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Rapid and selective determination of ammonium by fluorimetric flow injection analysis.

Selective and sensitive procedures for the determination of ammonium in river water and diluted urine were developed by using flow injection analysis equipment. The methods are based on the derivatization of ammonia with o-phthaldehyde (OPA) and thioglycolate under alkaline conditions. The formed isoindole derivative is detected fluorimetrically at an excitation wavelength of 415 nm and an emission wavelength of 485 nm. The derivatization only takes 15 to 20 s at room temperature to achieve the maximum sensitivity. The optimized OPA reagent shows a surprisingly high selectivity for ammonium in the presence of many primary amines. With respect to the analysis of turbid and fluorescent sample solutions the selectivity can be improved by separating the ammonia through a microporous membrane from the OPA reagent. Without this separation step ammonia can be detected in the range between 0.05 and 100 microM with excellent linearity. After the insertion of an optimized membrane separation cell ammonia can be determined in the linear range between 0.2 microM and 20 mM.

Amines↗

Automation of selective assays for on-line bioprocess monitoring by flow-injection analysis.

On-line analysis of one component in a complex media used for bioprocesses requires the application of selective tests such as enzymes assays. Because these assays are susceptible to interference by other medium components and have a limited detection range, automatic sample pretreatment is a prerequisite. The progress made with automatic sample pretreatment in flow-injection analysis makes this technique particularly suitable for on-line monitoring of bioprocesses. Moreover, newly developed software control systems may improve the necessary robustness of flow-infection analysis systems.

Animals↗

Flow injection analysis system for the supervision of industrial chromatographic downstream processing in biotechnology.

Sugar beet molasses is a natural resource for various products used in daily life, ranging from sucrose to amino acids for pharmaceutical industry. The separation of molasses into these high value components is performed on a large scale by ion exchange/exclusion chromatography. A biosensor system was set up for the "in time" analysis of serine and sucrose during molasses desugarisation. D-Serine was analysed with the multi-enzyme system D-serine dehydratase/lactic dehydrogenase and photometric detection of the NADH consumed. Sucrose was determined with invertase/mutarotase/glucose oxidase and the oxygen consumed was monitored amperometrically. An analysis could be performed within 2-5 min by directly injecting samples from the chromatographic process into the flow injection analysis system. The determination range for the sucrose analysis was 0-2.5 gl-1 and for the analysis of D-serine 0-0.5 gl-1. The standard deviation for the measurement of D-serine was 1.7%.

Biosensing Techniques↗

Sensitive and selective flow injection analysis of hydrogen sulfite/sulfur dioxide by fluorescence detection with and without membrane separation by gas diffusion.

Highly sensitive and selective FIA flow injection analysis procedures for the determination of sulfite/hydrogen sulfite/sulfur dioxide were developed on the basis of an in situ-generated o-phthalaldehyde (OPA)/ammonium reagent and fluorescence detection. The highest sensitivity was achieved at an excitation wavelength of 330 nm, an emission wavelength of 390 nm, and at pH 6.5. Sulfite concentrations between 2.5 nM and 5 microM can be determined with relative standard deviations between 10.5 and 1.0% (n = 5, confidence level alpha = 0.05) by utilization of a reagent that contains 0.2 mM OPA and 0.4 M NH4Cl in 50 mM potassium phosphate buffer. A concentration of 0.1 mM sulfite can be selectively detected in the presence of thiosulfate, thioglycolate, tetrathionate, cysteine, and ascorbate. The fluorometric sulfite detection was combined with a membrane gas diffusion step to improve the selectivity with respect to nonvolatile fluorescing substances. The total sulfite content can be quantitatively separated as sulfur dioxide into an acceptor solution before its flow detection. Between 40 nM and 0.1 mM sulfite can be determined. After 1,000-fold dilution, the total sulfite content can be determined in white and red wines.

Journal Article↗

Electrochemical enzyme immunoassay for phenytoin by flow-injection analysis incorporating a redox coupling agent.

Using phenytoin as a model analyte, we demonstrate an electrochemical enzyme immunoassay based on flow-injection analysis and incorporating 2,6-dichloroindophenol (DCIP) as a redox coupling agent. DCIP reacts with NADH to form NAD+ and DCIPH2, the reduced form of the coupling agent. The production of DCIPH2 is monitored at +250 mV vs Ag/AgCl. This low applied potential improves selectivity in the biological matrix, differentiating against components that are oxidizable at the more-positive potentials required for direct electrochemical detection of NADH. The kinetics-based assay also eliminates other common interferences, mainly from ascorbic acid and glutathione. This system does not require precolumns or analytical columns for isolation of the NADH response. Good agreement with a routine clinical laboratory procedure for phenytoin is obtained for clinical samples (r = 0.95), illustrating the feasibility of such an approach.

2,6-Dichloroindophenol↗

Copper determination in urine by flow injection analysis with electrochemical detection at platinum disk microelectrodes of various radii.

The incorporation of platinum disk microelectrodes of various radii (2.5-50 microns) in a well-jet flow cell offers reduced limits of detection for the determination of copper in urine by flow injection analysis compared with standard methods based on a conventional sized glassy carbon disk macroelectrode (radius 1.5 mm), in a thin-layer cell. The radius of the platinum disk microelectrode was found to be critical with respect to both the limit of detection and flow rate dependence. An optimal radius value of 28 microns was found with detection limits increasing with both larger and smaller electrode radii. In contrast, as theoretically expected, a diminished flow rate dependence was observed the smaller the radii of the platinum disk microelectrodes. Sample cleanup and preparation is conveniently achieved by the use of Sep-Pak cartridges and formation of a copper dithiocarbamate complex. The metal complex is easily oxidized at platinum disk microelectrodes in acetonitrile, which was the solvent used in the flow injection method of analysis.

Copper↗

Mid-infrared quantum cascade lasers for flow injection analysis

A Fabry-Perot quantum cascade laser (QCL) was used as a powerful light source for mid infrared (MIR) detection in flow injection analysis. The QCL lased at several wavelengths close to each other within a few wavenumbers (990-1010 cm-1), hence fitting well to the broad absorption bands of molecules in liquid phase. As compared with that of a state-of-the-art Fourier transform spectrometer, the signal-to-noise ratio could be improved by a factor of 50. Additionally, by using a QCL as the light source, optical path lengths of more than 100 microns could be used even in aqueous matrixes which reduced the danger of cell clogging. In the example shown here phosphate was determined in Diet Coke samples. The flow injection system used allowed the measurement of the sample at two pH values (5 and 13) at which the analyte was present as H2PO4- and PO4(3-), respectively. As the analytical readout the difference in IR absorption of H2PO4- and PO4(3-) at the laser wavelengths was taken. The FIA-QCL measurements were corroborated by ion chromatography which was used as a reference method.

Journal Article↗

Measuring the CO2 flux at the air/water interface in lakes using flow injection analysis.

The carbon dioxide flux at the air/water interface in lakes was calculated after the determination of H2CO3* (free CO2) and atmospheric CO2 using flow injection analysis (FIA) coupled to a conductometric detector. The method is based on the diffusion of CO2 through a hydrophobic membrane into a flow of deionized water, generating a gradient of conductivity proportional to the concentration of CO2 in the sample. Using one experimental set-up, the speciation of the inorganic carbon (H2CO3* and dissolved inorganic carbon) was accomplished by simply adjusting the sample pH. The determination of CO2 in the atmosphere was carried out by direct injection of the gaseous samples. The FIA apparatus was taken into the field and CO2 fluxes were evaluated in several Brazilian lakes. In these lakes, representing different eutrophic stages, the CO2 flux varied from -242 (invasive) up to 3227 (evasive) mumol CO2 m-2 h-1.

Air Pollution↗

Flow injection analysis of cholic acids in pharmaceutical preparations using a polymeric membrane ISE as detector.

The results reported in this paper regard the setting up of a polymeric membrane ISE that is selective for cholic acids (CA) and able to work in a flow system, especially in flow injection analysis (FIA), based on the exchanger (tetrakisdecylammoniumcholate, TDACh), which has proved effective, is of very simple but suitable structure and is above all easy to synthesise starting from commercially available chemicals. A complete analytical characterisation of the sensor was performed working both in batch conditions and in FIA, using in the latter case a 'wall jet' type of flow cell. The response toward different bile acid sodium salts such as the CA, deoxycholic (DCA), chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), taurocholic (TCA) sodium salts was checked. The application to the analysis of different commercial drugs by FIA was also performed to determine the UDCA or CDCA acid content of several pharmaceutical formulations. Lastly, a preliminary study is presented concerning the use of the investigated electrochemical sensor as high performance liquid chromatography (HPLC) detector.

Cholic Acids↗

On-line control of an immobilized hybridoma culture with multi-channel flow injection analysis.

An immobilized hybridoma cell line was cultivated at controlled glucose and glutamine concentrations. On-line analysis of the substrates was carried out with a multi-channel flow injection analysis system. The analysis system also determined on-line the lactate and ammonium concentration. The substrate concentrations were controlled using an adaptive-control strategy. This strategy consisted of the estimation of the real-time concentrations and volumetric substrate consumption rates by an Extended Kalman Filter, and a minimum variance controller, which used the estimated parameters to set the feed rates of the substrates. The closed-loop control was used to start-up two cultures with either glucose or glutamine as control-substrate for the medium feed rate. The controller kept the concentration of the control-substrate constant by enhancing the medium feed rate simultaneously to the increasing volumetric consumption rate of the substrate. When glutamine was used as control-substrate, the glucose concentration remained relatively constant, whereas the glutamine concentration decreased during the start-up at a constant glucose concentration. This indicates that glutamine is consumed faster than glucose and will be a better control-substrate to avoid limitation during the start-up of a culture with the applied hybridoma cell line. During the colonization of the microcarriers, the yield of ammonium on glutamine decreased from 0.80 to 0.55 (mol mol-1), indicating a change in the glutamine metabolism. The yield of lactate on glucose stayed constant for both experiments. During long-term culture of more than 800 h, the controller kept both the glucose and glutamine concentrations constant at perfusion rates between 0.50 h-1 and 0.15 h-1. The medium, glucose and glutamine feed rate were independently controlled. Both the specific glutamine and glucose consumption rates remained constant for all perfusion rates, which was probably as a result of the constant concentrations. The specific monoclonal antibody production rate decreased with the perfusion rate decreasing from 0.40 h-1 to 0.20 h-1. The immobilized-cell concentration decreased only at the lowest perfusion rate. Both effects could not be explained directly by the increasing ammonium and lactate concentrations nor by the decreasing amino-acid concentrations.

Animals↗

Characterisation of a thermophilic L-glutamate dehydrogenase biosensor for amperometric determination of L-glutamate by flow injection analysis.

Carbon paste wax electrodes incorporating thermophilic L-glutamate dehydrogenase, NADP and a polymeric toluidine blue O (poly-TBO) mediator have been characterised for the amperometric determination of L-glutamate at 313-318 K in a flow injection analysis (FIA) system. The biosensors exhibit good sensitivity, mechanical stability and reproducibilty, unlike carbon paste- or carbon wax-based electrodes under the same conditions. The carbon paste wax electrode responds linearly to L-glutamate up to 40 mM, the detection limit is 0.3 mM and the RSD (n = 10) for 5 mM L-glutamate was 7.6%. The response to some potential interferents has been quantified. Addition of finely ground hexaammineruthenium (III) trichloride ([Ru(NH3)6]Cl3) to the carbon paste wax electrodes decreases the FIA peak width and increases the peak current. The metal complex appears to accelerate the rate of oxidation of NAD(P)H by poly-TBO.

Biosensing Techniques↗

Electrochemiluminescent metallopolymer coatings: combined light and current detection in flow injection analysis.

The application of thin films of the metallopolymer [Ru(bpy)2PVP10]2+ for the electrochemiluminescent (ECL) detection of oxalate in a flow injection analysis system is reported, where bpy is 2,2'-bipyridyl and PVP is poly(4-vinylpyridine). Immobilization of the ECL reagent means that it can be regenerated in situ, eliminating the need to constantly deliver it to the reaction zone. Electrochemically generated Ru3+ reacts with the analyte to form the excited-state [Ru2+]*, which luminesces at 610 nm. The reaction is optimal at low pH, where the layer is swollen and homogeneous charge transport through the layer is more facile. Unlike traditional approaches, we simultaneously monitor both the amperometric and luminescent response of the modified electrode. The precision of both signals is similar at approximately 2% (n = 10). However, the ECL response has a larger dynamic range extending from the low-micromolar to high-millimolar range and a lower limit of detection, approximately 0.2 microM or 4 pmol of oxalate injected. The ECL approach displays excellent selectivity for oxalate over a wide range of potential interferences including oxygen, amines, iron sulfate, ammonium nitrate, urea, and glucose. Ascorbic acid represents the most significant ECL interference. However, the signal observed for a 1 mM solution of ascorbic acid is still only 2.6% of the response observed for the injection of a similar concentration of oxalate.

Journal Article↗

Immobilization of linamarase and its use in the determination of bound cyanide in cassava using flow injection analysis.

Extracts from the tubers (cortex and parenchyma) and leaves of Manihot esculenta Crantz (cassava) were analyzed for their releasable cyanide content using flow injection analysis incorporating an immobilized linamarase bioreactor. Linamarase was immobilized under very mild conditions to an activated 2-fluoro-N-methylpyridinium Fractogel support. The released cyanide, which was monitored spectrophotometrically at 525 nm using an alkaline picrate reagent, was found to be highest in the cortex and lowest in the parenchyma.

Calibration↗

[Construction and application of all-solid-state aconitine electrochemical detector in flow injection analysis].

A new kind of all-solid-state electrochemical detector for very toxic alkaloids such as aconitine, mesaconitine and hypaconitine has been studied. It exhibits Nernstian response for these alkaloids with a slope of 56 mV/decade over the concentration range of 3 x 10(-5)-1 x 10(-2) mol/L at pH 2-7 under the flow condition. Direct potentiometry for the determination of aconitine in Aconitum kusnezoffii Reichb., Aconitum carmichaeli Debx. and Xiaohuoluo Wan showed average recoveries of 98.5, 98.3 and 96.8% and relative standard deviations of 1.8, 2.4 and 3.5%, respectively. It can be used for the determination of very toxic alkaloids in the above mentioned samples by flow injection analysis. It also can be used for the study of the hydrolytic kinetics of aconitine.

Aconitine↗

Determination of L-glutamate using flow injection analysis with immobilized L-glutamate oxidase reactor.

L-Glutamate oxidase (GOD) and horseradish peroxidase (HRP) were covalently coupled on alkylamine pretreated controlled pore glass (CPG) by means of glutaraldehyde. The immobilized enzymes were packed into a teflon tube and used in flow injection analysis (FIA) system for L-glutamate determination. A good linearity range was obtained at 0.1-2.0 mM, and the coefficient of variation was 0.7% (n = 8). More than 80 samples were measured within an hour. The stability of the immobilized GOD reactor was good, retaining 50% of its initial activity after 4 months storage in buffer at 4 degrees C. When the concentration of L-glutamate remained lower than 2.5 mM, the determination of L-glutamate in this system was not affected by pH and temperature within the range of 6.0-8.0 and 20-35 degrees C, respectively. The system was applied to determine L-glutamate in broth samples during L-glutamate fermentation and good correlations were achieved between results obtained with the FIA system, L-glutamate oxidase kit and Warburg's method.

Amino Acid Oxidoreductases↗

Flow injection analysis and real-time detection of RNA bases by surface-enhanced Raman spectroscopy.

Surface-enhanced Raman scattering (SERS) spectroscopy has been successfully interfaced with a flow injection analysis system to detect RNA bases in real time. Four of the major base components of RNA, uracil, cytosine, adenine, and guanine, were introduced into the flow injection system and were mixed with a Ag sol prior to SERS measurements. Several experimental parameters including pH, temperature, flow rate, and tubing materials were examined, and their impact on the SERS spectra is presented here. The feasibility of interfacing flow injection based SERS detection methods with liquid or high-performance liquid chromatography for the detection of individual components in a complex mixture is also assessed.

Chromatography, High Pressure Liquid↗

Flow injection analysis of mercury(II) in pharmaceuticals based on enzyme inhibition and biosensor detection.

An enzymatic amperometric procedure for measurement of mercury(II) in pharmaceuticals, based on the inhibition of invertase and on a glucose electrode was studied. Analytical parameters for measurements in batch and flow injection analysis (FIA) have been optimised. Mercury(II) was detected in the 10-60 ppb range with RSD < or =2%. A sample throughput of 6 h(-1) for batch and 15 h(-1) for FIA was obtained. The total mercury(II) from thimerosal (thiomersal, sodium ethylmercurithiosalicylate) in eye-drop samples was measured with the amperometric procedure after oxidative cleavage treatment. Results for both batch and FIA procedures correlated well with atomic absorbtion spectroscopy (AAS) data.

Biosensing Techniques↗

Automated determination of antibody oxidation using flow injection analysis.

The oxidation of antibody carbohydrate residues is a common approach used for site-specific antibody immobilization or modification. In this study a flow injection analysis system (FIA) was developed for monitoring antibody oxidation. Antibodies were oxidized with periodate and the resulting aldehyde groups were labeled with Lucifer yellow CH (LyCH). The labeled antibodies were then injected onto an FIA system where the amount of LyCH label was determined by absorbance measurements at 428 nm and the amount of antibody was determined using an on-line bicinchoninic acid protein assay. The analysis time was 2 min per 20 microliters sample injection. The limits of detection for rabbit immunoglobulin G (IgG) and LyCH were 1 x 10(-8) and 4 x 10(-7) M, respectively. The dynamic ranges for IgG and LyCH extended to 2 x 10(-5) and 7 x 10(-3) M. The within-run precision was +/- 5% or less for both analytes. Studies with known LyCH/antibody mixtures indicated that the FIA system had greater accuracy than manual methods at high LyCH levels. One specific application studied for this system was its use in monitoring the time course of periodate-antibody oxidation.

Animals↗