Conductivity detection of polymerase chain reaction products separated by micro-reversed-phase liquid chromatography

In this paper, we describe the application of micro-reversed-phase high-performance liquid chromatography (mu-RP-HPLC) for the separation and/or purification of polymerase chain reaction (PCR) products with detection accomplished using a miniaturized conductivity detector. The conductivity detector used two Pt wires and a bipolar waveform applied to the electrode pair from which the conductivity of the bulk solution could be measured. In the mobile phase used for the mu-RP-HPLC separation of the PCR product, the mass detection limit for herring sperm DNA using conductivity was found to be 11 ng. Efficient separation of the PCR amplicon from the other reagents present in the PCR cocktail was achieved in less than 4 min with a capacity factor of 2.5 and separation efficiency of 9.1 x 10(3) plates. The separation was carried out using reversed-phase ion-pair chromatography with a triethylammonium acetate ion-pairing agent.     

Nanoband electrode for high-performance in-channel amperometric detection in dual-channel microchip capillary electrophoresis

A nanoband electrode detector integrated with a dual-channel polydimethylsiloxane microchip is proposed for in-channel amperometric detection in microchip capillary electrophoresis. Gold nanoband electrodes, which were fabricated on SU-8 substrates with a 100-nm-width gold layer, were introduced into the dual-channel microchip to be an electrochemical detector. Due to the nano-sized width of the detector, the noise of the amperometric detection was significantly reduced, and a high separation resolution was achieved for monitoring the analytes. The detection sensitivity of the system was improved by high signal-to-noise ratio, and a low detection limit on microchip was obtained for p-aminophenol (2.09 nM). Because of the high resolution in measuring half-peak width, the plate number that is used to evaluate the separation efficiency was 1.5-fold higher than that using 50-μm-width electrochemical detector. The effect of sample injection time and data acquisition time on separation efficiency was investigated, and an attractive separation efficiency was achieved with a plate number up to 17,500.     

Capacitance‐to‐digital: The upgrades of single chip detector

The capacitance‐to‐digital single chip detector was upgraded. The paper discusses hardware issues and benefits of the designed/upgraded detector. The device can be operated from rechargeable lithium‐ion battery as stand‐alone, portable system and is capable of transmitting real‐time data wirelessly. The detector and additional modules (battery, battery holder, microcontroller board, wireless module) weight is less than 85 g. Electrophoretic separation in low conductivity 20 mM MES/L‐His buffer, pH 6.1, was performed in order to evaluate detection parameters. The system is capable of quantification of potassium ions down to 0.31 μM. Investigation of differential signal acquisition configuration showed improved performance regarding external noise and temperature fluctuations. The system can be a solution for stand‐alone, field‐portable capillary format separation detector.     

Automated separation and quantitation of lipid fractions by high-performance liquid chromatography and mass detection

This report describes an improved separation and quantitation of lipid fractions in a total lipid extract by high-performance liquid chromatography using a modified solvent and gradient system delivered by dual pumps and incorporating a mass detector and autosampler. The detector responses for various lipid fractions (cholesteryl esters, triacylglycerols, free cholesterol, and seven major phospholipid classes) were fitted to a quadratic equation, y = ax2 + bx + c, and quantified after detector calibration by a computer. This new system has the advantage of automation and reproducible separation. The present method was applied to rat liver analysis.     

An optical rotatory detector for high-performance liquid chromatography using polarization modulation.

A sensitive and variable-wavelength optical rotatory (OR) detector for high-performance liquid chromatography is presented. This design is entirely different from that of conventional OR detectors consisting of a crossed polarizer pair. By placing a polarizing prism and a retardation plate into a commercial circular dichroism (CD) detector, the OR signal was obtained. The Mueller matrix approach was used to prove the principle of the OR signal appearance. Sugars and 4-androstene-3,17-dione were chosen as test compounds. The limit of detection was below 0.5 microg of injected sucrose at 260 nm, which was superior to that obtained with a conventional OR detector. For 4-androstene-3,17-dione, which is CD active, and shows a large anomalous OR dispersion curve, our detector gave a large OR signal with approximately half the intensity of the CD signal at 340 nm.     

丙烯酰胺与季铵类阳离子单体双水相共聚的二次相分离

以(2-甲基丙烯酰氧乙基)三甲基氯化铵(DMC)为例,用分光光度法研究了丙烯酰胺(AM)与季铵类阳离子单体在聚乙二醇(PEG)水溶液中双水相共聚的二次相分离.提出了二次相分离的机理,认为是阳离子单体单元的电离使得聚合物带上了正电荷,大分子间的静电排斥作用增加了其在PEG相中的溶解性(增容作用).讨论了反应条件对体系二次相分离的影响,发现二次相分离在一定的单体浓度和PEG浓度、以及适当的单体比例和PEG分子量条件下才能发生.总单体用量或PEG用量增加,第二次分相先出现后消失;PEG分子量增大,第二次分相逐渐明显;反应温度升高,两次分相更快完成.DMC摩尔分率为0.25～0.60时,第一次分相的临界转化率很小,第二次分相的临界转化率则随DMC摩尔分率的增加而增大;DMC摩尔分率超过0.60后,聚合反应主要在过渡区完成,第二次分相难以发生.     

High-speed LC Analysis Using Electrochemical Detection

Abstract

The chromatographic performance of an electrochemical detector incorporating a flowcell with improved dispersive charac- teristics has been evaluated for use in high-speed liquid chroma- tography. High-speed C18/3 um columns, 100 × 4.6 mn, i.d. were found to be well matched to this detector with respect to extra- column contributions to band broadening. The capabilities of this high-speed LC-EC system are demonstrated by a 3-minute separation of phenols and a 4-minute separation of catecholamines and ace- t ominophen.     

Analysis of polyphenols in white wine by CZE with amperometric detection using carbon nanotube-modified electrodes

A method for the simultaneous detection of five polyphenols (caffeic, chlorogenic, ferulic and gallic acids and (+)-catechin) by CZE with electrochemical detection was developed. Separation of these polyphenols was performed in a 100 mM borate buffer (pH 9.2) within 15 min. Under optimized separation conditions, the performance of glassy carbon (GC) electrodes modified with multiwalled carbon nanotube layer obtained from different dispersions was examined. GC electrode modified with a dispersion of multi-walled carbon nanotubes (CNT) in polyethylenimine has proven to be the most suitable CNT-based electrode for its application as amperometric detector for the CZE separation of the studied compounds. The excellent electrochemical properties of this electrode allowed the detection of the selected polyphenols at +200 mV and improved the efficiency and the resolution of their CZE separation. Limits of detection below 3.1 μM were obtained with linear ranges covering the 10?? to 10?? M range. The proposed method has been successfully applied for the detection (ferulic, caffeic and gallic acids and (+)-catechin) and the quantification (gallic acid and (+)-catechin) of polyphenols in two different white wines without any preconcentration step. A remarkable signal stability was observed on the electrode performance despite the presence of potential fouling substances in wine.     

Simultaneous separation of inorganic anions and cations using capillary electrophoresis with a movable contactless conductivity detector

A number of small inorganic anions and cations were separated after injection of the sample into both ends of a separation capillary. The ions were detected using a capacitively coupled contactless conductivity detector (CCCCD) which could be placed at various positions along the capillary length. Counter-directional migration of anions and cations occurs towards the detector, which is placed at an appropriate position along the capillary so that the migration order is determined by the respective effective separation capillary lengths for both anions and cations. As the CCCCD detector can be easily moved to any position along the capillary, virtually any effective separation length can be attained. Depending on the number of analytes in the sample, one can choose to obtain either electropherograms with inter-migrating zones of cations and anions or separations with distinct regions of anion and cation zones, respectively. A new term 'apparent separation selectivity' is introduced to describe the manner in which the position of the detector can be varied in order to determine the final separation.     

Wall-jet conductivity detector for microchip capillary electrophoresis

A new end-column ‘hybrid’ contactless conductivity detector for microchip capillary electrophoresis (CE) was developed. It is based on a “hybrid” arrangement where the receiving electrode is insulated by a thin layer of insulator and placed in the bulk solution of the detection reservoir of the chip, whereas the emitting electrode is in contact with the solution eluted from the channel outlet in a wall-jet arrangement. The favorable features of the new detector including the high sensitivity and low noise, can be attributed to both the direct contact of the ‘emitting’ electrode with the analyte solution as well as to the insulation of the detection electrode from the high DC currents in the electrophoretic circuit. Such arrangement provides a 10-fold sensitivity enhancement compared to currently used on-column contactless conductivity CE microchip detector as well as low values of noise and easy operation. The new design of the wall-jet conductivity detector was tested for separation of explosive-related methylammonium, ammonium, and sodium cations. The new detector design reconsiders the wall-jet arrangement for microchip conductivity detection in scope of improved peak symmetry, simplified study of inter-electrode distance, isolation of the electrodes, position of the wall-jet electrode to the separation channel, baseline stability and low limits of detection.     

A COMMENT ON SPECTRAL TRANSMITTANCE IN MAMMALIAN SKELETAL MUSCLE

Spectral transmittance studies, related to photoradiation therapy light dosage, were carried out in vitro on mammalian skeletal muscle with expanded beam, broad band, white light (420-1100 nm). Spectra were collected by an EG&G 550/555 spectroradiometer with a 550-2A silicon detector. Bovine striated muscle imposes heavy absorption and distinct spectral structure, reflecting tissue absorption maxima at short wavelengths. Transmission above 650 nm is improved by a 10' factor, relative to blue light, in 1 cm of muscle. Half value layers were 0.82 mm in the 630 nm region, and were sensitive to tissue composition. Bovine muscle exhibited spectral effects related to the cadaver studies done by Wan et at. (1981b) but show singular differences, due primarily to complex tissue composition encountered in cadaver wall measurements, as contrasted with simple muscle. Spectral transmittance is a function of wavelength, tissue type and thickness.     

Analysis of virtual two-dimensional gels based upon affinity capillary electrophoresis hyphenated to ion trap-mass spectrometry

Affinity capillary electrophoresis (ACE) is a robust tool for the study of noncovalent biomolecular interactions and to determine the binding constants. It is advantageous due to the speed of analysis, the high and reproducible separation efficiencies, the low consumption of analytes, the ability to study several interactions at the same time, and to cover a wide range of affinity. The use of an ion trap-mass spectrometer as a sensitive and specific detector, coupled on-line with a classical UV detector, permits extracting simultaneously the electropherograms corresponding to each ionic species. The mass spectra, acquired by scanning the results of a first separation due to ACE, were assimilated into a virtual two-dimensional (2-D) gel. We developed a software application, which was designed to create and analyze these virtual 2-D gels. The validity of this new analytical tool for probing biomolecular interactions has been demonstrated on mixtures of antibiotics of the vancomycin group and several dipeptide substrates. Using the dynamic equilibrium affinity electrophoresis approach, we have shown that molecular components interacting with a low affinity are easily located on the virtual 2-D gels, and that binding constants and stoichiometry of the interactions can be assessed. As the binding constants derived from ACE-electrospray ionization-mass spectrometry (ESI-MS) are unreliable, they must only be determined with the UV detector.     

The development of a staggered parallel separation liquid chromatography/tandem mass spectrometry system with on-line extraction for high-throughout screening of drug candidates in biological fluids

A new parallel liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been developed, in which the mass detector was shared between two staggered parallel chromatographic runs. Since the chromatography for biofluids assay generally requires good analyte retention and thus tends to leave large blank chromatographic windows, this parallel system allowed the efficient use of the mass detector during these blank windows, resulting in significantly improved sample throughput. Also, in order to remove the bottleneck in sample extraction for this parallel separation system, a high-flow extraction device was used to perform on-line extraction. This allowed for the direct injection of biofluids onto the system. The performance and capability of this system was evaluated in tests that contained a single analyte (oxazepam) and multiple analytes (12-in-1). The results indicated that the data generated from this system were comparable to those obtained on a conventional single-column system. An application of the system for high-throughput pharmacokinetic screening of drug candidates was also demonstrated.     

Electromagnetic induction detector for capillary electrophoresis and its application in pharmaceutical analysis

A new electromagnetic induction detector for capillary electrophoresis and its application are described. The detector is consisted of an inductor, a resistor, a high-frequency signal generator and a high-frequency millivoltmeter. The conditions affecting the response of the detector, including dimension of the magnetic ring, position of the capillary, number of coil turns, frequency, excitation voltage and value of the resistor were examined and optimized. The feasibility of the proposed detector was evaluated by detection of inorganic ions and separation of amino aids. Its quantification applicability was investigated by determination of aspirin and paracetamol in pharmaceutical preparation (Akafen powder). The primary factors affecting separation efficiency, which include variety of buffer, buffer concentration, injection time and injection height and separation voltage, were researched. Experimental results demonstrated that this new detector showed a well-defined correlation between sample concentrations and responses (r = 0.997-0.999), with detection limits of 30 μmol L⁻¹ for aspirin and 10 μmol L⁻¹ for paracetamol, as well as good reproducibility and stability. Compared with currently available detection techniques, this new detector has several advantages, such as simple construction, no complicated elements, ease of assembly and operation, and potential for universal applications. It can be an alternative to the traditional methods in the quality control of the pharmaceutical preparations.     

五氯苯酚测定方法研究进展

介绍了目前国内五氯苯酚常用的分析方法,对每种分析方法的原理、操作条件、检测限、线性范围及应用对象作了较为详细的论述.指出吸光光度法操作简单,仪器比较普及,但五氯苯酚吸收带较宽,分辨率低,共存有机物影响较大,其应用受到限制.高效液相色谱法样品无需气化,也不需衍生化,是高沸点、热稳定性较差的五氯苯酚较理想的分析方法.乙酰衍生化、毛细管柱分离、电子俘获检测器检测是目前测定五氯苯酚最常用的气相色谱法.吹扫捕集技术、固相微萃取技术以及质谱检测技术在气相色谱分析中的应用有效提高了测定五氯苯酚的准确度和灵敏度.离子选择性电极法测五氯苯酚仍处于研究阶段.     

Sol-Gel Processing by Aging and Pore Creator Addition for Porous Silica Antireflective Coatings

An antireflective film produced by incorporation of polyethylene glycol (PEG) in the sol is compared to one produced using an aged sol. Incorporation of organic polymer in the precursor solution is a more effective method of increasing the porosity of films and transmittance of the glass substrate. Combination of aging and PEG addition increases the potential for the creation of uniformly porous structures. Aging at high oxide concentration enhances condensation and forms large silica particles containing continuous isolated silanol sites which favor PEG adsorption via hydrogen bonding. Film morphology is affected by phase separation in both the sol and during drying of film. Coating uniformity was improved by minimising phase separation during drying. This was achieved by increasing PEG adsorption by silica sol. Using this technique, coated glass with maximum transmittance of 99.7% can be produced by a simple single step coating.     

Electrochemical detector based on sol-gel-derived carbon composite material for capillary electrophoresis microchips

An on-chip disk electrode based on sol-gel-derived carbon composite material could be easily and reproducibly fabricated. Unlike other carbon-based electrodes reported previously, this detector is rigid, convenient to fabricate, and amenable to chemical modifications. Based on the stable and reproducible characters of this detector, a copper particle-modified detector was developed for the detection of carbohydrates which extends the application of the carbon-based electrode. In our experiments, the performance of the new integrated detector for rapid on-chip measurement of epinephrine and glucose was illustrated. Experimental procedures including the fabrication of this detector, the configuration of separation channel outlet and electrode verge, and the performance characteristics of this new electrochemical detector were investigated.     

Simultaneous separation of hydrophilic and hydrophobic compounds by using an online HILIC-RPLC system with two detectors

In the present study, we developed a novel online hydrophilic interaction chromatography (HILIC)-RPLC separation system for simultaneous separation of both hydrophilic and hydrophobic solutes in a complex sample with one injection. A HILIC Si column and a C18 column were hyphenated with an interface including two electronic 2-position valves, a solvent pump, and a solute transfer column. By using column-switching technique, the nonretained hydrophobic solutes were eluted out of the HILIC column and transferred into the C18 column to perform further separation. The hydrophilic solutes were separated on the HILIC column at the same time. One detector was equipped for each column to record the individual chromatograms. By separating a standard mixture and a traditional Chinese medicine (TCM) extract, the developed HILIC-RPLC system demonstrates its potential for "entire-components" separation of complex samples with improved peak capacity and throughput compared with the common single-column LC.     

Use of contactless conductivity detection for non-invasive characterisation of monolithic stationary-phase coatings for application in capillary ion chromatography

A capacitively-coupled contactless conductivity detector (C4D) has been utilised as an on-capillary detector within a capillary ion chromatograph, incorporating a reversed-phase monolithic silica capillary column semi-permanently modified with a suitable ionic surfactant. The monolithic capillary column (150 x 0.1 mm i.d.) was modified using sodium dioctyl sulfosuccinate (DOSS), an anionic surfactant, for the separation of small inorganic and organic cations. With the use of the on-capillary conductivity detector, the longitudinal homogeneity and temporal stability of the coating were investigated. The approach allowed a detailed non-invasive observation of the nature of the ion-exchange coating over time, and an example of an application of the technique to produce a longitudinal stationary-phase charge gradient is shown. An investigation of the basis of the measured on-capillary conductivity was carried out with a counter ion study, clearly showing the on-capillary detection technique could also distinguish between chemical forms of the immobilised ion exchanger. The above method was used to produce a stable and homogeneously-modified monolithic ion-exchange capillary column, for application to the separation of inorganic alkaline earth cations and amino acids.