Adsorptive membrane chromatography for purification of plasmid DNA

Adsorptive membranes were investigated for the downstream processing of plasmid DNA by quantifying both separation efficiencies and adsorption uptake with the anion-exchange membranes. Separation efficiencies of the 10-ml Mustang-Q were measured using pulses of 6.1-kilo base pair plasmid DNA and lysozyme tracers, and comparing the responses for both conventional and reverse-flow operation. The plasmid exhibited nearly 200 plates/cm, almost as high efficiency as the protein despite the large difference in size. This behavior contrasts strongly with typical behavior for spherical porous particle packings, which predicted large decreases in efficiency with increases in tracer size. Batch adsorption isotherms for the 6.1-kilo base pair plasmid on small sheets of anion-exchange membranes at various ionic strengths showed high capacities for very large biomolecules. The maximum binding capacity for the membrane unit was calculated as 10 mg plasmid/ml, an order of magnitude greater than typical values reported for porous beads.     

Shrinking-bed model for percolation process applied to dilute-acid pretreatment/hydrolysis of cellulosic biomass

For many lignocellulosic substrates, hemicellulose is biphasic upon dilute-acid hydrolysis, which led to a modified percolation process employing simulated two-stage reverse-flow. This process has been proven to attain substantially higher sugar yields and concentrations over the conventional single-stage percolation process. The dilute-acid pretreatment of biomass solubilizes the hemicellulose fraction in the solid biomass, leaving less solid biomass in the reactor and reducing the bed. Therefore, a bed-shrinking mathematic kinetic model was developed to describe the two-stage reverse-flow reactor operated for hydrolyzing biphasic substrates, including hemicellulose, in corn cob/stover mixture (CCSM). The simulation indicates that the shrinking-bed operation increases the sugar yield by about 5%, compared to the nonshrinking bed operation in which 1 reactor volume of liquid passes through the reactor (i.e.,t = 1.0). A simulated optimal run further reveals that the fast portion of hemicellulose is almost completely hydrolyzed in the first stage, and the slow portion of hemicellulose is hydrolyzed in the second stage. Under optimal conditions, the bed shrank 27% (a near-maximum value), and a sugar yield over 95% was attained.     

Stereospecific high-performance liquid chromatographic determination of tocainide

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.     

Effect of moisture on the extraction efficiency of polycyclic aromatic hydrocarbons from soils under atmospheric pressure by focused microwave-assisted extraction

The performance of a large commercial chromatographic column was investigated using a short pulse of a tracer and an extension of the reverse-flow technique. This technique permits separate determination of the unavoidable irreversible microscopic processes and the reversible effects of flow maldistribution, and allows for the separation of flow maldistribution in the flow distributors from flow maldistribution inside the packed bed. This analysis was performed on a 0.44 m Millipore IsoPak column using Cellufine GC 700, cellulosic-based media with an average particle diameter of 75 microm, for the stationary phase. The column efficiency was quantified by analysis of the effluent curve from a short pulse of a 5% aqueous acetone tracer. The study examined behavior of beds of different lengths (10-24 cm) and beds packed from different slurry concentrations (10-75% v/v). The slurry-packed columns were very uniform, and no significant macroscopic flow maldistribution was observed inside the column. The observed bed plate heights conformed to the predictions of available one-dimensional continuum models. Dispersion in the flow distributors was significant, corresponding to 15-25% of the intracolumn dispersion when the full 24 cm available bed length was used and a proportionally larger increase for shorter bed lengths. Thus, the headers are shown to produce a significant increase in the observed plate height.     

Determination of chloride, sulfate and nitrate in groundwater samples by ion chromatography

Groundwater is a significant source of water for both domestic and agricultural use in some regions of the Maracaibo lake basin in Venezuela. Chemically suppressed ion chromatography with a Dionex Model 2000i/sp, lonpac AS11, ASRS-I system was used for the analysis of major inorganic anions in groundwater samples. About 50 samples of groundwater, taken over several months in three different locations, were analyzed after filtration and sometimes dilution. In all the samples, the separation between the peaks of chloride, nitrate and sulfate showed good resolution (symmetrical peaks, not broadened), even when the chloride concentration was as high as 850 mg l(-1) and reproducibility (RSD) was -2%. No other peaks (i.e. fluoride, nitrite and phosphate) were observed at selected experimental conditions. With the chosen parameters, the method is well-suited for the routine determination of these anions in groundwater samples, giving results in less than 10 min (including column clean-up). With an appropriate combination of detector output ranges (300 and 1,000 microS), only one set of calibration solutions was needed for all samples. In the Sierra Maestra location, the groundwater samples, were significantly different in total anion levels. Mean total chloride plus sulfate concentrations (approximately 525 mg l(-1)) were about 100 times higher than in the other sites. Some water quality implications of these groundwater samples are also discussed.     

Microchip electrophoresis with wall-jet electrochemical detector: influence of detection potential upon resolution of solutes

This report studies the electrochemical response of wall-jet detector for microchip electrophoresis (microCE). It shows that in wall-jet configuration, the electrochemical detector operates in coulometric mode and that there is an influence of detection potential upon peak width and therefore upon the resolution of solutes. Upon raising the detection potential from +0.3 to +0.9 V, the resolution between model analytes, dopamine and catechol, increases from 0.63 to 2.90. The reasons for this behavior originate in wall-jet detector design and in its typically significant higher detector volume than the volume of injected sample. The conversion efficiency of the wall-jet electrochemical detection cell was found to be 97.4% for dopamine and 98.0% for catechol. The paper brings deeper understanding of operations of wall-jet electrochemical detectors for microchip devices, and it explains previously reported significantly sharper peaks when electrocatalytic electrodes (i.e., palladium and carbon nanotube) were used in microCE-electrochemistry wall-jet detector.     

Solid-state light-emitting devices based on the tris-chelated ruthenium(II) complex. 4. High-efficiency light-emitting devices based on derivatives of the tris(2,2'-bipyridyl) ruthenium(II) complex

Light-emitting devices from the tris(2,2'-bipyridyl)ruthenium(II) complex [Ru(bpy)(3)(2+)] and new derivatives thereof were prepared. Due to the electrochemical nature of the device operation, single-layer devices in an ITO/ Ru(bpy)(3)(2+) complex + PMMA/Ag sandwich configuration achieved very high external quantum efficiencies. The derivatives of the Ru(bpy)(3)(2+) complex were designed and synthesized to inhibit self-quenching of the excited state by adding different alkyl substituents on the bipyridyl ligands. As a result, devices that contain these new Ru(bpy)(3)(2+) complexes show a higher photoluminescence and electroluminescence efficiency than devices made from the unmodified Ru(bpy)(3)(2+) complex. External quantum efficiencies up to 5.5% at brightnesses in the range of 10-50 cd/m(2) are reported. In addition, the response time of such devices (which is a result of the electrochemical operation) has been shortened dramatically. An "instantaneous" light emission is achieved for devices that employ smaller counterions such as BF(4)(-) to increase the ionic conductivity. Such a device shows a response time of less than 1 s to emit 10-20 cd/m(2) after the operating voltage of 2.4 V has been applied.     

Improved method for measurement of rhodanese activity using methanethiosulfonate as sulfur donor substrate and its application to human serum.

We have developed a sensitive method for the measurement of rhodanese activity in human serum which is based on the colorimetric method for the determination of thiocyanate produced from methanethiosulfonate and cyanide as substrates. Thiocyanate gives a red complex with ferric ion in an acidic condition. The present method is about 70-fold more sensitive than the conventional method using cyanide and thiosulfate as substrates and correlates well (r = 0.997) with the conventional method in bovine liver rhodanese. Within-run precision of the method is 0.91% for 420 units/l serum and the calibration curve is linear up to 1850 units/l. The normal value for human serum, determined by the present method on 31 healthy persons, was 20.9 +/- 20.0 units/l (mean +/- 2S.D.). Rhodanese activity was clearly elevated in some serum samples which were observed at abnormal values in some biochemical diagnostic tests and showed significant positive correlations with guanase activity (r = 0.728, p less than 0.01) and glutamic-oxalacetic transaminase activity (r = 0.625, p less than 0.01).     

Development of a chemiluminescent enzyme immunoassay for urinary 1-hydroxypyrene

A chemiluminescent enzyme-immunoassay for urinary 1-hydroxypyrene has been developed and optimized. The enzymatic activity of horseradish peroxidase-labeled tracer was measured with an enhanced chemiluminescent system and the results were compared with those from conventional colorimetric detection. The method fulfilled all the requirements of accuracy and precision and the detection limit was 0.001 pmol/well, which enabled analysis in less than 1 microL urine. Subjects working in the center of Bologna who were exposed daily to vehicular exhaust gas were studied. Their urinary 1-hydroxypyrene concentrations were compared with the levels of benzo( a)pyrene in air particulate matter. Urinary 1-hydroxypyrene, which ranged from 0.5 to 10 nmol L(-1), correlated poorly with the concentration of benzo( a)pyrene in air particulate matter, which ranged from 5 to 140 ng m(-3). No significant effect of vehicle exhaust gas exposure was observed among the different groups of subjects working in different areas of the town. Thus, at a relatively low level of exposure 1-hydroxypyrene does not seem to be a sensitive biomarker of exposure to polycyclic aromatic hydrocarbons.     

Fast thermal desorption spectroscopy study of morphology and vaporization kinetics of polycrystalline ice films

Fast thermal desorption spectroscopy was used to investigate the vaporization kinetics of thin (50-100 nm) H(2)O(18) and HDO tracer layers from 2-5 microm thick polycrystalline H(2)O(16) ice films at temperatures ranging from -15 to -2 degrees C. The isothermal desorption spectra of tracer species demonstrate two distinct peaks, alpha and beta, which we attribute to the vaporization of H(2)O(18) initially trapped at or near the grain boundaries and in the crystallites of the polycrystalline ice, respectively. We show that the diffusive transport of the H(2)O(18) and HDO tracer molecules in the bulk of the H(2)O(16) film is slow as compared to the film vaporization. Thus, the two peaks in the isothermal spectra are due to unequal vaporization rates of H(2)O(18) from grain boundary grooves and from the crystallites and, therefore, can be used to determine independently the vaporization rate of the single crystal part of the film and rate of thermal etching of the film. Our analysis of the tracer vaporization kinetics demonstrates that the vaporization coefficient of single crystal ice is significantly greater than those predicted by the classical vaporization mechanism at temperatures near ice melting point. We discuss surface morphological dynamics and the bulk transport phenomena in single crystal and polycrystalline ice near 0 degrees C.     

Head-space flow injection for the on-line determination of iodide in urine samples with chemiluminescence detection

A simple head-space (HS) flow injection (FI) system with chemiluminescence (CL) detection for the determination of iodide as iodine in urine is presented. The iodide is converted to iodine by potassium dichromate under stirring in the closed HS vial, and the iodine is released from urine by thermostatting and is carried in a nitrogen flow through an iodide trapping solution. The concomitant introduction of aliquots of iodine, luminol and cobalt(II) solutions by means of a time-based injector into an FI system allowed its mixing in a flow-through cell in front of the detector. The emission intensity at 425 nm was recorded as a function of time. The salting-out of the standard solutions affected the gas-liquid distribution coefficient of iodine in the HS vial. The typical analytical working graphs obtained under the optimized experimental conditions were rectilinear from 0 to 5 mg l(-1) iodine, achieving a precision of 2.3 and a relative standard deviation of 1.8 for ten replicate analyses of 50 and 200 microg l(-1) iodine. However, a second-order process becomes significant at higher iodine concentrations (from 10 to 40 mg l(-1)). The detection limit of the method is 10 microg l(-1) (80 ng) iodine when 8 ml samples are taken. Data for the iodide content of 10 urine samples were in good agreement with those obtained by a conventional catalytic method, and recoveries varied between 101 and 103% for urine samples spiked with different amounts of iodide. The analysis of one sample takes less than 20 min. In the present study the iodide levels found for 100 subjects were 86.8 +/- 19.0 (61-125) microg l(-1), which is lower than the WHO's optimal level (150-300 microg per day).     

An indirect spectrophotometric method for the determination of silicon in serum, whole blood and erythrocytes.

An indirect method for the determination of silicon in blood samples has been developed. The proposed method overcame interference from a large amount of salts and phosphate in blood samples, and enabled us to determine the silicon contents in serum and whole blood by the same operation. After blood samples were digested by microwave heating, silicon, present as silicate in the sample solution, was reacted with molybdate to form a silicomolybdate complex. The complex was then separated from unreacted molybdate by a cation-exchange resin column. The molybdate liberated from the complex was spectrophotometrically determined in place of silicon. Since the method is not affected the composition of matrices between serum and whole blood, it could achieve good precision and accuracy, and could also estimate the silicon contents in erythrocytes from those in serum and whole blood. The sensitivity of the method was almost equal to that of the conventional silicomolybdenum blue method, and the calibration curve was linear up to 50 micromol l(-1) of silicon with a detection limit of 1.1 micromol l(-1) in whole blood. The mean concentrations of silicon in five healthy subjects were 11 micromol l(-1) for serum, 28 micromol l(-1) for whole blood and 50 micromol l(-1) for erythrocytes. Thus, the obtained distribution ratio between serum and erythrocytes was in the range of 0.15-0.39, and was found to be included in a narrow range.     

One-step cleanup for PAH residue analysis in plant matrices using size-exclusion chromatography

A new one-step cleanup procedure, based on size-exclusion chromatography (SEC), usable for the extracts from accelerated solvent extraction (ASE), Soxhlet extraction, or ultrasonic extraction (USE), is described. The method is suitable for the determination of polycyclic aromatic hydrocarbons (PAHs), especially from very complicated plant matrices (e.g. pine needles, deciduous leaves, mosses). The main improvement compared with previous conventional procedures is that analyte peaks barely overlap with matrix peaks in the chromatograms and that it is a very rapid and simple one-step procedure with clearly improved analytical performance. Essential advantages of this SEC procedure are the sharper GC-MS chromatograms for the PAH fraction at retention times between 9.2 and 12.0 min, distinctly separated substance peaks resulting in better analysis, shorter running times, and lower solvent consumption.     

Amperometric determination of acetylsalicylic acid in drugs by batch injection analysis at a copper electrode in alkaline solutions

This paper describes the determination of acetylsalicylic acid (ASA) as salicylate (SA) in pharmaceutical formulations by using amperometric detection with copper electrodes in 0.10 mol l(-1) NaOH solution. Batch injection analysis (BIA) was explored for this application. The system exhibited sharp current response peaks, rapid washout and excellent repeatability. A large linear dynamic range from 1 to 1000 mumol l(-1) was obtained by using an injected volume of 100 mul, with a detection limit of 0.48 mumol l(-1). R.S.D. of 0.37% for 30 repetitive (1x10(-4) mol l(-1)) injections and sampling frequency of 60 h(-1) were achieved. The results obtained using this system for ASA determination in seven different drug samples compared well with those found by spectrophotometry (Trinder test).     

掺杂正八面体AgBr乳剂介电频谱的研究——I-离子的影响

本文研究了用不同量的I~-(1×10~(-3)-4×10~(-2)mol I~-/mol AgBr)进行表面掺杂的正八面体AgBr乳剂的介电吸收频谱, 并用强X射线光源相应作了多晶X射线物相分析。当I~-的加入量小于1×10~(-2)mol/mol AgBr时, 介电吸收峰随加入I~-量的增加而逐渐向高频方向位移。吸收峰弥散, 分布很宽。当I~-的加入量大于1×10~(-2)mol/mol AgBr时, 样品的介电吸收峰不再明显向高频方向移动, 峰形亦相对比较尖锐。已有证据表明, 在掺杂I~-以后, 在AgBr微晶的表明层内除生成Ag(Br, I)混晶外, 还附生有一薄层β-AgI。分散相颗粒表面层大约2.0 nm范围内的组成与结构对非均匀电介质中的界面极化效应有重要影响, 此外, 对界面极化效应应用的可能性作了初步探讨。     

Application of gas chromatography coupled to chemical ionisation mass spectrometry following headspace solid-phase microextraction for the determination of free volatile fatty acids in aqueous samples

Gas chromatography coupled to positive and negative ion chemical ionisation mass spectrometry was evaluated for the determination of free volatile fatty acids (VFAs) from aqueous samples by headspace solid-phase microextraction. Negative ion chemical ionisation in the selected ion monitoring mode using ammonia as reagent gas provided acceptable sensitivity and the highest selectivity for the determination of C2-C7 fatty acids using a polydimethylsiloxane-Carboxen fibre. Detection limits in the range of 150 microg l(-1) for acetic acid and from 2 to 6 microg l(-1) for the remaining carboxylic acids were achieved. The reproducibility of the method was between 9 and 16%. The developed analytical procedure was applied to the analysis of VFAs in raw sewage. The absence of interfering peaks provided a more accurate determination of acetic, propionic, butyric and isovaleric acids than a similar analytical scheme but using a flame ionisation detector.     

Ultra-performance liquid chromatographic determination of manufacturing intermediates and subsidiary colors in D&C Red No. 34 and its lakes

An ultra-performance liquid chromatography (UPLC) method was developed to determine the manufacturing intermediates and subsidiary colors in the monosulfo monoazo color additive D&C Red No. 34 and its lakes. This method is currently used for batch certification of the color additives by the U.S. Food and Drug Administration to ensure that each lot meets published specifications for coloring drugs and cosmetics. The new UPLC method has replaced an HPLC method for determining the intermediates and a TLC method for determining the subsidiary colors. The intermediates are 2-amino-1-naphthalenesulfonic acid (Tobias acid) and 3-hydroxy-2-naphthalenecarboxylic acid (3-hydroxy-2-naphthoic acid). Subsidiary colors are positional isomers of the major dye component or related compounds containing lower numbers of substituent groups. The analytes are identified by comparison of their UPLC retention times and UV or visible absorption spectra with those of standards. Validation studies showed that peak area calibrations for the analytes were generally linear (R > 0.999), and recoveries were 98-103%. The LODs were 0.002-0.02%, and the RSDs at the specification levels were 0.7-2.2%. Survey analyses of 12 samples of certified D&C Red No. 34 straight colors and lakes from six domestic and foreign manufacturers yielded results for the intermediates by UPLC and HPLC that were consistent within experimental error. The UPLC analyses yielded results for the subsidiary colors that were consistently lower than results previously obtained by TLC, which we attribute to limitations of the TLC method. The new UPLC method provides sharper peaks, better peak separation, and faster analysis times than the formerly used HPLC method and is more accurate, much faster, and much less labor-intensive than the formerly used TLC method.     

A simple, rapid and high-throughput fluorescence polarization immunoassay for simultaneous detection of organophosphorus pesticides in vegetable and environmental water samples

A simple, rapid and high-throughput fluorescent polarization immunoassay (FPIA) for simultaneous determination of organophosphorus pesticides (OPs) using a broad-specificity monoclonal antibody was developed. The effects of tracer structure, tracer concentration, antibody dilution, methanol content and matrix effect on FPIA performance were studied. The FPIA can detect 5 OPs simultaneously with a limit of detection below 10 ng mL(-1). The time required for the equilibrium of antibody-antigen interaction was less than 10 min. The recovery from spiked vegetable and environmental samples ranged from 71.3% to 126.8%, with the coefficient of variations ranging from 3.5% to 14.5%. The developed FPIA was applied to samples, followed by confirmation with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. The developed FPIA demonstrated good accuracy and reproducibility, and is suitable for rapid and high-throughput screening for OP contamination with high-efficiency and low cost.     

Simultaneous spectrofluorimetric determination of europium, dysprosium, gadolinium and terbium using chemometric methods

A spectrofluorimetric method for the simultaneous determination of dysprosium, europium, gadolinium and terbium in ternary and quaternary mixtures by the use of pyridine-2,6-dicarboxylic acid as a chelating agent was developed. The influence of chemical variables affecting the analytical reaction was evaluated. A partial least-squares procedure and PC Quant software were used to assess data obtained from a variable number of calibration solutions and wavelengths. The ensuing method was validated by applying it to the analysis of synthetic ternary (Eu-Dy-Tb) and quaternary mixtures (Eu-Dy-Gd-Tb) over the concentration ranges 60-550 mug Eu l(-1), 30-400 mug Dy l(-1) and 30-400 mug Tb l(-1) in the former, and 20-220 mug Eu l(-1), 20-235 mug Dy l(-1), 25-230 mug Gd l(-1) and 75-230 mug Tb l(-1) in the latter. The results obtained by using the two quantitation procedures are compared. The relative errors in the determinations were less than 8% in most cases.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.