FIA System Research Articles

Composites: A novel alternative to construct solid state Ag/AgCl reference electrodes

An Ag/AgCl solid-state reference electrode is developed by means of a graphite–AgCl–Silver dag-epoxy resin composite. The response of the composite reference electrode (CRE) to chloride ions is evaluated; a linear non-nernstian response is observed associated to the following equation E = −15.15 (±1.10) − 44.05 (±0.38) log[Cl −]. Comparing the CRE's response potential versus a saturated commercial Ag/AgCl reference in KCl 0.1 M, a mean of 40.7 ± 0.4 mV of the distribution of potential versus time data is observed over a period of 1 h. The performance of the CRE as reference for a glass membrane electrode by means of direct pH measurements and quantitative determination of acids by acid–base titrations is evaluated obtaining statistically stable, precise and exact results compared with those obtained using a combined glass electrode. The typical cylindrical configuration of the CRE is changed to adapt it to a FIA system for the determination of ammonium ion, obtaining a sensitivity 50.30 ± 0.26 mV/log[NH 4 +] and a linear range 8.5 × 10 −5 to 0.1 M, which are analytical parameters statistically equivalent to those presented by the classical determination system.

Preparation and assay performance of supramolecule of cyclophane-complexed polyoxometalates supported on the gold surface

A novel supramolecular complex of octamolybdates and cyclodextrin was simply prepared for the first time by self-assembled method under room temperature. Successful formation of the functional material was demonstrated by carrying out analysis on its thin films supported on the gold surface by various electrochemical techniques, based on the redox probe of octamolybdates contained in the assembly. Sensing and assay performance of the final functional films were evaluated in FIA system by determining oxo anions in samples based on the electroactivity of surface-confined molybdenum catalyst.

High-Throughput Nanoliter Sample Introduction Microfluidic Chip-Based Flow Injection Analysis System with Gravity-Driven Flows

In this work, a simple, robust, and automated microfluidic chip-based FIA system with gravity-driven flows and liquid-core waveguide (LCW) spectrometric detection was developed. The high-throughput sample introduction system was composed of a capillary sampling probe and an array of horizontally positioned microsample vials with a slot fabricated on the bottom of each vial. FI sample loading and injection were performed by linearly moving the array of vials filled alternately with 50-microL samples and carrier, allowing the probe inlet to enter the solutions in the vials through the slots sequentially and the sample and carrier solution to be introduced into the chip driven by gravity. The performance of the system was demonstrated using the complexation of o-phenanthroline with Fe(II) as a model reaction. A 20-mm-long Teflon AF 2400 capillary (50-microm i.d., 375-microm o.d.) was connected to the chip to function as a LCW detection flow cell with a cell volume of 40 nL and effective path length of 1.7 cm. Linear absorbance response was obtained in the range of 1.0-100 microM Fe(II) (r2=0.9967), and a good reproducibility of 0.6% RSD (n=18) was achieved. The sensitivity was comparable with that obtained using conventional FIA systems, which typically consume 10,000-fold more sample. The highest sampling throughput of 1000 h-1 was obtained by using injection times of 0.08 and 3.4 s for sample and carrier solution, respectively, with a sample consumption of only 0.6 nL for each cycle.

Integration of a glucose biosensor based on an epoxy-graphite-TTF·TCNQ-GOD biocomposite into a FIA system

An amperometric glucose biosensor based on graphite and non-conducting epoxy resin biocomposite was constructed. Glucose oxidase (GOD) and the tetrathiafulvalene–tetracyanoquinodimethane (TTF·TCNQ) conducting organic salt were incorporated into the bulk of the composite to form a renewable biosensor. Several graphite-TTF·TCNQ ratios (w/w) were studied in order to select the best biosensor to be integrated in a FIA system for the automated detection of glucose. The optimal amount of GOD in the composite was studied as well. The selection was based on the analytical response of the electrodes. Best results were obtained by an electrode whose composition was 5% GOD, 76% polymer, 9.5% graphite and 9.5% TTF·TCNQ. An especially designed flow amperometric cell was constructed so that the biosensor could be integrated into a FIA system and glucose in beverage samples could be determined.

A BIPOTENTIOMETRIC FIA SYSTEM FOR DETERMINATION OF VITAMIN C

A bipotentiometric FIA system has been developed for the determination of vitamin C. The system was a single stream manifold, which consisted of a peristaltic pump, a self made injector with internal loop and two Pt-electrodes connected to a PC by serial ADC or parallel ADC interface card. One of the Ptelectrodes was located in the injector before the internal loop, while the other electrodes at the end of the TEFLON reaction tube. The PC was supported with a self-made software for controlling the pump, signal acquisition, processing, and visualisation, and further peak determination, followed by calibration of standard solutions and calculation of sample concentrations. The above FIA-system configuration was applied to determine vitamin C by the injection of sample solution into a stream of potassium hexacyanoferrate (III) solution. The optimum result was achieved with knitted tube reaction of 0.8 mm i.d., 50 cm length, at flow rate 0,64 ml/minute, and consumed 3.6 ml of carrier solution per five injection, and gave linearity range at 0.001 – 0.100 N vitamin C, with the coefficient of correlation 0.9999, RSD peak to peak better than 3.0% and the error of artificial samples were less than 3.4 %. Key Words : Bipotentiometric-FIA, injector with internal loop, module-program, vitamin C

A Sequential Enzymatic Microreactor System for Ethanol Detection of Gasohol Mixtures

A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single micro reactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 +/- 2.3% and 63.2 +/- 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1-30 % v/v (10.0-238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034-0.087 g ethanol/L working with the on-line dilution integrated to the biosensor FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated.

Determination of As(III) and As(V) ions by chemiluminescence method

A new chemiluminescence (CL) method for the selective determination of As(III) and As(V) ions in aqueous solution has been studied using a FIA system. The method is based on the increased CL intensity with the addition of As(V) ion into a solution of lucigenin and hydrogen peroxide. The addition of As(III) ion into the solution did not change the CL intensity. Total concentration of As ions was determined after pre-oxidation of As(III) to As(V) with hydrogen peroxide in basic solution. The As(III) content was estimated by subtracting the content of As(V) ion from total As concentration. The effects of concentrations of KOH and H 2O 2, and flow rates of reagents on CL intensity have been investigated. The calibration curve for As(V) ion was linear over the range from 1.0×10 -2 to 10 μg/g, the coefficient of correlation was 0.997 and the detection limit was 5.0×10 -3 μg/g under the optimal experimental conditions.

Micro-analytical GO/HRP bioreactor for glucose determination and bioprocess monitoring

A bi-enzymatic micro-analytical bioreactor integrated in a FIA system for glucose measurements is described. Its robustness and small dimensions (working volume of about 70 μl containing approximately 1.2 mg GO and 0.26 mg HRP) make it easy to operate. The column is based on immobilisation of glucose oxidase (GO) and horseradish peroxidase (HRP) on alkylamine controlled pore glass (CPG) beads. The column has excellent shelf life (no significant loss of activity after 1 year if kept at 4 °C), and a very high operational stability that was demonstrated through extensive usage for glucose determinations over 1 year period during which the column retained almost all of its activity. More importantly, this operational stability allows glucose monitoring in the culture media without a decay of signal over the experiment time and consequently no signal correction or re-calibration is needed. This high operational stability was also confirmed by continuous glucose conversion with 30% activity loss after converting quantity of glucose equivalent to 21600 FIA injections of 20 μl with 1.7 mM glucose. Such good performance is a result of an optimised immobilisation method and moreover of the implementation of in situ enzyme stabilisation strategy which consisted on promoting the instantaneous H 2O 2 consumption produced by the GO. This strategy has the additional advantage of allowing concomitant assay of the H 2O 2 based on the HAP catalysed co-oxidation of phenol-4-sulphonic acid (PSA) in the presence of 4-aminoantipyrine (4-AAP). The glucose measurements are reproducible with high precision against the standard HPLC method. Linear range and sensitivity depend on sample injection volume; the upper limit is about 1.1 g/l. Lower detection limit is 10 mg/l. The column performance has been validated for E. coli and S. cerevisiae fermentation monitoring, and glucose measurements in an animal cell culture (rat Langerhans islets).

Improved FIA-ABTS Method for Antioxidant Capacity Determination in Different Biological Samples

In order to evaluate the actual antioxidant features of foods, beverages and also plasma from patients, a number of assays have been developed in the last few years to determine the so called total antioxidant activity (TAA), intended as the cumulative capacity of a biological sample to scavenge free radicals. Most of the assays partially failed in obtaining a good reproducibility when using plasma because it is composed of a large number of substances, some of which are present at very high concentrations and possess masking features. For these reasons we have improved the widely known ABTS method by means of a FIA system where both temperature and dispersion of sample and reagent were strictly controlled. We found that temperature may be a critical aspect in the measurement of plasma TAA whilst its influence may be less important in the assay of non-complex biological samples. We demonstrated that also the reaction time may be critical, depending on the nature of the substance employed. Data confirmed the high TAA of a methylsalicylate-containing mouthrinse as well as the negligible TAA offered by the chlorhexidine containing one. White wines (Verdicchio) also displayed interesting TAA values. The improved method was useful to screen rapidly, without dilution, with very limited handling of the sample and with high repeatability the TAA of plasma in addition to chemical products, beverages and non-complex biological mixtures.

Catalase-based thin-layer enzyme cell used in organic-phase FIA system for determination of moisture in oily foods

The aim of our present work was to study the possibility of constructing a biosensor based on immobilized catalase enzyme (EC 1.11.1.6.) in organic-phase solutions. The catalase enzyme was immobilized by glutaraldehyde on a natural protein membrane in a thin-layer enzyme cell, connected to a stopped-flow injection analyser (SFIA) system with an amperometric detector. Adding FMCA to acetonitrile, the optimal concentration was 7.5 mg l−1, while with TBATS it was 2.7 mg l−1. The optimal pH value of the immobilized enzyme in buffer was about 6.0. On studying the role of the buffer solution used in the carrier solvent, the activity of the enzyme changed dramatically. The signal was highest when there was no buffer added to the carrier solution and decreased rapidly when the content increased (0–1.5%). Utilizing these results, a quick analytical method was developed to monitor indirectly the water content (activator) in various butter and margarine samples by maintaining a fixed substrate concentration. The water content of samples was compared with the results obtained by the gravimetric reference method (AOAC Method 920.116); the correlation coefficient (r) was 0.993.

Development of Methodologies for Different Degrees of Resolution of Linear Alkylbenzene Sulfonates in Groundwater and Wastewater by Liquid Chromatography Using Sodium Dodecyl Sulphate

Linear alkylbenzenesulfonates (LAS) are used extensively as surfactants in consumer formulations as a complex mixture of homologues and isomers. Separation of homologues and isomers of LAS is important in industrial and environmental samples in order to establish their behaviour. Here we present a HPLC methodology with fluorescent detection (FD) for the determination of the homologues and isomers as well as the sum of LAS present. The quantification of total LAS was achieved without a column, using the liquid chromatograph as a FIA system. The different homologues were separated using a Lichrospher-100 RP-8 column of 125 × 4 mm using a linear gradient of methanol and 30.0 mM sodium dodecyl sulphate (SDS), increasing the methanol content linearly from 55% to 70% in 16 min. The resolution of isomers was carried out by using two coupled Lichrospher-100 RP-18 columns of 250 × 4 mm. Elution of isomers was done with a gradient of flow rate from 1.0 to 0.25 mL min−1 using a linear gradient of acetonitrile and 5.0 mM SDS increasing the acetonitrile content linearly from 20% to 40% in 160 min. The methods were validated and applied satisfactorily to the determination of LAS in urban wastewater and ground water.

On-line monitoring and control of substrate concentrations in biological processes by flow injection analysis systems

Concentrations of substrates, glucose, and ammionia in biological processes have been on-line monitored by using glucose-flow injection (FIA) and ammonia-FIA systems. Based on the on-line monitored data the concentrations of substrates have been controlled by an on-off controller, a PID controller, and a neural network (NN) based controller. A simulation program has been developed to test the control quality of each controller and to estimate the control parameters. The on-off controller often produced high oscillations at the set point due to its low robustness. The control quality of a PID controller could have been improved by a high analysis frequency and by a short residence time of sample in a FIA system. A NN-based controller with 3 layers has been developed, and a 3(input)-2(hidden)-1(output) network structure has been found to be optimal for the NN-based controller. The performance of the three controllers has been tested in a simulated process as well as in a cultivation process ofSaccharomyces cerevisiae, and the performance has also been compared to simulation results. The NN-based controller with the 3-2-1 network structure was robust and stable against some disturbances, such as a sudden injection of distilled water into a biological process.

Focussed microwave-assisted sample preparation: total phenol determination in petroleum refinery effluents by flow injection spectrophotometry

This paper reports the development of a new approach for total phenol distillation using a focussed microwave oven, aiming its determination in petroleum refinery effluents and sour waters. In the procedure, 25 ml of sample is distilled during 15 min at 210 W power. At these conditions, recoveries as high as 95% are obtained, making possible the determination of total phenol in the samples without any interference and in a time significantly lower than that required by the reference method. In the course of the research, the influence of the distilled volume was investigated and a Doehlert matrix was employed for the multivariate optimization of the irradiation power and time. Quantification of phenol was spectrophotometrically performed in a FIA system, exploring classical reaction of phenol with 4-amineantipyrine and ferricyanide. Aqueous standards solutions of phenol could be directly injected in the FIA system for calibration purposes, making the procedure very simple and low time-consuming. A detection limit of 10 μg l −1 was achieved as well as a quantification limit of 33 μg l −1, becoming the procedure very suitable to be applied in the control of total phenols in effluents from petroleum industry.

A novel strategy to verification of adulteration in alcoholic beverages based on Schlieren effect measurements and chemometric techniques

A novel strategy to evaluation of adulteration in alcoholic beverages based on the measurement of the Schlieren effect using an automated FIA system with photometric detection is proposed. The assay is based on the Schlieren effect produced when beverage samples are injected in a single-line FIA system that uses water as carrier stream and a light-emitting diode–phototransistor photometer controlled by microcomputer as detector. The flow system presents limited mixing conditions which make possible to create gradients of refractive index (Schlieren effect) in the injected sample zone. These gradients are reproducible, characteristic of each alcoholic beverage and undergo specific modifications when adulterations with water or ethanol are imposed. Schlieren effect data of brandies, cachaças, rums, whiskies and vodkas were treated by SIMCA to elaborate class models applied in the evaluation of alcoholic beverages adulteration. Samples of the original matrix of each sort of beverages were adulterated in laboratory by adding water, methanol and ethanol in levels of 5% and 10% (v/v). These samples were used as test set to validate SIMCA class models. The verification of authenticity using Schlieren effect measurements presented good results making possible to identify 100% of the beverages samples adulterated in laboratory and 93% of the actual adulterated alcoholic beverages with confidence levels of 95%. As principal advantage, the automated system does not use reagents to carry out the analysis.

Sequential determination of iron(II) and iron(III) in pharmaceutical by flow-injection analysis with spectrophotometric detection.

A flow injection procedure for the sequential spectrophotometric determination of iron(II) and iron(III) in pharmaceutical products is described. The method is based on the catalytic effect of iron(II) on the oxidation of iodide by bromate at pH = 4.0. The reaction was monitored spectrophotometrically by measuring the absorbance of produced triiodide ion at 352 nm. The activating effect for the catalysis of iron(II) was extremely exhibited in the presence of oxalate ions, while oxalate acted as a masking agent for iron(III). The iron(III) in a sample solution could be determined by passing through a Cd-Hg reductor column introduced in the FIA system to reduce iron(III) to iron(II), which allows total iron determination. Under the optimum conditions, iron(II) and iron(III) could be determined over the range of 0.05 - 5.0 and 0.10 - 5.0 microg ml(-1), respectively with a sampling rate of 17 +/- 5 h(-1). The experimental limits of detection were 0.03 and 0.04 microg ml(-1) for iron(II) and iron(III), respectively. The proposed method was successfully applied to the speciation of iron in pharmaceutical products.

A Study for the Validation of Spectrophotometric Methods for Detection, and of Digestion Methods Using a Flow Injection Manifold, for the Determination of Total Phosphorus in Wastewaters1

A study for the validation of orthophosphate determination using an FI-manifold/spectrophotometric detection set-up is made. Under optimized conditions, the method with the largest valid range (0–50 mg L–1), with the possibility of extending it even higher) is the molybdate/ascorbic acid method; its reproducibility and interferences are similar to other tested methods. A study among the conventional digestion methods is then made to test the adaptability of the method to FI systems. The persulfate digestion method was found to be more suitable for use with flow systems. The persulfate digester was connected in-line with the FI-manifold; under optimized conditions, the sensitivity of the method is decreased, especially when used with organic phosphorus compounds, but it is still reasonable for use. The sensitivity is also dependent on the nature of the phosphate species attached to the organic residue. In addition, it was found that colloid precipitate phase in the sample decreases the sensitivity. Finally, real wastewater samples were tested using the UV radiation-assisted, persulfate digester-implemented FI-system method, and using the batch method. It was found from the correlation of the two methods that the fraction of P-recovery in the FIA system was 2/3 of the total phosphate, suggesting that the rest was due to the polyphosphate content.

Automatic Determination of Uric Acid in Urine in a FIA System with a Tubular Amperometric Detector

This paper describes the use of a FIA system for the automatic determination of uric acid in urine, using a tubular amperometric detector. In this system, glassy carbon electrodes of tubular configuration were activated by successive cyclical scans from - 0.30 to 2.00 V versus Ag/AgCl reference electrode in 0.50 mol.L -1 sulphuric acid solution. Samples were diluted in supporting electrolyte before analysis and no other pre-treatment was employed. Such dilution enabled the matrix effects to be reduced and therefore improving the sensitivity provided by the activation step that allows the uric acid (UA) to be detected at lower concentration levels after dilution. Uric acid determination was performed in a single channel FIA manifold, which provided reproducibility of sample transport to the detector and enabled a sampling rate of 120 samples per hour to be achieved. The tubular configuration of the cell offered robustness to the system since it was rigidly fixed to the manifold, allowing the manifold to be generalised for routine analysis application. The results obtained with the FIA system for UA determination in urine were compared with those from the enzymatic method used in clinical analysis laboratories. No statistical difference between methods was found at the 95% confidence level. Relative deviations between both methods were within ± 4% and the proposed system showed good repeatability (about 3%, n = 10).

A miniaturized FIA system for the determination of residual chlorine in environmental water samples.

The present study was aimed at developing a portable miniaturized FIA system with a two-electrode based amperometric detector for on-site monitoring of residual chlorine in environmental samples. The electrode potential control and current data collection were conducted with a home-designed potentiostat with a size of 150 x 100 x 40 mm and the weight of 350 g. It was confirmed that the amperometric response increased in proportion to the chlorine concentration up to 5 microg/ml, and the detection limit for chlorine was 0.05 microg/ml. Besides a drastic reduction in the size of the analytical system, such miniaturization achieved the advantages of increased speed, minimal mobile-phase consumption, and high sensitivity. The proposed method was successfully applied to the determination of residual chlorine in samples of pool water and tap water. The results were well agreed with those obtained by the N,N-diethyl-p-phenylenediamine (DPD) photometric method.

Flow injection analysis system for on-line cutinase activity assay

A FIA system based on a micellar system for a substrate ( p-nitrophenylbutyrate) with low stability in aqueous phase was built to monitor cutinase activity in bioprocesses. All samples were previously diluted with 50 mM phosphate buffer pH 7.0 containing 11.6 mM sodium cholate. The cutinase activity in this diluted solution enhances about 40% in relation to phosphate buffer. Furthermore, the enzyme adsorption and consequent blocking/fouling of injector and tubes of the FIA system was eliminated due to excellent properties of sodium cholate as surface active agent. The cutinase activity is based in following the hydrolysis of p-nitrophenylbutyrate to p-nitrophenol in the reaction stream through the formation of an absorbance peak at 400 nm proportional to enzyme activity. The compositions of reaction stream as well as its stability were studied in order to minimize non-enzymatic hydrolysis of p-nitrophenylbutyrate and maximize cutinase activity assay reproducibility. An excellent correlation was obtained between the FIA system and off-line method for determination of cutinase activity in the culture media, and during separation of Saccharomyces cerevisiae cells and cutinase concentration by micro and ultrafiltration, respectively.

Digital Pulse ac Voltammetry for the Simultaneous Analysis of Electroactive and Electrosorptive Species in Flow Systems

A digital two-step and three-step pulse potential ac voltammetry system was proposed and applied for the simultaneous analysis of electrosorptive and electroactive species in flow systems. To perform the ac polarography function, a PC was interfaced to a potentiostat to mimic all the necessary hardware functions of an analog ac polarograph. From the measurement of the change of phase-selective charging current and the zero-order current, I(3)(-), Br(-) specifically adsorbed and Cd(2+), Pb(2+) reduced at a hanging mercury drop electrode can be determined simultaneously in a FIA and IC system. With the digital pulse ac voltammetry-coupled IC, detection limits as low as 5.0 microM and linear dynamic ranges from 5.0 to 100 or 200 microM with linear correlation coefficients better than 0.9990 were found for the analysis of I(3)(-), Br(-), and S(2)O(3)(2)(-).