An enzymatic flow injection method for the determination of oxygen

An enzymatic flow injection method for the determination of dissolved oxygen is described. Oxygen from the sample is reduced quantitatively to hydrogen peroxide in a packed-bed reactor containing immobilized glucose oxidase β-d-Glucose is used as a cosubstrate. The effluent is mixed with a stream containing chromogens and fed into a second reactor containing immobilized peroxidase. The coloured product formed is monitored spectrophotometrically. The response is linear from the detection limit (2–5 μM O₂ to air-saturated samples (0.3 mM O₂) when the peak areas are plotted versus the O₂ content in the samples (50 μl). The maximum speed for 1% carry-over is 60 samples per hour and the results are available 25 s after the start of sampling. Broadening of the peak is caused by adsorption of the coloured product in the peroxidase reactor.     

Biparametric multicommutated flow analysis system for determination of human serum phosphoesterase activity

A multicommutated flow analysis (MCFA) system constructed of microsolenoid valves and pumps offering simultaneous determination of activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) in human serum samples has been developed. The MCFA system is based on optoelectronic flow-through detector made of two light emitting diodes and operating according to paired emitter detector diode (PEDD) principle. This photometric PEDD device has been dedicated for detection of p-nitrophenol (NP) generated in the course of enzymatic hydrolysis of p-nitrophenyl phosphate and optimized for the determination of NP in human serum samples. The developed PEDD-based MCFA system allows independent optimization of conditions for reaction and detection steps of photometric ACP and ALP bioassays. Moreover, it allows elimination of photometric interferences from serum matrix components according to two-points kinetic mode of measurement. The single measurement cycle takes 12 min, consists of four measurements (two for each phosphoesterase) and enables determination of serum ACP and ALP activities at physiological and pathological levels. The real analytical utility of the developed MCFA system has been confirmed by analysis of control sera as well as real human serum samples from healthy persons and oncological patients.     

Immobilization of xanthine oxidase and its use in the quantitation of hypoxanthine in fish muscle tissue extracts using a flow injection method

Fish muscle extracts (Scomberomorus— brasiliensis- carite) were analyzed for their hypoxanthine content using a flow injection system incorporating an immobilized xanthine oxidase bioreactor. The xanthine oxidase was immobilized under mild conditions to a 2-fluoro-1-methylpyridinium Fractogel support. The uric acid produced from the oxidation of hypoxanthine by the immobilized xanthine oxidase at pH 7.0 and 35‡C was monitored at 290 ran. Hypoxanthine concentrations as low as 4.4 Μmol/L can be detected. Up to 30 samples per hour can be analyzed at a flow rate of 1 mL/min, using 150 ΜL sample volumes and a bioreactor dimension of 1.0 cm x 2.0 mm id. Recovery yields were between 92 and 99%. Both within day and between day precisions gave CVs < 5.00% (n = 30). Good correlation (r = 0.998) is obtained when 78 fish samples were analyzed for their hypoxanthine content both by this FI method and a reference HPLC method.     

Multicommutated flow system for the determination of glucose in honey with immobilized glucose oxidase reactor and spectrophotometric detection

A new automated method for the determination of glucose in honey is proposed. The method is based on multicommutated flow analysis (MCFA) and employs an immobilized glucose oxidase reactor and spectrophotometric detection at 505 nm of the red quinoneimine formed (Trinder's method).The calibration curve obeyed a second order equation in the range 0-0.14 g L⁻¹ (h = −2.2199 C² + 1.3741C + 0.0077, r² = 0.9991, where h is the peak height (absorbance) and C the concentration in g L⁻¹). The method was validated analyzing eight commercial samples, both by the AOAC 954.11 and 977.20 official methods. According to Student's t-test of mean values, at the confidence level of 95% the results obtained with the proposed method were in agreement with those obtained by the official methods. Precision (s_r(%), n = 10) was 3% and the sampling frequency of the system was 20 samples h⁻¹.     

Immobilization of L-glutamate oxidase and peroxidase for glutamate determination in flow injection analysis system

Streptomyces SP.N 14, isolated from soil samples, produced extracellular L-glutamate oxidase (GOD) in liquid culture. After a two-step ammonium sulfate purification and dextran G-150 chromatography, the specific activity was reached at 28.2 U/mg. The partial purified enzyme and horseradish peroxidase (HRP) were covalently coupled to alkylamine controlled pore glass (CPG) by means of glutaraldehyde. About 200–300 U/g of immobilized GOD and 300–400 U/g of immobilized HRP were obtained. The immobilized enzymes were packed into a teflon tube and used in flow injection analysis (FIA) for glutamate in broth. A good linear range was observed for this immobilized enzyme system at 0.1–2.0 mM, and the precision was 2.8% (n = 25). More than 80 samples were measured within an hour. One enzyme column with about 4 U of immobilized GOD and 5 U of immobilized HRP, applied for 50 assays/d, has been used for more than 50 d. The concentration of L-glutamate remaining lower than 2.0 mM, the determination of glutamate in this system was not affected by pH and temperature within the range of 6.0–7.0 and 25–35‡C, respectively. The system was applied to determine L-glutamate in broth samples during L-glutamate fermentation, and good correlation was achieved between results obtained with the system and with the Warburg’s method.     

Chemiluminescence determination of carbofuran at trace levels in lettuce and waters by flow-injection analysis

A simple, fast chemiluminescence (CL) flow-injection (FI) method based on the reaction of luminol with KMnO₄ in alkaline medium has been described for the direct determination of carbofuran. The method is based on the enhancing effect in the emission light from the oxidation of luminol produced in presence of carbofuran. The optimisation of instrumental and chemical variables influencing the CL response of the method has been carried out by applying experimental design, using the proposed flow-injection manifold. Under the optimal conditions, the CL intensity was linear for a carbofuran concentration over the range of 0.06-0.5 μg ml⁻¹, with a detection limit of 0.02 μg ml⁻¹. The method has been successfully applied to the determination of carbofuran residues in spiked water and lettuce samples.     

Flow-injection method for the determination of serine using immobilized enzyme

A flow-injection method for the determination of serine using a mini-column containing immobilized serine dehydratase isolated and purified from rat liver is described. Ammonia produced from the enzymatic reaction is coupled with hypochlorite and phenol in an alkaline medium yields a blue product due to the indophenol anion formation, which is the basis of a spectrophotometric detection at 640 nm. The limit of detection (2×blank noise) is 0.01 mM with a sample throughput of 25 h⁻¹. Calibration graph is linear in the range 0.2–1.0 mM, with relative standard deviation 0.6–1.0%.     

Immobilized enzymes - A simple calorimetric method for the determination of the catalytic activity

The results communicated in this paper show that rapid and reliable information about the activity of immobilized enzymes follows from calorimetric measurements. The study was done using spherical and plain carriers as well as different enzymes (urease, glucose-oxidase, invertase). The enzyme thermistor developed by Danielsson et al. was used as a measuring system. This measuring system was applied to investigate the activity of enzyme carrier complexes produced by the sol-gel technique. The influence of processing parameters could be pointed out at complexes of different forms (xerogel, gel on ceramic carrier, thin gel layers on foil, etc.). With the described calorimetric method, a fast and reliable technique for comparative determination of the activity of immobilized enzymes is available. A special advantage of this method is its variability in carriers and the generally applicable thermal measuring principle. Therefore, it seems useful for the development of new immobilization techniques.     

Development of the DET technique for high-resolution determination of soluble reactive phosphate profiles in sediment pore waters

The DET (diffusive equilibrium in thin films) technique is developed to measure soluble reactive phosphate (SRP) profiles in sediment pore waters at a millimetre resolution. The analytical procedure includes equilibration of the gels in sediments, section of the gels after retrieval from the sediments, back elution of phosphorus in the gels, and analysis of SRP in the eluents. Recovery of phosphorus is improved from back elution with 0.25?M nitric acid relative to deionised water. SRP concentrations in pore waters of different sediments measured by DET probes agree well with those directly measured by the colorimetric method. Pore water profiles obtained simultaneously using gel probes and other techniques (including Rhizon and dialysis peeper) also show comparability at similar resolutions. The DET probes were used to investigate pore water SRP profiles in the sediments of two contrasting regions (algal-dominated and macrophyte-dominated) in Lake Taihu. An increasingly upward movement of SRP was observed in subsurface pore waters of the algal-dominated region coupled with an increase in water temperature from March to May. Peak-shape distribution of SRP and horizontal heterogeneity was observed in pore waters of the macrophyte-dominated region, which is most likely caused by the activity of submerged macrophyte roots.     

A multisyringe flow injection method for the automated determination of sulfide in waters using a miniaturised optical fiber spectrophotometer

In this paper, a fully software-controlled multisyringe flow injection (MSFIA) spectrophotometric system is proposed for the determination of sulfide in environmental and waste waters. The implementation of ancillary solenoid valves into the flow network allows a multitude of injection modalities to be explored, the selected modality being directly dependent on the aim of the assays. The multicommuted sandwich-type approach is introduced in this work as an efficient means to warrant high sensitivity for the particular assay with excellent repeatabilities and a considerable reagent saving. Moreover, a high injection frequency may be easily attained by carrying out a multiple injection modality during a single forward displacement of the piston driver bar. The interfacing of the robust and versatile multisyringe piston pump with an optical fiber plug-in spectrophotometer furnished with a light emitting diode enables the miniaturization of the flow analyzer, which is thus readily adaptable to in-situ and real-time monitoring schemes. The flow method is based on the coupling Fischer’s reaction of sulfide with N,N-dimethyl-p-phenylenediamine in the presence of Fe(III) as oxidizing reagent in a 0.7 M HCl medium. Careful selection of the physical and chemical variables enabled coefficients of variations better than 1.5% (n = 10) at the 1 mg l⁻¹ level for both injection modalities. Dynamic working ranges of 0.2–2.0 and 0.5–5 mg l⁻¹ sulfide for sandwich and multiple injection techniques, and detection limits of 0.09 and 0.15 mg l⁻¹, respectively, were obtained. Furthermore, the sandwich modality featured an average slope of 0.43 ± 0.02 l mg⁻¹ calculated from 10 day-to-day calibration plots. This result reveals better sensitivity than other flowing stream methods described in the literature. The multiple injection technique allowed an improvement of the injection throughput up to 80 h⁻¹, although a decrease of sensitivity was concomitantly observed (average slope of 0.17 ± 0.01 l mg⁻¹).

The multisyringe flow method was successfully applied to the determination of sulfide in different spiked water matrices (namely, mineral, tap, freshwater, seawater and wastewater) with recoveries ranging from 96 to 104%. Good agreement was also found in water samples between the MSFIA results and those of the batch APHA-standard method.     

Validation of an environmental friendly segmented flow method for the determination of phenol index in waters as alternative to the conventional one

Phenolic compounds are a sort of common pollutants in water. Phenol index becomes an expedite indicator for the evaluation of the contamination level of water samples, in spite of the knowledge of the individual phenol and its derivatives are also important.In this work, an environmental friendly method for the determination of phenols, using a segmented flow system based on the conventional method's reactions without the liquid-liquid extraction step, was validated.Three linear dynamic ranges using C₆H₅OH: 1-10 μg l⁻¹, 10-200 μg l⁻¹ and 0.2-2.5 mg l⁻¹, with a coefficient of variation lower than 2%, were obtained. Several method's performance parameters were determined: limits of detection, limits of quantification, precision through duplicate analysis and trueness using the reference materials purchased from LGC Promochem, RTC no. QCI-043-2 Lot:P1. Measurement uncertainty was evaluated using an interlaboratory approach based on proficiency testing data. Relative combined uncertainty for phenols in water samples, , of 0.054 were obtained, in according to those imposed by the Portuguese Legislation: target for 1 μg l⁻¹ of phenol (surface waters) and target for 500 μg l⁻¹ of phenol (wastewaters).A high efficiency reduction and elimination of reagents and wastes, reduction of analysis time and exposition of the analyst were also obtained.     

纳米银增敏鲁米诺液相化学发光分析法测定4-乙酰氨基酚的研究

研究发现在碱性条件下,纳米银对鲁米诺-铁氰化钾液相化学发光体系发光信号具有明显的增敏作用,而4-乙酰氨基酚对该体系具有强烈的抑制作用。结合流动注射技术,建立了流动注射化学发光分析法测定对乙酰氨基酚的新方法。该方法测定对乙酰氨基酚的线性范围为9.0×10-12～1.0×10-10g/mL(0.9947)和1.0×10-1...     

Spectrophotometric determination of chloride in waters using a multisyringe flow injection system

A multisyringe flow injection system (MSFIA) with spectrophotometric detection is proposed as a fast, robust and low-reagent consumption system for the determination of chloride (Cl⁻) in waters. The system is based in the classic reaction of Cl⁻ with Fe³⁺ and Hg(SCN)₂, but due to the hazardous properties of this last reagent, the proposed methodology has been developed with the aim to minimize the consumption of this one, consuming less than 0.05 mg of Hg for a Cl⁻ determination, being the system of this type with the lowest Hg consumption. The linear working range was between 1 and 40 mg L⁻¹ Cl⁻ and the detection limit was 0.2 mg L⁻¹ Cl⁻. The repeatability (RSD) was 0.8% for a 10 mg L⁻¹ Cl⁻ solution, and the injection throughput was 130 h⁻¹. The proposed system is compared with other chloride monitoring flow systems, this comparison is realized with a point of view of the equilibrium between the obtained analytical features and produced residues toxicity. The proposed system was applied to the determination of Cl⁻ in mineral, tap and well water.     

An enzymatic method for TLC detection and determination of fenitrothion in water

A simple enzymatic method is described for field TLC detection and determination of fenitrothion as fenitrooxon in water, with pig liver acetone powder as enzyme source. By this method, fenitrothion can be detected as fenitrooxon at ng levels and amounts ranging from 5 to 50 ng can be estimated.     

高效液相色谱指纹图谱法分析南方红豆杉药材的氯仿提取物

建立了南方红豆杉药材氯仿提取物的高效液相色谱(HPLC)指纹图谱分析方法。采用Eurospher 100 C18色谱柱(250 mm×4 mm, 5 μm),以甲醇和水为流动相进行梯度洗脱,流速为1 mL/min,检测波长为232 nm,柱温为30 ℃。以10-脱乙酰巴卡亭III(10-DABIII)为参照物,在相同的色谱条件下测定了10批不同产地的南方红豆杉药材氯仿提取物的指纹图谱,获得了11个共有指纹峰,并利用主成分分析法(PCA)对指纹图谱进行统计分析。结果表明南方红豆杉药材的质量与种植区域有关。该方法稳定、可靠,可用于南方红豆杉药材的质量控制。     

Method development based on all injection analysis for the determination of phosphorus in soil and sediment extracts

In this work, we have modified the technique of all injection analysis (AIA) by changing the position of the detector. The detection is then located as a part of the circulatory loop. With this new detector position, we could monitor for many numbers of circulation. The sensitivity was improved by using the cumulative signal data obtained when the number of circulation rounds was increased. The dilution effect using this new detector location was also less than that with the previous system. We employed a four-channel peristaltic pump to aspirate four types of liquids into the system together at one time. The AIA method was then developed for determination of phosphorus in soils and sediment extracts. The method was optimized for the new harmonized scheme of extraction that has been developed by the European Commission.     

Micro-extraction procedures for the determination of Ra-226 in well waters by SF-ICP-MS

The radium-226 (t_1/2 = 1622 years) content of highly alkaline well water collected from the United Arab Emirates (UAE) was measured by double focusing sector-field inductively coupled plasma-mass spectrometry (SF-ICP-MS) after separation of the radium from other alkaline earth elements using a newly developed procedure. The results were comparable with those obtained by α-spectrometry for samples with concentrations ranging from 6.75 to 459 pg/L (0.25 to 17 Bq/L). Instrumental sensitivity on matrix-free samples was compared for two sample introduction systems, i.e. an Apex-Q high sensitivity system and a concentric nebulizer. A 12-fold improvement in sensitivity (instrumental detection limit = 1.5 pg/L or 55 mBq/L) was found when the Apex-Q system was used. Two chromatographic methods were tested for the sequential separation of the alkaline earth elements contained in the well water samples in order to reduce matrix and polyatomic interference effects. Optimal elution parameters were determined and used for the separation and pre-concentration of Ra-226 in those samples. A method detection limit of 0.189 pg/L (7 mBq/L), which corresponds to a mass of 0.38 fg of Ra-226 in the sample, was achieved. Only 2 mL of sample is necessary when a combination of 50 W-X8 and Sr*Spec resin, which are reusable, are utilized for the separation. This new analytical protocol significantly reduces sample preparation time resulting in a throughput rate of approximately 20 samples in only 8 h; faster than the other published extraction procedures.     

Sequential molecular vapor elution analysis for the separation and determination of LiCl and NaCl in river waters

The determination of alkali metal chloride in river waters by sequential molecular vapor elution analysis (SMVEA) using a molecular absorption detector (MA) is reported. An improved molybdenum column was developed for SMVEA. An optimum flow rate of carrier gas (pure argon or nitrogen) for separation of metal vapors was 12.0 ml min⁻¹. Lithium chloride and sodium chloride peaks completely separated from potassium, rubidium, and cesium chlorides but CaCl₂, FeCl₂, MgCl₂, and MnCl₂ peaks did not appear at a column temperature of 1000 °C and vaporization temperature of 1000 °C. The appearing order of these metal chlorides was LiCl, NaCl, KCl, RbCl, and CsCl. It was not understood by considering the boiling points of these metal chlorides. The delay of appearing time is due to an interaction between the molecular vapors and inside surface of the column. Under the experimental conditions, the number of theoretical plates was 247 for LiCl and 268 for NaCl in the improved column. Under the optimal experimental conditions, river waters were analyzed for lithium and sodium chlorides. The analytical results agreed well with the recoveries were in the range of 94–105%. By SMVEA, it was found that lithium and sodium chlorides in waters were determined without interference of matrix elements, no chemical treatment for river water samples.     

An improved fluorometric coupled enzymatic method for the determination of pyrophosphate in plasma and platelets

A fluorometric coupled enzymatic method for the determination of pyrophosphate is described. The kinetic method can determine pyrophosphate in amounts as low as 0.5 pmol. Intra- and interassay CV's were both less than 1% and recoveries were found to be quantitative. No significant interference was observed from EDTA, heparin, magnesium, or calcium. Correlation with an established method was significant (r = 0.87).Clinical studies of the applicability of the procedure were demonstrated by the measurement of pyrophosphate in plasma and platelets. Mean pyrophosphate levels in plasma and platelets of “normal” subjects were found to be 3.27 ± 0.2 μM and 2.40 ± 0.10 nmol/10⁸ platelets or 17.59 ± 1.44 nmol/mg platelet protein, respectively.Pyrophosphate levels in plasma and platelets of patients on hemodialysis were determined and found to be significantly lower than the “normal” population. This finding may in part explain the occurrence of metastatic calcification seen in this patient population.