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.     

An automatic system for acidity determination based on sequential injection titration and the monosegmented flow approach

An automatic sequential injection system, combining monosegmented flow analysis, sequential injection analysis and sequential injection titration is proposed for acidity determination. The system enables controllable sample dilution and generation of standards of required concentration in a monosegmented sequential injection manner, sequential injection titration of the prepared solutions, data collecting, and handling. It has been tested on spectrophotometric determination of acetic, citric and phosphoric acids with sodium hydroxide used as a titrant and phenolphthalein or thymolphthalein (in the case of phosphoric acid determination) as indicators. Accuracy better than |4.4|% (RE) and repeatability better than 2.9% (RSD) have been obtained. It has been applied to the determination of total acidity in vinegars and various soft drinks. The system provides low sample (less than 0.3 mL) consumption. On average, analysis of a sample takes several minutes.     

Stopped-flow injection analysis. The influence of diffusion on dispersion of normal and reverse flow injection

A relationship is derived to enable the comparison of the dispersion heights of normal and reverse flow injection analysis (FIA). A single channel flow system is employed in the absence of a chemical reaction. The stopped-flow injection method is used to probe the influence of molecular diffusion on the overall dispersion of normal and reverse FIA, which appeared to demonstrate fundamentally different diffusion behaviors. Small discrepancies are observed between the dispersion heights, which are enhanced by the stopped-flow period, especially when unmatched matrix ionic compositions of the indicator and counter solutions were involved. For these conditions, the diffusion flux rate is enhanced considerably, displaying a peak, in addition to the transient, for both methods. The influence of diffusion on the dispersion characteristics of normal and reverse FIA is discussed theoretically. Diffusion in the proposed model is postulated to oppose dispersion by convection. The latter initiates concentration gradients in the injection zone and propagates it with flow time over the dispersion zone profile. The diffusion flux then reacts in order to confine the indicator dispersion for normal FIA and to enhance it for reverse FIA. This model is consistent with the experimental results and accounts for most of the phenomena encountered. Probably owing to the influence of secondary flow phenomena, the use of coiled tubes has suppressed the effects of diffusion on the overall dispersion behavior.Part of the experimental work was performed at IMI Institute for Research and Development, Haifa, Israel.     

Time-based multisyringe flow injection system for the spectrofluorimetric determination of aluminium

A time-based multisyringe flow injection procedure with spectrofluorimetric detection is proposed in this paper for the determination of aluminium in drinking water. The flow methodology is based on the simultaneous or sequential injection of sample and chelating reagent (viz. 8-hydroxyquinoline-5-sulphonic acid) plugs using a multicommutation approach so that three successive injections may be performed with a sole displacement of the piston driver bar of the burette. Thus, an injection throughput as high as 154 h⁻¹ is achieved by sampling a 182 μl sample zone. In order to enhance the luminescence, the reaction is carried out in micellar medium using hexadecyltrimethylammonium chloride as surfactant. The influence of geometric and hydrodynamic variables as well as several parameters such as multicommutation timing, ligand and surfactant concentration and reagent pH was assessed.Under the selected working conditions, a linear dynamic range from 10 to 500 μg l⁻¹ Al(III), a 3σ detection limit of 0.5 μg l⁻¹ and a coefficient of variation of 0.6% at the 30 μg l⁻¹ level were obtained. The analytical features were compared with those reported in previous flow injection and sequential injection methods. The multisyringe technique was successfully applied to the determination of aluminium in drinking water at low mineralisation levels, validating the results by inductively coupled plasma atomic emission spectrometry.     

Optimization of a flow injection analysis system for tartaric acid determination in wines

The objective of this work is the development and optimization of a method for tartaric acid analysis in wines that does not require any sample pre-treatment and with adequate accuracy. A flow injection analysis manifold with three channels, using a dialysis unit to eliminate sample matrix interferences and to accomplish on-line dilution, is proposed for the spectrophotometrical determination of tartaric acid in wines making use of its reaction with vanadate. The proposed method is fast, accurate, simple, economic and does not require any sample pre-treatment. Preliminary studies using factorial designs were performed to determine which operational parameters should be included in the optimization stage. The optimization was performed using a modified simplex algorithm with a response function that included sensitivity, deviation from linearity at low concentrations and residence time, used as an inverse measure of sampling rate. The most relevant analytical parameters of the method are presented, including a comparison between the results provided by the proposed method and by an alternative procedure in the analysis of a set of wine samples from Portugal, with tartaric acid values in the range 0.5–4 g l⁻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.     

Development of xylitol oxidase-based flow injection analysis for monitoring of xylitol concentrations

A flow injection method for monitoring xylitol was developed using xylitol oxidase (XYO) immobilized on a VA-Epoxy Biosynth E3-support. The immobilized XYO cartridge had a good operational lifetime (up to 24 h) and storage stability (up to 1 month). The XYO-FIA system with an oxygen electrode was investigated systematically regarding the factors that can affect enzyme activity, such as pH, reaction temperature, carrier solution and sample matrix. In order to attain high activity of the immobilized XYO, potassium phosphate solution (1 M) with 0.5 g l⁻¹ Triton X-100 adjusted to pH 8.5 was used as the carrier solution. Sample matrix effects on the immobilized XYO were also investigated. High concentrations of some components (arabinose, 20 g l⁻¹; xylose, 30 g l⁻¹; NaCl, 30 g l⁻¹) in the sample had significant inhibitory effects on the response of the XYO-FIA system. The performance of the XYO-FIA system was tested by using different sample injection volumes (75-250 μl) and carrier flow rates (0.7-2.0 ml min⁻¹).     

Potentiometric multi-syringe flow injection system for determination of exchangeable potassium in soils with in-line extraction

A multi-syringe flow injection system for the potentiometric determination of exchangeable potassium in soil samples is proposed. Firstly, a manifold was devised to allow determination in soil extracts prepared off-line. It was possible to analyze samples prepared in extractants with different composition (Mehlich or Morgan) without physical or chemical modification of the manifold. A linear dynamic concentration range of 6–391 mg L^− 1 was obtained, allowing the direct introduction of soil extract without dilution. A determination frequency of 50 h^− 1 was achieved, with good repeatability for 10 consecutive injections of soil extracts (RSD < 3.0%). The in-line preparation of soil extract was implemented by automatic addition of extractant solution to a previously weighed portion of soil, followed by in-line filtration. Good repeatability was attained as the variance of the extraction procedure was not significantly different from the variance obtained in consecutive measurements of the same extract. Furthermore, results comparable to those obtained by off-line extraction and determination by flame emission spectrometry were attained for the two soil samples tested. Using this procedure, a determination frequency of 13 h^− 1 and a sampling rate of 4 h^− 1 were achieved.     

流动注射化学发光法测定佐米曲谱坦

在碱性条件下,佐米曲谱坦对鲁米诺-K3[Fe(CN)6]化学发光体系有较强的抑制作用,据此建立了佐米曲谱坦的流动注射化学发光分析法。该法的化学发光抑制值ΔI与佐米曲谱坦质量浓度在2.0×10-6~1.2×10-4g/mL范围内,呈良好的线性关系,检出限为7.6×10-7g/mL。对2.5×10-5g/mL佐米曲谱坦测定的相对标准偏差为1.2%(n=11)。方法适用于佐米曲谱坦片中佐米曲谱坦的测定。     

Microchemical determination of lodate and iodide in sea waters by flow injection analysis

A flow injection analysis method for iodate and iodide in sea water is described. The system involves spectrophotometric detection based on the catalytic, fading effect of either iodate or iodide on the indicator reaction of iron (III) thiocyanate and nitrite. With and without an anion-exchange column in the flow conduit, the system allows the determination of iodate and total iodine, respectively; iodide can be found by difference. Both iodate-iodine and total iodine can be determined in the range 0.75 to 150 g/1 on the sea water basis with analysis times of 20 min for iodate-iodine and 9 min for total iodine. The RSDs are within 1.3% for both iodate and iodide. Results are presented for the determination of iodate and iodide in sea waters and some brines associated with natural methane gas evolution.     

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.     

流动注射化学发光法测定那格列奈

在碱性介质中,那格列奈对Luminol-H2O2体系的化学发光有很强的抑制作用,据此建立了流动注射化学发光抑制法测定那格列奈的新方法.该法的化学发光抑制值△I与那格列奈的质量浓度在2.0×10-8～1.0×10-6 g/mL范围内,呈良好的线性关系,检出限为1.4×10-8 g/mL;对4.0×10-7 g/mL那格列奈连续进行11次平行测定,相对标准偏差为1.0%;通过对荧光光谱的研究,对机理进行了初步探讨.     

Novel analytical applications of porphyrin to HPLC post-column flow injection system for determination of the lanthanides

A novel indirect substitution spectrophotometric methodology using porphyrin is developed for the determination of some metals that do not react with porphyrin directly. The methodology is concerned with a multistep reaction system, which consists of 3 complexation reactions that occur in a sequence of EDTA with metal, EDTA with Cu(II) and porphyrin with Cu(II). The complexation reaction of Cu(II) with a cationic porphyrin, meso-tetrakis (4-N-trimethylaminophenyl)-porphine (ttmapp), which has a high molar absorptivity about 5 × 10⁵ l mol⁻¹ cm⁻¹, serves for spectrophotometric detection. This methodology was verified through individual determinations of 14 species of lanthanides following a batchwise procedure, which was further developed to an automatic procedure on a HPLC post-column flow injection system for simultaneous analysis of the lanthanides. Compared with the conventional post-column derivatization method using 4-(2-pyridylazo) resorcinol (PAR), the analytical sensitivity was greatly improved in this method and the results also showed good linearity.     

Multi-syringe flow injection solid-phase extraction system for on-line simultaneous spectrophotometric determination of nitro-substituted phenol isomers

In this paper, a time-based multi-syringe flow injection (MSFI) approach is proposed for automated disk-based sorbent extraction of three nitro-substituted phenol isomers (2-, 3-, and 4-nitrophenol) followed by on-line simultaneous determination of individual species by diode-array spectrophotometry. The method involves the on-line enrichment of the targeted analytes from an acidic medium containing 0.1 mol L⁻¹ HCl onto a co-polymeric sorbent material, and the concurrent removal of potentially interfering matrix components. The nitrophenol isomers are subsequently eluted with an alkaline solution (0.7 mol L⁻¹ NaOH), whereupon the eluate is delivered to a diode-array spectrophotometer for recording of the spectral data in the UV-vis region. Deconvolution of strongly overlapped spectra was conducted with multivariate regression models based on multiple linear regression calibration. The analytical performance of the chemometric algorithm was characterized by relative prediction errors and recoveries.The MSFI manifold was coupled to a multiposition selection valve to set a rugged analyzer that ensures minimum operational maintenance via exploitation of membrane switching protocols. As compared with earlier methods for isolation/pre-concentration of nitro-substituted phenols based on liquid-liquid extraction, the proposed flow-through disk-based system should be regarded as an environmentally friendly approach because the use of harmful organic solvents is circumvented. Under the optimized chemical and physical variables, the 3σ_blank detection limits for 2-, 3-, and 4-nitrophenol were 1.2, 3.2 and 0.3 μmol L⁻¹ for a sample loading volume of 1.5 mL, and the relative standard deviations were ≤5.0%. The flowing system, which is able to handle up to 135 samples automatically, was proven suitable for monitoring trace levels of the target isomers in mineral, tap, and seawater.     

Liposome-based flow injection enzyme immunoassay for theophylline

Preliminary results are presented on the development of a sensitive, quantitative immunoassay based on a regenerable, flow injection analysis system incorporating a double-amplification approach. The double amplification is achieved by means of liposome-encapsulated peroxidase enzyme molecules which are released subsequent to a competitive immunological reaction with analyte molecules for immobilized antibodies. The released peroxidase enzymatically cleaves, from an organofluorine substrate, fluoride ions which are then potentiometrically measured. The entire process is carried out in a flow injection analysis system. The competition between the analyte molecules (theophylline) and theophylline-derivatized liposomes for immobilized antibody sites in flow-through immunoreactor column results in unbound liposomes being carried downstream where they are ruptured in the presence of hydrogen peroxide andp-fluorophenol. The peroxidase molecules released react enzymatically to produce fluoride ions which are measured with an ion-selective electrode. The immunoreactor column is then regenerated with a chaotropic agent and the next sample or calibration solution is injected. By means of column regeneration and calibration, accurate quantitation can be achieved; a feature missing from conventional batch-type immunoassays. By means of this liposome/enzyme double-amplification approach, theophylline was determined over a range of concentrations from 0.2 to 4000 ng/ml. The detection limit of 200 pg/ml corresponds to about 100 femtomole of theophylline measured in the 100 l sample injected.Dedicated to Professor W. Simon on the occasion of his 60th birthday     

Downscaling a multicommuted flow injection analysis system for the photometric determination of iodate in table salt

In this work a downscaled multicommuted flow injection analysis setup for photometric determination is described. The setup consists of a flow system module and a LED based photometer, with a total internal volume of about 170 μL. The system was tested by developing an analytical procedure for the photometric determination of iodate in table salt using N,N-diethyl-henylenediamine (DPD) as the chromogenic reagent. Accuracy was accessed by applying the paired t-test between results obtained using the proposed procedure and a reference method, and no significant difference at the 95% confidence level was observed. Other profitable features, such as a low reagent consumption of 7.3 μg DPD per determination; a linear response ranging from 0.1 up to 3.0 m IO₃⁻, a relative standard deviation of 0.9% (n = 11) for samples containing 0.5 m IO₃⁻, a detection limit of 17 μg L⁻¹ IO₃⁻, a sampling throughput of 117 determination per hour, and a waste generation 600 μL per determination, were also achieved.     

An environmental friendly method for the automatic determination of hypochlorite in commercial products using multisyringe flow injection analysis

A multisyringe flow injection analysis system was used for the determination of hypochlorite in cleaning agents, by measurement of the native absorbance of hypochlorite at 292 nm. The methodology was based on the selective decomposition of hypochlorite by a cobalt oxide catalyst giving chloride and oxygen. The difference of the absorbance of the sample before and after its pass through a cobalt oxide column was selected as analytical signal. As no further reagent was required this work can be considered as a contribution to environmental friendly analytical chemistry. The entire analytical procedure, including in-line sample dilution in three steps was automated by first, dilution in a stirred miniature vessel, second by dispersion and third by in-line addition of water using multisyringe flow injection technique. The dynamic concentration range was 0.04-0.78 g L⁻¹ (relative standard deviation lower than 3%), where the extension of the hypochlorite decomposition was of 90 ± 4%. The proposed method was successfully applied to the analysis of commercial cleaning products. The accuracy of the method was established by iodometric titration.     

Application of amperometric sol-gel biosensor to flow injection determination of glucose

A glucose biosensor with enzyme immobilised by sol–gel technology was constructed and evaluated. The glucose biosensor reported is based on encapsulated GOX within a sol–gel glass, prepared with 3-aminopropyltriethoxy silane, 2-(3,4-epoxycyclohexyl)-ethyltrimetoxy silane and HCl. A flow system incorporating the amperometric biosensor constructed was developed for the determination of glucose in the 1×10⁻⁴–5×10⁻³ mol l⁻¹ range with a precision of 1.5%. The results obtained for the analysis of electrolytic solution for iv administration and human serum samples showed good agreement between the proposed method and the reference procedure, with relative error <5%.     

Conductometric determination of ammonium by a multisyringe flow injection system applying gas diffusion

A multisyringe flow injection system (MSFIA) coupled to a gas-diffusion cell has been developed for the conductometric determination of ammonium in different water samples. Operation strategies, membrane, reagent concentrations, and flow rates have been studied to optimize the sensitivity of detection and to fit the required working range. The proposed MSFIA system has been compared with former FIA and SIA systems using gas diffusion. The system was applied to the determination of ammonium in water samples of different matrices in order to evaluate its performance. These samples were coastal waters, pond waters, and compost aqueous extracts. Good recoveries of 102?±?13% were obtained and no significant differences with the reference methods were found. The system can be used for a wide concentration range of ammonia, from 0.075 to 360?mg?L^?1, without sample dilution and with a precision better than 2% of RSD. The throughput of the method was 32 injections per hour.     

Improved method for shipboard determination of iron in seawater by flow injection analysis

A flow injection analysis (FIA) catalytic spectrophotometric method for the determination of dissolved iron in seawater was further developed to yield a more sensitive assay with a low detection limit. The method employs an initial sample acidification step followed by an iron pre-concentration step involving an in-line 8-hydroxy-quinoline (8-HQ) metal-chelating resin column. The copper capacity and elution efficiency, as well as the iron FIA performance of three trace-metal clean resins were compared, resulting in the selection of a clean silica gel support for the 8-HQ ligand. The concentrated sample is eluted from the resin with an acidic carrier and mixed with reagents, initiating an iron-catalyzed, color-forming reaction. Increasing the reaction temperature from 18 to 30 °C doubled the sensitivity; reaction temperature control was necessary to obtain good reproducibility in the field. Reagent blanks were as low as 0.05 nM and a detection limit of 0.016 nM was obtained from three times the S.D. of a 0.06 nM seawater sample repeated six times. A 0.06 nM detection limit was calculated from shipboard experiments where total dissolved iron was determined for 10 different samples from the same station. The instrumental sensitivity and precision evolved to the point where the blank associated with the technique is the major factor influencing its detection limit.