Flow Injection Analysis Set-up Research Articles

Investigation of bovine serum albumin/tropicamide interaction using a quartz crystal microbalance sensor

This study describes the kinetics and evaluation of thermodynamic parameters of bovine serum albumin (BSA) and tropicamide (TPA) interactions, by using quartz crystal microbalance (QCM) technique, under similar physiological status. BSA was immobilized on the gold electrode surface of a QCM by using cystamine and glutaraldehyde as crosslinking agent. The immobilizing steps were followed by cyclic voltammetry technique. A flow injection analysis set-up was applied for injection of the drug solutions into the fluid system. Kinetics of the interaction was interpreted by zero- and pseudo-first-order kinetic models, depending on TPA concentration and temperature. The binding constants (k1 = 9.12, 6.55 and 6.8 mol−1 L s−1), dissociation rate constants (k−1 = 0.0028, 0.0049 and 0.0102 s−1), the binding equilibrium constant (K = 3257, 1337 and 635 mol−1 L) and the thermodynamic parameters of the interaction were estimated at different temperatures (293, 303 and 310 K, respectively). The results revealed that hydrogen bonding and van der Waals forces are the major types of interactions of between BSA and TPA.

Completely automated in-syringe dispersive liquid–liquid microextraction using solvents lighter than water

This paper describes the development of a new multisyringe flow injection analysis set-up that enables the complete automation of the dispersive liquid-liquid microextraction (DLLME) technique using solvents lighter than water. Its hyphenation with a liquid chromatographic separation is implemented using a single multisyringe pump obtaining a compact, simple, easy to operate, and fast instrument. DLLME is carried out with a throughput of 42 h(-1) and DLLME for the extraction of benzo(a)pyrene and its subsequent chromatographic determination can be carried out with an analysis throughput of 7 h(-1).

Flow Injection Analysis for the Direct Quantification of the Total Polyphenol Content in Coffee Brews

A flow-injection analysis (FIA) procedure with colorimetric detection was developed, to determine the total polyphenol content (TPP) in coffee brews with no need for sample work-up. A comparison of three different officially accepted colorimetric methods was performed and the Folin-Ciocalteu reaction was found to be the most suitable assay with respect to sensitivity, selectivity, lack of matrix effects and cost. Furthermore, the FIA setup adopted here was characterized by its simple experimental design, relatively inexpensive equipment and for providing results that are excellent in view of rapidity, accuracy and precision. FIA key parameters were optimised for sensitivity, linearity, working range and matrix effects, and calibrated against gallic, caffeic, ferulic and tannic acid equivalents. The method was successfully tested against false-positive adsorptions from non-polyphenol components. The method was applied first to brews from two different C. Arabica (Coffea arabica L.) and C. Robusta (Coffea canephora var. robusta L) coffees (light roast). Robusta showed 25% higher TPP content compared to Arabica. Secondly, coffees roasted to three different roast degrees, each along three different time-temperature roasting profiles, were compared: High-temperature with short roasting times showed higher TPP content in the brew than low-temperature with longer roasting times, an observation that indicates the potential for optimising the roasting process to increase polyphenol content.

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 IO 3 −, a relative standard deviation of 0.9% ( n = 11) for samples containing 0.5 m IO 3 −, a detection limit of 17 μg L −1 IO 3 −, a sampling throughput of 117 determination per hour, and a waste generation 600 μL per determination, were also achieved.

A Novel Type of Tri-Colour Light-Emitting-Diode-Based Spectrometric Detector for Low-Budget Flow-Injection Analysis

In this paper we describe a low-cost spectrometric detector that can be easily assembled in a laboratory for less than €80 with a minimal number of optical components and which has proved sensitive and flexible enough for real-life applications. The starting point for the idea to construct this small, compact low-cost spectrometric detector was the decision to use a tri-colour light-emitting diode (LED) of the red-green-blue (RGB) type as a light source with the objective of achieving some flexibility in the selection of the wavelength (430 nm, 565 nm, 625 nm) but avoiding the use of optical fibres. Due to the dislocation of the emitters of the different coloured light, the tri-colour LED-based detector required an optical geometry that differs from those that are described in literature. The proposed novel geometry, with a coil-type glass flow-through cell with up to four ascending turns, proved useful and fit for the purpose. The simplicity of the device means it requires a minimal number of optical components, i.e., only a tri-colour LED and a photoresistor. In order to make a flow-injection analysis (FIA) with the spectrometric detector even more accessible for those with a limited budget, we additionally describe a low-cost simplified syringe-pump- based FIA set-up (€625), the assembling of which requires no more than basic technical facilities. We used such a set-up to test the performance of the proposed spectrometric detector for flow-injection analyses. The tests proved its suitability for real-life applications. The design procedures are also described.

Bienzymatic analytical microreactors for glucose, lactate, ethanol, galactose and l-amino acid monitoring in cell culture media

A new alternative method for bioprocess monitoring based on bienzymatic analytical microreactors integrated in a flow injection analysis (FIA) system is described. Glucose-, alcohol-, lactate-, galactose- and l-amino acid oxidases (GO, AO, LacO, GalO and LAAO) and horseradish peroxidase (HRP) are immobilized on controlled pore glass (CPG) and used for the development of glucose, ethanol, lactate, galactose and amino acid sensors. The analytical methodology is based on HRP catalysed reaction of hydrogen peroxide produced by oxidases with phenol-4-sulfonic acid and 4-aminoantipyrine. The immobilized enzymes are characterized and used for preparation of the packed bed analytical microreactors. Shelf life and operational stability of the microeactors are determined. GO/HRP, AO/HRP and LAAO/HRP microreactors showed excellent shelf life, they could be stored and reused for more than 6 months with no or very little activity loss, while GalO/HRP and LacO/HRP could be stored for shorter periods of time (10–20 days). Operational stability of GO and LacO microreactors was very good: an equivalent to 16,900 FIA injections of 25 μl to a LacO microreactor resulted in loss of half of its activity, immobilized GO was so stable that it was impossible to evaluate enzyme halflife. Immobilized GalO and LAAO lose their operational activity much faster: approximately 1400 and 8000 FIA injections of the respective substrate solution in a FIA set-up resulted in 50% activity loss. The methods with all the described microreactors were successfully validated using off-line samples from S. cerevisiae, E. coli and mesenchymal stem cell cultures with HPLC as the reference method.

Two microcell flow-injection analysis (FIA) platforms for capacitive silicon-based field-effect sensors

pH- and penicillin-sensitive electrolyte-insulator-semiconductor (EIS) sensors (Al/Si/SiO 2/Ta 2O 5 and penicillinase) have been successfully integrated into a commercial flow-injection analysis (FIA) system. For the FIA set-up, a miniaturised home-made flow-through cell of plexiglass with a variable internal volume from 12 to 48 μl has been developed. As a second platform, an SU-8 based miniaturised flow-through cell, also combined with a pH-sensitive EIS structure, has been fabricated at wafer level. The application of SU-8 as a photoresist with good qualities (distinct chemical resistance, high aspect ratio, good mechanical and dielectric behaviour) is favoured. Different channel/sensor layouts have been realised with sensitive areas of the EIS sensors of 1.7–8.2 mm 2 and sample volumes of 0.3–4 μl, respectively. FIA parameters (sample volume, flow rate, distance between injection valve and sensor) have been optimised in terms of high throughput, minimum dispersion, fluid consumption, sensitivity and reproducibility.

Chlormequat Selective Electrodes: Construction, Evaluation and Application at Fia Systems

A flow injection analysis (FIA) system having a chlormequat selective electrode is proposed. Several electrodes with poly(vinyl chloride) based membranes were constructed for this purpose. Comparative characterization suggested the use of membrane with chlormequat tetraphenylborate and dibutylphthalate. On a single-line FIA set-up, operating with 1 2 10 m 2 mol L m 1 ionic strength and 6.3 pH, calibration curves presented slopes of 53.6 - 0.4 mV decade m 1 within 5.0 2 10 m 6 and 1.0 2 10 m 3 mol L m 1 , and squared correlation coefficients > 0.9953. The detection limit was 2.2 2 10 m 6 mol L m 1 and the repeatability equal to - 0.68 mV (0.7%). A dual-channel FIA manifold was therefore constructed, enabling automatic attainment of previous ionic strength and pH conditions and thus eliminating sample preparation steps. Slopes of 45.5 - 0.2 mV decade m 1 along a concentration range of 8.0 2 10 m 6 to 1.0 2 10 m 3 mol L m 1 with a repeatability - 0.4 mV (0.69%) were obtained. Analyses of real samples were performed, and recovery gave results ranging from 96.6 to 101.1%.

Development of a confined wall-jet flow-through cell for simultaneous electrochemical and surface plasmon resonance applications

A flow-through cell of the confined wall-jet type for electrochemical surface plasmon resonance (ESPR) applications has been developed. The geometrical cell volume of the electrochemical (EC) detection is adjusted to the surface plasmon resonance (SPR) cell volume by reducing the electrode diameter. An electrode diameter of 3 mm gives the best similarity of the SPR and EC responses, using hexacyanoferrate(II) in a flow-injection analysis (FIA) setup. Further hydrodynamic characterization is done by continuous-flow analysis. The dependence of the limiting Faradaic current on the volume flow rate ( φ v) and the spacing ( b) between working and auxiliary electrode have been determined. The exponents found for φ v are in the range from 0.29 to 0.35 and for b in the range from −0.37 to −0.57 for typical flows (10–1000 μl min −1) and spacings (0.13–0.50 mm), respectively. Preliminary results of an impedimetric/liposome-enhanced SPR immunosensor for interferon-γ (INF-γ), based on an acetylcysteine self-assembled monolayer, show the characteristics and applicability of this new flow-through cell for ESPR research in a FIA setup.

Indirect Determination of the Pesticide Dimethoxydithiophosphate by Flow Injection Analysis with Liquid-Liquid Extraction and Atomic Absorption Spectrometry with Hydride Generation

Abstract We developed a new procedure for the indirect determination of the organophosphorus pesticide dimethoxydithiophosphate (DDTP) by coupling, in continuous mode, flow injection analysis (FIA) with liquid-liquid extraction and atomic absorption spectrometry with hydride generation in the organic phase. Both continuous-mode extraction of the species Bi(DDTP)3 in isobutylmethylketone (IBMK) organic medium and the process of hydride(BiH3) generation in the same organic medium were optimized. The most suitable solvent of the reducing agent, NaBH4, was a 1 % solution of N,N-dimethylformamide in glacial acetic acid medium. Phase separation was accomplished with an original design that has been patented for use (Spain patent 10,118,929 Y). The hydride generated is transported by the FIA setup in continuous mode to the quartz cell where the analytical signal was measured. The procedure developed was rapid, continuous, and involved little manipulation, thus avoiding possible errors and contamination. Moreover, only small amounts (250 μL) of sample were required in the injection of the aqueous sample, and a volume of 30 μL was used in the injection of the organic phase extracted. The values obtained for the detection limit (5 ppb DDTP in the aqueous phase injected) and for precision (3.0%, expressed as coefficient of variation) are very acceptable in comparison with other procedures described in the literature. The accuracy obtained was in agreement with the imprecision of the method when it was applied to the aqueous samples. The procedure can be used for the analysis of surface waters subject to different types of treatment prior to their use for human consumption. For this type of water, European legislation permits pesticide concentrations up to 5 ppb, which is the value of the detection limit obtained in the proposed procedure.

Flow-injection determination of compounds containing sulfur by using potentiometric tubular sensor

A flow-injection analysis (FIA) setup with flow-through tubular solid-state AgI-based membrane detector, sensitive to the sulfhydryl group, is examined. Application of a flow-injection system is exemplified by the determination of different compounds containing sulfur in 0, 1 M HClO_4 as a supporting electrolyte. For compounds with -SH group, a rectilinear calibration graph is obtained by plotting the peak height versus cencentration of RSH. The experimental slope is in good agreement with the theoretical (59 mV {;p(RSH)};^-1) value postulated on an precipitation process and formation of RSAg into the carrier stream or at the sensing part of the detector. The described FIA setup with utilized sensor can be applied for calculation of the solubility product of RSAg or stability constant of Ag(SR)_n^(1-n)+ complex.

Detection and Characterization of Phospholipase D by Flow Injection Analysis

A precisely working automated system for the investigation of phospholipases D (PLDs, EC 3.1.4.4) from plant and microbial sources with flow injection analysis (FIA) has been developed. The two versions of the FIA setup described are based on the oxidation of choline liberated from phosphatidylcholine by PLD action and catalyzed by choline oxidase and the chemiluminescence detection of hydrogen peroxide produced by this reaction. The correlation between this chemiluminescence signal and the PLD activity was linear in the range between 1 and 100 mU/ml PLD. The sampling frequency was 12 samples per hour. This method was used to compare three different PLDs from cabbage and microbial sources with respect to their pH optima, temperature stability, effectors, and v/[S]-characteristics.

Direct and simultaneous determination of uric acid and glucose in serum with electrochemical filter/biosensor flow-injection analysis system

A flow-through electrolysis cell, named an electrochemical filter (ECF), with a three-electrode configuration was developed for the elimination and determination of electrochemically active components in biofluids, which give generally a nonspecific response to electrochemical biosensors. The use of a reference electrode ensured the precise control of the working electrode potential of the ECF and thus the quantitative current efficiency of the electrolysis. A flow-injection analysis setup was constructed by connecting two ECFs and a flow-through amperometric biosensor for glucose in series for the direct and simultaneous determination of glucose and uric acid in serum. The first ECF coulometrically determined uric acid, and the second one eliminated interferents with the biosensor: the sample was thus treated on-the-fly and then delivered to the biosensor, so that it determined glucose amperometrically without interference. The concentrations of glucose and uric acid measured in human control sera agreed well with the values determined with conventional methods. The system exhibited the wide dynamic range in the high glucose concentration region, thus can be applied to the analysis of serum of hyperglycaemic patients.

Detection of mixtures of organic pollutants in water by polymer film receptors in fibre-optical sensors based on reflectometric interference spectrometry

A measurement system, which includes a polymer based sensor, a flow injection analysis set-up and a diode array spectrometer, was used to detect binary mixtures of organic pollutants in water. For our present examination, the organic pollutants are toluene, dichloromethane, 1,2-dichloroethane, and 1,1,1-trichloroethane. The response characteristics of the sensor to pure and mixed organic substances in water was investigated. By including the dynamics of the polymer/analyte interaction, i.e., the difference between transient signal profile shapes, the concentration of the components in binary mixtures was determined by multivariate techniques. The results of principal component regression, partial least-squares regression and multivariate linear regression were compared. The relative errors for the prediction of new samples were below 6%.

Chemiluminometric enzyme sensors for flow-injection analysis

Chemiluminometric monoenzyme sensors were developed for the flow-injection analysis (FIA) of hydrogen peroxide, xanthine and hypoxanthine in the concentration range between 10 −3 and 10 −6 M. Both a fibre optic set-up with a photomultiplier tube (PMT) and a photodiode with an integrated preamplifier were investigated to detect the chemiluminescence. Microbial peroxidase or xanthine oxidase was immobilized covalently on a preactivated membrane, which was placed in the 7.5 μl flow detector cell. The photodiode and the PMT based H 2O 2 sensor achieve detection limits of 10 −6 and 10 −7 M, respectively. To develop bienzyme sensors for glutamate, lysine and xanthine the microbial peroxidase was coimmobilized with the corresponding oxidases on the sensor membrane. These analytes can be detected in the range between 10 −3 and 10 −6 M. The fibre optic H 2O 2 sensor was combined with packed bed enzyme reactors to determine, e.g., glucose, lactate, glutamine, glutamate, ammonia, xanthine, hypoxanthine and phosphate in the range between 10 −3 and 10 −7 M. On this basis a fully automated FIA set-up with 5 parallel arranged enzyme reactor channels was developed for the ondashline monitoring of animal cell cultures.

An automated system for multichannel flow-injection analysis

An automated multi-channel flow-injection analyzer was developed both for new flow-injection analysis (FIA) procedures and for the process monitoring of animal and microbial cultivations. Up to six different analytes can be determined sequentially. The FIA set-up is working with up to six enzyme reactors configurated in parallel but with only one fluorescence detector and one pair of injection valves. By means of an automated sampling device the analyzer can be coupled on-line to a fermentation process. All active hardware components are computer controlled independently from each other guaranteeing a high flexibility. The software package, Flow Injection Analysis Control and Configuration (FIACCO), was developed for the hardware control, signal evaluation and long term recording of the on-line calculated results. Single and double step stopped flow procedures were developed to control the analyte conversion in packed bed enzyme reactors without an essential increase of the peak widths. Two carrier solutions are mixed to adjust the optimum pH for each applied enzyme also within one cycle of analysis. An enzymatic and a nonenzymatic procedure was developed to determine ammonia/ammonium after its gas dialytic separation from the sample solution. The application of the FIA set-up to the on-line process monitoring of an animal cell culture is demonstrated with respect to the five key components, glucose, ammonia, glutamine, glutamate and lactate.

Determination of intracellular dehydrogenase activities using flow injection analysis

Reproducible rapid methods for cell disintegration in miniaturized flow cells were developed. The disintegration of yeast cells by ultrasonics and ultraturrax in a miniaturized flow through chamber was compared with the mixer mill disintegration. The flow through chamber was combined with a sterile on-line sampling device to take samples from yeast cell suspensions and with an automated flow injection analysis (FIA) set-up, to determine the released enzyme activities. The FIA set-up was tested with the sequential determination of malate dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase in mixtures. The automated FIA determination of the intracellular formate dehydrogenase of yeast cells ( Hansenula polymorpha) was performed.

On-line monitoring of intracellular enzyme activities with flow-injection analysis

Micromethods for rapid and reproducible cell disintegration were combined with flow-injection analysis (FIA) to determine intracellular enzyme activities. Flow through chambers were developed for cell disintegration by the action of ultrasonic, ultraturrax and glass microball-mixer mills. A membrane device was incorporated into the FIA set-up to separate the cell debris and the released enzyme from the disintegrated sample solution. After mixing with the corresponding enzyme substrate the assay reaction takes place. The reaction product is transferred into the inlet tube of a fluorescence detector. Dehydrogenase activities were determined in the range of 0.001–2 U/ml with relative standard deviations < 1% ( n = 4, α = 0.05). The measuring set-up was completed by a chemically sterilized on-line sampling device connected to a bioreactor. Intracellular formate dehydrogenase and malate dehydrogenase were on-line determined to monitor yeast cultivation processes.