Development of a fully integrated microdistillation flow injection system for the determination of trace level ammonia

An in-housed designed computerised flow injection system comprised a fully integrated microdistillation flow injection (MDFI) system for low level ammonia analysis was reported. In this system, the microdistillation separation step was incorporated into the flow injection manifold and the ammonia gas sensing probe sensing element was replaced by a flow-through micro-pH electrode which sensed the change in pH of a flowing collector solution caused by the dissolution of distilled ammonia gas, in a process analogous to that occurring in the internal solution of the gas sensing probe. A computerised control and data acquisition system was constructed for this system using a commercially available data acquisition card which offered many advantages such as improved data acquisition rates and control over the system components, as well as good graphics display and data processing options. The system was optimised using a multi-variable simplex optimisation technique.     

Selective determination of lithium in biological fluids using flow injection analysis

The use of flow injection analysis to automated extraction methods for the determination of lithium ion utilizing crown ethers or cryptands is demonstrated. The ion-pair extraction of cryptand 211, lithium, and resazurin exhibits a linear range for lithium ion of 70 ppb to 2.1 ppm. This method could tolerate up to 1000 ppm sodium ion. The chromogenic crown ether, 1-(2-hydroxy-5-nitrobenzyl)-1-aza-4,7,10-trioxacyclododecane, exhibits a linear range for lithium ion of 0.3 to 2 ppm. A sodium ion concentration of 230 ppm can be tolerated. Both extraction systems were used in the automated determination of lithium in blood serum and urine. Both methods agreed well with the known and/or atomic absorption values.     

On-line determination of biomass in a microalga bioreactor using a novel computerized flow injection analysis system

A flow injection analysis (FIA) device has been developed, which is able to assay successfully for biomass in a microalga bioreactor. The device is fully computerized and is operated via diluting small aliquots of the culture followed by measuring optical density (OD); this figure is then accurately correlated with biomass, in terms of both cell number and ash-free dry weight, during the entire culture time. Furthermore, the device is not expensive, is highly versatile, and is easy to operate owing to specifically developed, user-friendly software. The growth rate and biomass productivity of Pavlova lutheri, cultivated under batch and semicontinuous modes, were monitored as experimental testing model.     

An amperometric bi-enzyme sensor for determination of formate using cofactor regeneration

A biosensor for detection of formate at submicromolar concentrations has been developed by co-immobilizing formate dehydrogenase (FDH, E.C. 1.2.1.2), salicylate hydroxylase (SHL, E.C. 1.14.13.1) and NAD(+) linked to polyethylene glycol (PEG-NAD(+)) in a poly(vinyl alcohol) (PVA) matrix in front of a Clark-electrode. The principle of the bi-enzyme scheme is as follows: formate dehydrogenase converts formate into carbon dioxide using PEG-NAD(+). Corresponding PEG-NADH produced is then oxidized to PEG-NAD(+) by salicylate hydroxylase using sodium salicylate and oxygen. The oxygen consumption is monitored with the Clark-electrode. The advantages of this biosensor approach are the effective re-oxidation of PEG-NADH, and the entrapment of PEG-NAD(+) resulting in avoiding the addition of expensive cofactor to the working medium for each measurement. This bi-enzyme sensor has achieved a linear range of 1-300 microM and a detection limit of 1.98 x 10(-7) M for formate (S/N=3), with the response time of 4 min. The working stability is limited to 7 days due to the inactivation of the enzymes. Only sodium salicylate was needed in milli-molar amounts.     

Fluorometric determination of urea by flow injection analysis.

Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in alkaline medium to give a highly fluorescent compound that has an excitation wavelength of 372 nm and an emission wavelength of about 430 nm. The method is more selective to ammonia than the one which uses mercaptoethanol in place of the sulfite. Urease was immobilized to a Pall Immunodyne membrane which is commercially available. The immobilization occurs through covalent bonding which results in a highly stable enzyme preparation. The enzymatic membrane was fitted in a 5 cm long, 0.125 inch o.d. Teflon tubing which served as the enzymatic reactor. The system is difficult to use for the analysis of urea in serum because some compounds normally present in serum fluoresce at the same wavelength. This results in higher values for urea. If the reaction system is to be used for the evaluation of urea in serum, a blank should be run so that urea concentration can be calculated by difference.     

Chemiluminescence inhibition assay for folic acid using flow injection analysis

A new flow injection method for the determination of folic acid is described. A fast oxidation reaction occurred when folic acid was mixed with potassium ferricyanide generating ferrocyanide which then inhibited the chemiluminescent reaction of ferricyanide and luminol in alkaline medium. The decrease of chemiluminescence intensity was correlated with the folic acid concentration in the range 0.1-21 microg/mL; the detection limit for the assay was 0.03 microg/mL (3sigma). A complete analysis of folic acid, including sampling and washing, could be performed within 2 min with a relative standard deviation of less than 4.0%. The proposed method has been applied successfully to the determination of folic acid in pharmaceutical preparations.     

A flow injection chemiluminescence method for the determination of lincomycin in serum using a diperiodato-cuprate (III)-luminol system

In this paper, the novel trivalent copper–periodate complex {K₅[Cu(HIO₆)₂], DPC} has been applied in a luminol‐based chemiluminescence (CL) reaction. Coupled with flow injection (FI) technology, the FI‐CL method was proposed for the determination of lincomycin hydrochloride. The CL reaction between luminol and DPC occurred in an alkaline medium. The CL intensity could be greatly enhanced by lincomycin hydrochloride. The relative CL intensity was proportional to the concentration of lincomycin hydrochloride in the range of 1 × 10^?8 to 5 × 10^?6 g mL^?1 and the detection limit was at the 3.5 × 10^?9 g mL^?1 level. The relative standard deviation at 5 × 10^?8 g mL^?1 was 1.7% (n = 9). The sensitive method was successfully applied to the direct determination of lincomycin hydrochloride (ng mL^?1) in serum. A possible mechanism of the lumonol–DPC CL reaction was discussed by the study of the CL kinetic characteristics and the spectra of CL reaction. The oxidability of DPC was studied by means of its electrochemical response. Copyright © 2010 John Wiley & Sons, Ltd.     

A flow injection analysis system for fermentation monitoring and control

An automated analysis system for on-line fermentation monitoring is presented. The modular system consists of an in-line sterilizeable crossflow microfilter, a selection valve that allows injection of sample or standards, a degassing unit, a dilution module, and a FIA manifold with a spectrophotometric UV/VIS detector. In the dilution module samples are conditioned and diluted depending upon concentration of analyte and the working range of the analyzer. Methods for the monitoring of glucose, ethanol, ammonia and phosphate are described. Results from the monitoring of glucose and their use in fermentation control are presented. The maximal analysis frequency is 30 samples per hour including the dilution of 1 : 200. Detection limits are 5 mg/L for ethanol and glucose, 1 mg/L for phosphate and 50 mg/L for ammonia.     

Chemiluminescent assays for choline and phospholipase-D using a flow injection system

A flow injection chemiluminescent method is described for the determination of choline. The method is based on the production of hydrogen peroxide from choline using on-line covalently bound immobilized choline oxidase column. The product is mixed downstream and detected via the cobalt catalyzed chemiluminescent oxidation of luminol. The detection limit is 1×10⁻⁷ mol/L, with rsd 1.8 to 2.8% in the range 2–10×10⁻⁵ mol/L. The sample throughput is 30 per hour. The method was applied to the determination of choline produced off-line from phosphatidylcholine using phospholipase-D isolated from cabbage. © 1997 John Wiley & Sons, Ltd.     

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.     

Flow injection chemiluminescence determination of isoniazid using the lucigenin-periodate system.

A weak chemiluminescence (CL) signal was observed during the mixing of isoniazid with lucigenin in alkaline aqueous solution. The CL signal was enhanced more than 100 times in the presence of potassium periodate. This CL system was developed for the determination of isoniazid using a flow injection mode. The CL intensity is proportional to the concentration of isoniazid in the range 0.005-1.0 mg/L. The limit of detection is 0.0034 mg/L and the relative standard deviation is 2.0% for 0.2 mg/L isoniazid solution in 11 repeated measurements. The method was applied to the determination of isoniazid in pharmaceutical preparations and satisfactory results were obtained.     

Methanol determination in Pichia pastoris cultures by flow injection analysis

An automated reverse flow injection analysis (r-FIA) system using stop-flow technique for quantifying methanol based on the enzymatic reactions of alcohol oxidase and peroxidase was developed. The system permitted methanol analysis in a linear range of 0.006-0.1 g methanol l^–1 without external dilution, and with a sampling frequency of 12 analyses per hour, with a relative standard deviation of 1.16%. The analyser was validated analysing samples from a Pichia pastoris fermentation producing a heterologous protein.     

Simultaneous determination of L- and D-methotrexate using a sequential injection analysis/amperometric biosensors system

A sequential injection analysis (SIA) is proposed for the simultaneous determination of L- and D-methotrexate (Mtx) using amperometric biosensors as detectors. A SIA system is proposed due to the highest precision and accuracy and lower consumption of sample and buffer. The amperometric biosensors used as detectors in SIA system were based on L-amino acid oxidase (L-AAOD) or/and L-glutamate oxidase (L-Glox) and horseradish peroxidase (HRP) for the assay of L-Mtx and D-amino acid oxidase (D-AAOD) and HRP for the assay of D-Mtx were selected. The linear concentration ranges are of pmol/l to nmol/l magnitude order, with very low limits of detection. The SIA/biosensors system can be used reliably on-line in synthesis process control, for the simultaneous assay of L- and D-Mtx with a frequency of 34 samples per hour.     

An improved microfluorometric enzymatic assay for the determination of ammonia.

A simple, rapid microfluorometric enzymatic method for the determination of ammonia is described. The basis for the assay is the enzymatic conversion of ammonia and α-ketoglutarate to glutamate by glutamate dehydrogenase and the measurement of disappearance of NADH. The assay sensitivity is 1–50 nmol of ammonia.     

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.     

Increasing glucose determination range by flow injection analysis (FIA) using glucose oxidase immobilised on polyaniline

A Flow Injection Analysis (FIA) for glucose using glucose oxidase (E.C.1.1.3.4) was developed. The enzyme was immobilised on polyaniline chemically synthetized on internal surface of silicon tube (2.0 mm of diameter and length from 1 to 5 meters), using glutaraldehyde as a bifunctional agent. The system was able to measure glucose at levels from 5 to 500mM with a response time of 2min using 250l of sample. The system has been operated satisfactorily for one month with more than 300 assays with loss of 60% of activity.     

Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically.Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute.The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic- spectrophotometric procedure.An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.Abbreviations GPC sn-glycerol-3-phosphorylcholine - GPE sn-glycerol-3-phosphorylethanolamine - GPS sn-glycerol-3-phosphorylserine - GPA sn-glycerol-3-phosphoric acid - PDE glycerophosphorylcholine-phosphodiesterase - GPA-Ox glycerophosphate oxidase - Cho-Ox choline oxidase     

Measurement of protein using flow injection analysis with bicinchoninic acid

We have used the bicinchoninic acid reagent developed by Pierce Chemical Co. to measure proteins in a simple flow injection analyzer. The sensitivity is comparable to that of the Lowry method and no pipetting of reagents is needed. Results are obtained in less than 1 min and samples may be run at a rate of 60/h. The response is linear over a range of protein concentration (0-10 micrograms) and sample size (5-20 microliters) convenient for most analytical requirements. A peristaltic pump, a controlled-temperature water bath, and a spectrophotometer with flow cuvette are the only special apparatus required.