Determination of biogenic amines by capillary electrophoresis with pulsed amperometric detection

The biogenic amines, putrescine, cadaverine, spermidine and spermine were separated and quantified by capillary electrophoresis with pulsed amperometric detection. Detection potential of the pulsed amperometric detection was optimized as 0.6 V Optimal separation of the biogenic amines was achieved using a separation buffer of 30 mM citrate at pH 3.5, while keeping the buffer in the detection cell as 20 mM NaOH. Using these conditions, the four biogenic amines were baseline separated. Extrapolated limits of detection for putrescine, cadaverime, spermidine and spermine were 400, 200, 100 and 400 nM for the standard mixture (polyamines dissolved in running buffer), respectively. These are lower than ultraviolet detection and comparable or even lower than laser-induced fluorescence detection results as reported in the literature. The number of theoretical plates was maintained at the 105 level, which is absolutely higher than any reported method. When applying capillary electrophoresis-pulsed amperometric detection to milk analysis, only spermidine was found in amounts varying between 0.1 and 0.5 mg/kg.

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.

Programmed cell death in Laminaria japonica (Phaeophyta) tissues infected with alginic acid decomposing bacterium

TdT-mediated dUTP-biotin nick end labeling (TUNEL) is a sensitive and valid method for detecting DNA cleavage in programmed cell death (PCD). Using this method, DNA cleavage was observed in Laminaria japonica sporophytic tissues, which were infected with alginic acid decomposing bacterium. It was found that DNA cleavage occurred 5 min after the infection, the fragments with 3'-OH groups of cleaved nuclear DNA increased with time of infection and spread from the infection site. Although no typical DNA ladder (200 bp/ 180 bp) was detected by routine agarose gel electrophoresis, the cleavage of nuclear DNA fragments of 97 similar to 48.5 kb could be detected by pulsed field gel electrophoresis (PFGE). By using CaspGLOW(TM) fluorescein active caspase-3 staining method, caspase-3 activity has been detected in response to the infection of alginic acid decomposing bacterium. Our results are similar to the observations in hypersensitive response (HR) of higher plant, suggesting that the rapid cell death of L. japonica infected by alginic acid decomposing bacterium might be involved in PCD, and indicating that the occurrence of PCD is an active defense process against the pathogen's infection.

Determination of barbituric acid and 2-thiobarbituric acid with end-column electrochemical detection by capillary electrophoresis

Capillary electrophoresis (CE) with end-column electrochemical detection (EC) of barbituric acid (BA) and 2-thiobarbituric acid (TA) has been described. Under optimum condition, BA and TA were separated satisfactorily, and a response of high sensitivity and stability was obtained at a detection potential of 1.25 V versus Ag/AgCl. Optimized end-column detection provides detection limit as low as 0.5 and 0.1 mu M for BA and TA, respectively. The calibration graph was linear over three orders of magnitude. The relative standard deviations (n = 10) of peak currents and migration times obtained for both BA and TA were 3.4, 3.7, and 1.7, 1.2%, respectively. The proposed method has been applied to analyze water sample with satisfactory results. (C) 2000 Elsevier Science B.V. All rights reserved.

End-column amperometric detection of aesculin and aesculetin by capillary electrophoresis

Determination of aesculin (AL) and aesculetin (AT) by capillary electrophoresis with end-column amperometric detection using a 33 mu m microdisk carbon fiber electrode is described. The HDVs, the effect of pH, buffer concentration, injection voltage, injection time and separation voltage on the peak current response (i(p)) of the analytes and the number of theoretical plates (N) were studied. The method has high sensitivity and good reproducibility. Under the optimum condition - 10 mM, pH 9.00 phosphate buffer, 4 s at 9 kV injection, separation at 15 kV and +1.0 V as the detection potential - low detection limits (S/N = 3) of 0.06 and 0.3 mu M were obtained for AL and AT, respectively. The calibration curve was linear over three orders of magnitude. The relative standard deviations (n = 15) of peak current and migration time were 3.9% and 4.6%, and 0.96% and 0.75% for 15 consecutive injections of 5 mu M AL and AT, respectively. The use of this method for the separation and detection of the two compounds present in the traditional Chinese medicine and human urine samples is also reported. (C) 1999 Elsevier Science B.V. All rights reserved.

Determination of sulfadiazine and sulfamethoxazole by capillary electrophoresis with end-column electrochemical detection

Capillary electrophoresis (CE) with end-column electrochemical detection (EC) of sulfadiazine (SDZ) and sulfamethoxazole (SMZ) is described. Under the optimum conditions, SDZ and SMZ were separated satisfactorily, and a highly sensitive and stable response was obtained at a potential of 1.1 V versus Ag/AgCl. Optimized end-column detection provides detection limits as low as 0.1 mu M for both compounds, which corresponds to 0.024 and 0.021 fmol with peak efficiencies of 394000 and 335000 theoretical plates for SDZ arid SMZ, respectively. The calibration graph was linear over three orders of magnitude. The relative standard deviations (n = 12) of peak currents and migration times were 2.3 and 2.7%, and 0.8 and 1.3%, respectively, for the two compounds. The proposed method was applied to the analysis of tablets and human urine samples with satisfactory results.

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.

Evaluation of a sol-gel derived carbon composite electrode as an amperometric detector for capillary electrophoresis

In this paper, we report the construction and application of a sol-gel derived carbon composite electrode (CCE) as an amperometric detector for capillary electrophoresis. The electrochemical properties were characterized and compared with those of conventional carbon fiber and carbon paste electrode (CPE). Experimental results show that peak-to-peak noise of CCE was about 20% of CPE and electrode capacitance was comparatively low. When applied to the detection of dopamine and epinephrine, the optimal detection potential for CCE was 0.1 V lower than CPE under the same separation conditions; CCE with diameter of 75 and 100 mum could achieve a low detection limit of 3.10(-8) and 6.10(-8) M for the detection of epinephrine, which approaching that of the 33-mum diameter carbon fiber electrode. Also, the linearity for epinephrine at CCE was more than two orders of magnitude, which was slightly wider than that of carbon fiber electrode. Applications to real sample analysis were tested by the determination of betahistine dihydrochloride in tablets and human urine. Using CCE with diameter less than or equal to100 mum as an amperometric detector after capillary electrophoresis separation, a low detection limit and a wide linear range combined with excellent reproducibility were obtained. This CCE possesses of many advantages, namely, convenience, ease of fabrication, low cost and high stability.

Numerical simulation of temperature field evolution of metallic alloy surface due to pulsed laser treatment

This paper performed a numerical simulation on temperature field evolution for the surface layer of a metallic alloy subjected to pulsed Nd:YAG laser treatment. The enthalpy method was adopted to solve the moving boundary problem, I.e. Stefan problem. Computational results were obtained to show the temperature field evolution. Effects of latent heat and mushy zone width on the temperature field were investigated. The results also show very high values of temperature gradient and cooling rate, which are typical characteristics during the solidification process.

Diffraction integral formulas of the pulsed wave field in the temporal domain

To resolve the diffraction problems of the pulsed wave field directly in the temporal domain, we extend the Rayleigh diffraction integrals to the temporal domain and then discuss the approximation condition of this diffraction formula. (C) 1997 Optical Society of America.

Magnetic field modulated direct overwrite with pulsed laser irradiation for TbFeCo magneto-optical disks

WE have designed a dual-beam magneto-optical (MO) storage system to test the dynamic storage properties of MO disks. The characteristics of this dual-beam system are demonstrated. Magnetic field modulated direct overwrite, which is a promising technique for highspeed MO storage, is realized on TbFeCo MO disks with this dual-beam MO system. The effect of light intensity, magnetic field intensity, and linear velocity of the disk and the modulating frequency variation on carrier-to-noise ratio is investigated. (C) 1997 Society of Photo-Optical Instrumentation Engineers.

Spatiotemporal heterogeneity of plankton communities in Lake Donghu, China, as revealed by PCR-denaturing gradient gel electrophoresis and its relation to biotic and abiotic factors

The 16S and 18S rRNA genes of planktonic organisms derived from five stations with nutrient gradients in Lake Donghu, China, were studied by PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, and the relationships between the genetic diversity of the plankton community and biotic/abiotic factors are discussed. The concentrations of total nitrogen (TN), total phosphorus (TP), NH4-N and As were found to be significantly related (P < 0.05) to morphological composition of the plankton community. Both chemical and morphological analyses suggested that temporal heterogeneity was comparatively higher than spatial heterogeneity in Lake Donghu. Although the morphological composition was not identical to the DGGE fingerprints in characterizing habitat similarity, the two strongest eutrophic stations (I and II) were always initially grouped into one cluster. Canonical correspondence analysis suggested that the factors strongly correlated with the first two ordination axes were seasonally different. The concentrations of TN and TP and the densities of rotifers and crustaceans were generally the main factors related to the DGGE patterns of the plankton communities. The study suggested that genetic diversity as depicted by metagenomic techniques (such as PCR-DGGE fingerprinting) is a promising tool for ecological study of plankton communities and that such techniques are likely to play an increasingly important role in assessing the environmental conditions of aquatic habitats.

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregrarn is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.