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Flow injection analysis with immobilized enzymes for process control of pullulan production by fermentation.

A flow injection system is described for the parallel determination of pullulan and glucose during a fermentation of the fungus Aureobasidium pullulans. The polysaccharide was hydrolyzed by pullulanase and amyloglucosidase, immobilized to controlled-pore glass (CPG). The glucose produced was oxidized by glucose dehydrogenase and the NADH formed determined photometrically. The pullulan concentration was calculated from the difference to the response obtained for free glucose. The calibration curves for monomer and polymer were both linear between 2 mg dm-3 and 20 mg dm-3. Analysis of one sample for the determination of glucose and pullulan took about 10 min.

Enzymes, Immobilized↗

Electrochemical flow injection analysis study of ion partitioning at high surface area carbon fiber electrodes.

Charge-selective electrochemistry was previously shown to occur at high surface area carbon fibers that were produced by fracturing the outer periphery with anodic current or positive potential. The cyclic voltammetric behavior of electroactive species observed at these fibers exhibited a distinct pH dependence related to the protonation/deprotonation of oxygen-containing functional groups at the surface of the carbon fiber. In this paper, electrochemical flow injection analysis (EC-FIA) is used to probe ion partitioning in to and out of the interior microstructure of the treated carbon fiber, for both electroactive and electroinactive species. It was found that the extent of partitioning was the result of both ion charge and hydrated ionic radius, in addition to the level of fracture. It was further observed that the direction of movement for an injected ionic species could be controlled relative to the ion concentration, the pH of the carrier solution, or both. EC-FIA allowed the simultaneous observation of current due to ion movement and that due to electron transfer to a redox-active species. The results presented are consistent with a model in which fixed negatively charged sites in the interior of fractured fibers govern ion partitioning with positively charged ions in the carrier solution, with counterions located in the interior "free" volume.

Journal Article↗

Flow-injection analysis with fluorescence detection for the determination of trace levels of ammonium in seawater.

A method using flow-injection, gas-diffusion, derivatisation and then fluorescent detection has been established for ammonium ion determination in seawater. The fluorescent derivative formed by reacting ortho-phthaldialdehyde (OPA) and sulfite with ammonia gives high sensitivity while removing potential interferences. This is required to measure the low concentrations of ammonium often seen in the open ocean. The experimental conditions (flow-rate, reagent concentrations, membrane configurations, etc.) were manipulated to improve performance. For a sample throughput of 30 samples h(-1), the limit of detection was 7 nM, the coefficient of variation was 5.7% at 800 nM, and the calibration curve was linear to at least 4 micromol L(-1). Interferences were minimised by a gaseous diffusion step. Volatile small molecular-weight amines as interferents were discriminated against by this method. They neither passed through the membrane as efficiently as ammonia, nor reacted as readily with OPA when sulfite was the reductant. Contamination by ammonia from laboratory and shipboard sources complicates application of the method to natural waters, especially measurement of low concentrations (<100 nM) in open-ocean waters. Steps to overcome contamination are described in detail. Some results are presented for ammonium determination in Southern Ocean and Huon Estuary (Tasmania) waters.

Amines↗

[Micro-determination of fluoride in biological samples by pyrohydrolysis and flow-injection analysis using a fluoride ion-selective electrode].

An apparatus has been developed for the isolation of fluoride in biological samples through pyrohydrolysis. With this apparatus, it is possible to determine both organic and inorganic fluorocompounds with a recovery close to 100% and precision within 5%. The high recovery rate can be expected even for highly heat-resistant compounds such as CaF2, without using WO3 as a catalyst. For determination of the isolated fluoride, a separate apparatus was developed in which flow-injection analysis was used in conjunction with a fluoride ion-selective electrode as a detector. With this apparatus, fluoride in a sample solution with a volume as small as 0.2 ml, and at a concentration as low as 0.5 microgram/l, can be determined within 3 minutes with a precision of several percent. Combined use of the two apparatuses makes it possible to determine fluoride in different biological samples within 10-15 minutes with a precision of several percent, free from external contamination. By selecting suitable conditions for analysis and using a 1 g sample, it is possible to determine fluoride at a concentration as low as 5 ng/g. By employing these apparatuses, the fluoride content in different biological samples has been determine and the effectiveness of their use confirmed.

Adult↗

Flow-injection analysis with electrochemical detection of reduced nicotinamide adenine dinucleotide using 2,6-dichloroindophenol as a redox coupling agent.

The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow-injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 microM and with a detection limit of 0.38 microM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.

2,6-Dichloroindophenol↗

Simultaneous determination of nitrate and nitrite in biological samples by multichannel flow injection analysis.

An automated method for the simultaneous determination of nitrite and nitrate in biological samples by using a multichannel flow injection analyzer has been developed. The method was based on the reaction of nitrite with Greiss reagent. The sample solution was injected and equally divided into two channels; channel one (1) represented total nitrite obtained by cadmium reduction of nitrate to nitrite while channel two (2) represented only nitrite. The absorbance of the color product was measured by photometric detectors with 540-nm filters. This method combines high reproducibility of sample introduction via flow injection and sensitivity of spectrophotometric detection. The detection limit is 25 nM for both nitrite and nitrate. The chemistry manifolds are constructed of Teflon tubing which, along with a low-pressure Flowfit connector system, provides for low maintenance, ease of use, and high sample throughput. We demonstrated that the system can be used for the determination of both nitrate and nitrite in a variety of biological samples as well as a comparison of the results from this system and the HPLC system.

Animals↗

Determination of L-cysteine in amino acid mixture and human urine by flow-injection analysis with a biamperometric detector.

Based on the electrocatalytic oxidation of cysteine at pretreated platinum electrode and the flow-injection biamperometry for irreversible couple, a novel electrochemical detector is proposed for the selective determination of cysteine in amino acid mixtures and human urine samples. A thin-layer flow through cell was used to achieve large electrode surface area to volume ratio. Two identical pretreated platinum electrodes were mounted in the cell with an applied potential difference of 10 mV. By coupling two independent and irreversible electrode processes, namely, the oxidation of cysteine and the reduction of platinum oxide, the biamperometric detection scheme has been established. The resulting current is linear to cysteine over the range 4 x 10(-7)-4 x 10(-5) M with the detection limit 1 x 10(-7) M (15 pmol). The selectivity of the detector is tested by 55 foreign species including 26 ions, 11 amino acids, 6 vitamins, and 12 other compounds possibly found in urine. The detector performs well as a routine assay, showing high efficiency (180 samples/h) and good reproductivity shown by a RSD of 0.6% for eight repeated determinations of 2 x 10(-6) M cysteine. The urine samples are detected directly without the need of pretreatment or adding other reagents.

Amino Acids↗

Enzymatic determination of bicarbonate in serum by flow injection analysis.

An automated method for the determination of bicarbonate in human serum based on the enzymatic reaction between the analyte and phospho(enol)pyruvate (PEP) in the presence of PEP carboxylase is proposed. The analytical reaction is coupled with a derivatization reaction in which the NADH consumed is fluorimetrically monitored (lambda ex = 340 nm, lambda em = 460 nm). A stopped-flow/flow-injection approach is used in which the enzymes (PEP carboxylase and malate dehydrogenase) are immobilized on controlled-pore glass. The linear determination range is between 25 and 300 mmol/l (r2 = 0.9973). The %C.V. for the within- and between-run studies, performed at three concentration levels, ranges between 1.0 and 3.6% and the sampling frequency is 20 per h.

Analysis of Variance↗

Polyvinyl chloride matrix membrane electrodes for manual and flow injection analysis of chloroquine in pharmaceutical preparations.

Two types of polyvinyl chloride (PVC) matrix membrane electrodes responsive to the antimalarial drug chloroquine have been constructed, electrochemically evaluated, compared and used in pharmaceutical analysis. Type 1 is the classic PVC model with chloroquine-tetraphenylborate (TPB) sensor; Type 2 is a coated silver disk without internal filling solution. Both electrode types exhibited rapid linear potentiometric response to the logarithmic concentration of diprotonated chloroquine cation in the 10(-1) - 10(-6)M range with calibration slopes 28-30 mV/concentration decade over the pH range 1.8-6.2. These electrodes were sensitive enough to permit determination of chloroquine phosphate at concentrations as low as 5 microgram/mL with good accuracy and precision. Determination of chloroquine in various pharmaceutical preparations using direct potentiometry and potentiometric titration with NaTPB gave an average recovery of 98.8% of the nominal values (SD 0.5%). The Type 2 electrode was also assessed in a flow-through sandwich cell for flow injection analysis. Results were compared with data obtained by the U.S. Pharmacopeia method.

Chloroquine↗

Screening for ochratoxin A in blood by flow injection analysis.

A micromethod for ochratoxin A detection in human sera by flow injection technique is described. The method requires 50 microliter of sera, and it is designed to distinguish samples containing less than 10 ng ochratoxin A per ml. The method is based on fluorescence measurement following a simple extraction procedure for which very small amounts of chemicals are needed. Since the method is not confirmatory, all samples showing fluorescence above a certain intensity have to be reanalysed with some other method where a confirmation step in included. Because of the small amount of serum needed and the rapid procedure (less than 15 min), a large number of samples can be analysed very quickly. The method may therefore be applicable for large screening campaigns conducted to determine the presence of ochratoxin A in blood. This conclusion is based on 1675 samples and 147 standards analysed concurrently by the flow injection technique and an earlier published enzymic method. The method is also suitable for monitoring ochratoxin A levels in the blood of experimental animals.

Chromatography, High Pressure Liquid↗

Flow-injection analysis of dopamine in injections with a periodate-selective electrode.

Dopamine determination in pharmaceutical preparations based on its oxidation with periodate (IO(4)(-)) using a new IO(4)(-)-selective electrode under flow conditions is presented. An electrode with a tubular configuration, no internal reference solution, and a PVC (31. 2%) membrane, with metaperiodate bis(triphenylphosphoranylidene)ammonium (1.3%) as ion exchanger and 2-nitrophenyloctylether (67.5%) as mediator solvent, was used. Optimization procedures were directed at potentials versus dopamine readings instead of potential versus the remaining IO(4)(-). This approach was achieved by selecting a 50-cm reactor and an overall flow of 7 mL/min, and injecting 70 microL of dopamine standards in a 3.0 x 10(-4) M IO(4)(-) solution. Under these conditions, a linearity range of 8.0 x 10(-3) to 2.7 x 10(-1) g/L, with a slope of 310.1 +/- 7.4 mV L g(-1) and a reproducibility of +/-0.4 mV, were recorded (n = 8). Interference from common excipients was negligible. Under these conditions, analysis of dopamine injections (n = 12) presenting 200 mg/injection gave average and standard deviation values of 201.0 and 3.3 mg/injection, respectively. A simple and inexpensive flow-injection analysis (FIA) manifold, with a good potentiometric detector, enabled the analysis of 200 samples/h without requiring pretreatment procedures. Comparison with the dopamine injection analysis in the United States Pharmacopoeia monograph showed good accuracy, with a relative deviation of -0.2%.

Dopamine↗

Determination of Henry's law constants of phenols by pervaporation-flow injection analysis.

A novel dynamic nonequilibrium technique for the determination of Henry's law constant (HLC) of phenols based on pervaporation-flow injection (PFI) is described. A linear relationship between HLC and the amount of phenol measured by a detector in the acceptor line of a PFI system was demonstrated. This relationship was constructed using five frequently encountered phenols (phenol, 2,4-dimethylphenol, 2,4-dichlorophenol, 2-chlorophenol, and 2,3-dimethylphenol) and used for the determination of the HLC of three other phenols (2,4,6-trichlorophenol, 2-methylphenol, and 3-methylphenol). The HLC of all eight phenols were also determined by the single equilibrium static technique (SEST). Fairly good agreementwas observed between both techniques regarding the HLC of 2,4,6-trichlorophenol, 2-methylphenol, and 3-methylphenol. On the basis of the results obtained, it was concluded that the PFI technique offers considerable advantages over SEST in terms of precision, speed, labor intensity, and possibilities for automation.

Air↗

Determination of picomole amounts of thiamine through flow-injection analysis based on the suppression of luminol-KIO(4) chemiluminescence system.

A continuous flow sensor for the determination of thiamine was constructed by using controlled-reagent-release technology in a FIA-CL system. The analytical reagents, luminol and KIO(4), were both immobilized on an anion-exchange column. The CL signal produced by the reaction between luminol and KIO(4), which were eluted from the column through H(2)O injection, was decreased in the presence of thiamine. The decreased CL intensity was linear with thiamine concentration in the range 3.3 pmol ml(-1)-6.7 nmol ml(-1); and the limit of detection was 1.0 pmol ml(-1) (3.). The whole process, including sampling and washing, could be completed in 0.5 min with a relative standard deviation of less than 3.0%. The flow sensor showed remarkable stability and could be easily reused over 80 h. The sensor proposed was tested in determination of thiamine in pharmaceutical preparation and human urine samples.

Calibration↗

Fluorometric determination of ethanol in liquor samples by flow-injection analysis using an immobilized enzyme-reactor column with packing prepared by coupling alcohol oxidase and peroxidase onto chitosan beads.

A flow-injection system was developed for the determination of ethanol with an immobilized enzyme-reactor column. This system, which consisted of hand-made reactor columns packed with alcohol oxidase and horseradish peroxidase immobilized onto chitosan beads, and a fluorometric detector, was applied to the determination of ethanol in liquor samples. Under the recommended conditions, the ethanol, which was present in the pretreated samples, was converted to hydrogen peroxide when it was passed through the immobilized alcohol oxidase (AOD) column with 0.1 mol/dm3 phosphate buffer (pH 7.0). A sample can be analyzed with this system in <10 min. The calibration curve for ethanol was linear from 2.0 to 0.1 mg/dm3. The determination limit, which was defined by the difference between the sample peak and blank peak, was estimated to be 50 microg/dm3 for ethanol. Interferences from some substances present in actual liquor samples decreased the analytical response and activity of the immobilized AOD-reactor column, but they were removed by dilution and pretreatment with an octyldecylsilane cartridge.

Alcohol Oxidoreductases↗

[Amperometric detection in the determination of drugs by non-separating flow methods--flow injection analysis and sequential injection analysis].

The present review dealing with the use of various electrochemical detection systems in flow methods of analysis of drugs (such as FIA and SIA techniques). The review covers the period of 1988 to 1998 and involves 78 references. The drugs determined are arranged according to the functional groups undergoing electrochemical transformation; for all the analytes data on the detection conditions, detection limits and ranges of quantitation are included. Advantages and drawbacks of amperometric detection of drugs in flow systems are discussed.

Electrochemistry↗

Simple and selective method for determination of iodide in pharmaceutical products by flow injection analysis using the iodine-starch reaction.

This work exploited the well-known iodine-starch reaction for development of a simple flow-injection (FI) method for determination of iodide in pharmaceutical samples. Iodide in an injected zone was oxidized to iodine. A gas diffusion unit enables selective permeation of iodine through a hydrophobic membrane. Detection was made very selective for elemental iodine by employing formation of the I(3)(-)-starch complex. The detection limit (3 S/ N) of the system was 1 mg I L(-1). For a liquid patent medicine used for asthma treatment we suggested modification of the system. Direct injection of this sample, which contains a particularly high concentration level of iodide (ca. 9000 mg I L(-1)), can be achieved by coupling a dialysis unit to the FI system. This has increased the working range to 6000-10,000 mg I L(-1) without employing complicated nanoliter injection.

Flow Injection Analysis↗

Flow-injection analysis of cobalt(II) utilizing enhanced lophine chemiluminescence with hydroxylammonium chloride.

A rapid and convenient flow-injection method is described for the determination of Co(II). The method utilized the phenomenon that the lophine-Co(II)-H2O2 chemiluminescence (CL) reaction is enhanced in alkaline media by the addition of hydroxylammonium chloride. The calibration curve was linear over the range 2 x 10(-7) to 2 x 10(-5) M with a detection limit of 4.5 x 10(-8) M (0.9 pmol) at a signal-to-noise ratio of 2. The method was applied to the assay of cyanocobalamin in commercially available eye lotions.

Chromatography, Liquid↗