chamber can be reduced by about 96% when r increased from 4 to 128, according to both theoretical and experimental results. Moreover, the separation resolution for a dye mixture was enhanced by a factor of 4 when r increased from 16 to 128, which corresponded to an 80% reduction in sample initial bandwidth. Furthermore, a similar enhancement on amino acids separation was obtained by using injection control in the reported µFFE device and readily integrated into online/offline sample preparation and/or downstream analysis procedures.     

Multi-purpose capillary electrophoresis system with concentration gradient detection

A robust, inexpensive and versatile capillary electrophoresis (CE) system for routine and rapid analysis is reported, which consists of a rugged cartridge holding a 20-mum i.d. 15-cm long capillary, and an inexpensive, universal and sensitive concentration gradient detector. The design of the cartridge simplifies the sample introduction process and makes it possible to perform many separation modes, including moving boundary capillary electrophoresis (MBCE), capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP) and capillary isoelectric focusing (CIEF), on the same system. This arrangement provides more information about a sample's components since analytes can be separated by different modes performed on the same CE system. The detector only consists of a low-power HeNe laser, or laser diode, and a photodiode position sensor. Amino acids and proteins of 10(-6)-10(-3)M concentration can be separated by different capillary electrophoretic modes, and detected directly by the detector. The universal detector shows particularly good sensitivity when applied to CE separation modes having self-concentration and focusing effects. Femtomoles of proteins were separated and detected with CIEF. In addition, a short and narrow capillary allows use of high electrical fields which facilitate rapid separations. Four amino acids at millimolar concentrations were fully separated and detected in less than 80 sec by the MBCE mode when a high electric field was applied. The physical size of the whole system is much smaller than that of conventional CE instruments with UV absorbance or fluorescence detector.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。