University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Controlled pore glass

Classification: Solid phase -> Controlled pore glass

Citations 196

"In-situ Immobilisation Of Glucose Oxidase On A Novel Microporous Silica Support"
Analyst 2000 Volume 125, Issue 2 Pages 237-239
Natalie G. Wilson, Tom McCreedy and Gillian M. Greenway

Abstract: Glucose oxidase has been covalently immobilized onto a highly porous, but robust microporous silica structure. The porosity of this microporous structure was adjusted by the inclusion of acetamide to improve the enzyme immobilization procedure. The covalent enzyme attachment was achieved by an on-line immobilization procedure in which the reagents were pumped through the support that had been placed in-situ in the reactor tube. Glucose oxidase was immobilized with glutaraldehyde cross-linking to the 3-aminopropyltriethoxysilane treated support. The narrow tube enzyme reactor produced was then incorporated into a flow injection system and its activity tested using the determination of glucose with spectrophotometric detection at 436 nm. A linear calibration range was obtained for glucose between 5 x 10^-4 and 5 x 10^-3 mol L-1 with a limit of detection of 1.4 x 10^-4 mol L-1. The %RSD was 4% at the 5 x 10^-4 mol L-1 level.
Glucose

"Monitoring Of The Bioconversion Of Glycerol To Dihydroxyacetone With Immobilized Gluconobacter Oxydans Cell Using Thermometric Flow Injection Analysis"
Process Biochem. 2001 Volume 36, Issue 11 Pages 1045-1052
Marián Navrátil, Ján Tkáč, Juraj Švitel, Bengt Danielsson and Ernest Šturdík

Abstract: The bioconversion of glycerol to dihydroxyacetone (DHA) by Gluconobacter oxydans cells was monitored by flow microcalorimetry. DHA was produced in an air-lift reactor using bacterial cells immobilized in calcium pectate gel. Glycerokinase and galactose oxidase were immobilized on controlled pore glass and used for the determination of glycerol (substrate) and dihydroxyacetone (product), respectively. Concentration values obtained by flow microcalorimetry were in a good correlation with a spectrophotometric reference method. Furthermore, flow microcalorimetry can be used for on-line monitoring of the process. (C) 2001 Elsevier Science Ltd. All rights reserved.

"Determination Of Ethanol In Wines By Flow Injection Spectrophotometry Using Gas-diffusion And An Immobilized Enzyme Reactor"
Am. J. Enol. Vitic. 1999 Volume 50, Issue 3 Pages 259-263
António O. S. S. Rangel and Ildikó V. Tóth

Abstract: A flow injection analysis (FIA) system for the spectrophotometric determination of ethanol in wines using an immobilized alcohol dehydrogenase reactor was developed. A gas diffusion unit was used to achieve a large dilution and to separate ethanol from the matrix. This way, the contact of the sample with the reactor was avoided and possible interferences in the spectrophotometric measurements were significantly reduced. The alcohol dehydrogenase enzyme was immobilized on alkylaminated controlled pore glass, and the consumption of the NAD cofactor was minimized (0.6 µmol per assay) by using the merging zone technique. The detection limit was 0.4% (v/v). Good precision was achieved, with relative standard deviations less than 2.2% (n = 9). For 20 samples of different types of table and Port wines, the results showed good agreement with the OIV usual procedure, the relative deviation being less than 4.4%. Thirty determinations per hour can be carried out within the ethanol concentration range of 5% to 25% (v/v), without any sample pretreatment.
Ethanol Port Spectrophotometry

"Enzymatic Determination Of Ethanol And Glycerol By Flow Injection Parallel Multi-site Detection"
Anal. Chim. Acta 2000 Volume 416, Issue 2 Pages 205-210
António O. S. S. Rangel and Ildikó V. Tóth

Abstract: A flow injection method was developed for the sequential enzymatic determination of ethanol and glycerol in wines, using immobilized ethanol dehydrogenase and glycerol dehydrogenase, respectively. The enzymes were immobilized separately on alkylaminated controlled pore glass. A multi-site spectrophotometric detection system was used in parallel configuration to monitor the absorbance change in the two independent analytical channels. A 50-fold dilution of the samples was necessary before injection. The working range was between 0.05 and 0.5% (v/v) for the ethanol and between 0.03 and 0.3 g L-1 for the glycerol determination, with corresponding detection limits of 2 x 10^-3% (v/v) and 2 x 10^-3 g L-1. Relative standard deviations (RSD) (n=9) lower than 2.3% for the ethanol and 2.1% for the glycerol determination were found. For 13 samples of different types of table and Port wines, the results showed good agreement with the corresponding reference procedures; a two level recovery study also showed good accuracy for the developed methods. The sampling rate was 10 hr-1, corresponding to 20 determinations per hour.
Ethanol Glycerol Port Wine Spectrophotometry

"Determination Of Glucose In Blood By Flow Injection Analysis And An Immobilized Glucose Oxidase Column"
Anal. Chim. Acta 1984 Volume 166, Issue 1 Pages 111-118
M. Masoom and Alan Townshend

Abstract: Glucose oxidase was immobilized on controlled-porosity glass and used in a glass column (25 mm x 2.5 mm). The H2O2 (1 µM) produced by the enzymatic reaction between the glucose oxidase and glucose was detected by the current produced in a flow-through cell with two platinum electrodes having a p.d. of 0.6 V. The carrier stream (2 mL min-1) was 0.1 M phosphate buffer (pH 4.5) and a fixed sample volume (~20 µL) was delivered into the stream by means of a rotary valve. Glucose (0.2 mM) could be determined in plasma with either a dialyser or a column (25 mm x 2.5 mm) of Cu diethyldithiocarbamate on controlled-porosity glass inserted before the enzyme column. Results agreed well with those obtained by a conventional analyzer. system and no deterioration of the enzyme was observed over a 10-month period.
Glucose Blood Plasma Amperometry Electrode

"Simultaneous Determination Of Sucrose And Glucose In Mixtures By Flow Injection Analysis With Immobilized Enzymes"
Anal. Chim. Acta 1985 Volume 171, Issue 1 Pages 185-194
M. Masoom and Alan Townshend

Abstract: Sucrose was determined in a flow injection system by using a manifold comprising columns of β-D-fructofuranosidase - aldose 1-epimerase and glucose oxidase (each immobilized on controlled-pore glass) in sequence, followed by amperometric detection of the H2O2 produced. At pH 6.8, max. sensitivity was achieved, the enzyme maintained activity for ~12 weeks, and the detection limit was 10 µM-sucrose. Incorporation of a controlled by-pass around the first column allowed one sample to traverse both columns, thereby allowing the sequential determination of sucrose and glucose (0.1 to 10 mM), while the next sample passed through only the glucose oxidase column; accurate determinations were possible within 25 s. The simultaneous determination of sucrose and glucose was also possible (in 2 min) by means of a single injection, which was split in the apparatus, thus allowing a sucrose - glucose and a glucose peak to be obtained in sequence. For five samples, the coefficient of variation was 1.8% for the combined peak and 2.9% for the glucose peak.
Sucrose Glucose Amperometry

"Solid-state Chemiluminescence Detector For Hydrogen Peroxide Based On An Immobilized Luminophore. Application To Rain-water"
Anal. Chim. Acta 1985 Volume 174, Issue 1 Pages 151-161
P. van Zoonen, D. A. Kamminga, C. Gooijer, N. H. Velthorst and R. W. Frei, G. Gübitz

Abstract: The construction and use of a H2O2 detector, based on peroxyoxalate chemiluminescence, is described. The detector consists of a two-bed reactor packed with solid bis-(2,4,6-trichlorophenyl) oxalate(I) and 3-aminofluoranthene as luminophore, immobilized on controlled-pore glass beads. Optimum conditions for pH, solvent and purity of I for flow-independent operation, and four detector configurations are discussed. Samples can be injected into a moving stream or directly into the monitor to provide a manually operated field monitor. The peak height is directly proportional to the concentration. of H2O2 and the log. - log. calibration graph is rectilinear with a slope of 1 from the detection limit (15 nM, signal-to-noise ratio of 3) up to 10 mM. The coefficient of variation (n = 10) for manual injections of samples was 3% at the 0.5 µM level, with a limit of detection of 40 nM. Sample throughputs are 100 h-1 in the flow injection mode and 40 h-1 for manual injection. Results are presented for determination of H2O2 in rain-water.
Hydrogen peroxide Rain Chemiluminescence

"Determination Of Cholesterol By Flow Injection Analysis With Immobilized Cholesterol Oxidase"
Anal. Chim. Acta 1985 Volume 174, Issue 1 Pages 293-297
M. Masoom and Alan Townshend

Abstract: The injected sample passes at pH 7 through a column of cholesterol oxidase, immobilized on controlled-porosity glass by cross-linking with glutaraldehyde, and the H2O2 produced is detected with an amperometric flow-cell detector. The calibration graph of peak height vs. concentration. is rectilinear for up to 80 mg dl-1 with a coefficient of variation of 1 to 3%, the detection limit is 0.2 µg, and the achievable sample throughput is 80 h-1. The method was applied to the determination of cholesterol in blood serum, wax-wool alcohol and a butter extract and gave results in good agreement with those obtained by established procedures.
Cholesterol Wax wool Blood Serum Food Amperometry

"Preconcentration Of Copper(II) On Immobilized 8-quinolinol In A Flow Injection System With An Ion-selective Electrode Detector"
Anal. Chim. Acta 1986 Volume 179, Issue 1 Pages 509-514
Lars Risinger

Abstract: The cations are bound to the exchanger, containing quinolin-8-ol bound to controlled-pore glass, while the other components pass to waste without contacting the Cu(II)-selective electrode. The bound ions are eluted with 0.5 M HNO3 and the solution is buffered to pH 5.0 with 1 M Na acetate before arrival at the electrode. The recoveries are >95% and the detection limits are 0.1 and 0.03 µM for samples of 5 and 25 ml, respectively.
Copper(II) Water Electrode Potentiometry

"Flow Injection Determination Of Sulfite And Assay Of Sulfite Oxidase"
Anal. Chim. Acta 1986 Volume 179, Issue 1 Pages 399-405
M. Masoom and Alan Townsend

Abstract: The SO32- in formaldehyde-stabilized solution is determined in a carrier of 0.1 M phosphate buffer, pH 8.5. The solution is passed through a column of immobilized sulfite oxidase (on controlled-pore glass) and the H2O2 liberated is measured amperometrically. The concentration. range is from 10 µM to 0.8 mM and the coefficient of variation at 0.4 mM is 2.2% (n = 3). Sulfite oxidase is determined in a stream of 2 mM Na2SO3 stabilized with formaldehyde in 0.1 M phosphate buffer, pH 8.0. The concentration. range is 5 to 100 iu l-1.
Sulfite Enzyme, sulfite oxidase Amperometry

"Conversion Techniques In Flow Injection Analysis. Determination Of Sulfide By Precipitation With Cadmium Ions And Detection By Atomic Absorption Spectrometry"
Anal. Chim. Acta 1986 Volume 184, Issue 1 Pages 165-172
Bo A. Petersson, Zhaolun Fang, Jaromir Rika and Elo H. Hansen

Abstract: Aqueous solution of Na2S and Cd(NO3)2, both containing ammonium acetate buffer, were injected into the flow injection apparatus (details given), mixed and passed to an ion-exchange column (2.6 cm x 2.5 mm) of quinolin-8-ol azo-immobilized on controlled-pore glass (125 to 177 µm; pore size 50 nm). The pptd. CdS passed through the column and Cd was determined by AAS at 228.8 nm; the excess of Cd(II) was retained and later eluted with 1 M HNO3. Calibration graphs were rectilinear up to 2 mg L-1 of S2-, with a detection limit of 10 µg L-1 and a sampling rate of 100 h-1; the coefficient of variation was 1.2%. Phosphate interfered.
Sulfide Water Ion exchange Spectrophotometry

"Cyclic Regeneration Of Nicotinamide Adenine Dinucleotide With Immobilized Enzymes. Flow Injection Spectrofluorimetric Determination Of Ethanol"
Anal. Chim. Acta 1986 Volume 185, Issue 1 Pages 49-55
M. Massom and Alan Townshend

Abstract: Aqueous samples containing ethanol were injected into a stream of 1.5 mM NAD+, 1 mM 2-oxoglutaric acid and 1 mM ammonium acetate in 0.1 M phosphate buffer (pH 8.0) flowing at 2.5 mL min-1. The stream was then fed through a glass column (5 cm x 2.5 mm) containing yeast alcohol dehydrogenase immobilized on controlled-pore glass beads (CPG-240) and the NADH formed was determined fluorimetrically at 460 nm (excitation at 360 nm). After regeneration in a similar column containing CPG-240-immobilized glutamate dehydrogenase the NAD+ solution was returned to its reservoir. Calibration graphs based on peak height were rectilinear for 0.05 to 0.5% of ethanol in aqueous solution with coefficient of variation of 1.7%. With such recycling, the same 50 mL NAD+ carrier stream could be used for 4 days at 50 determinations per day.
Ethanol Fluorescence

"Flow Injection Determination Of Adenosine And Inosine In Blood Plasma With Immobilized Enzyme Columns Connected In Series And Fluorimetric Detection"
Anal. Chim. Acta 1986 Volume 186, Issue 1 Pages 131-137
Yohji Hayashi, Kiyoshi Zaitsu and Yosuke Ohkura

Abstract: Samples were mixed with HClO4, and centrifuged, the supernatant solution was treated with K2CO3 followed by centrifugation, and urate oxidase and catalase, were added to the supernatant solution. After 30 min at 37°C, the solution was treated as above, the supernatant solution was diluted with Tris - HCl buffer of pH 8.0 and an aliquot was analyzed on successive columns (4 cm x 0.86 mm) of adenosine deaminase, purine nucleoside phosphorylase, xanthine oxidase, urate oxidase and horse-radish peroxidase, immobilized on amino-derivatized controlled-pore glass AMP-CPG 1400 (120 to 200 mesh; 140 nm pore size). The carrier and reagent solution contained 0.15 M NaCl - 50 mM Na2HPO4 - 10 mM Na2EDTA and 0.1 M Tris - HCl buffer (pH 8.0); the reagent solution also contains 5 mM 3-(4-hydroxyphenyl)propionic acid(I),flow rates were 0.25 mL min-1, and the H2O2 formed was detected fluorimetrically after reaction with I at 405 nm (excitation at 305 nm). Calibration graphs were rectilinear from 0.5 to 500 pmol of adenosine or inosine, with recoveries of ~100% and coefficient of variation of 1% (n = 10).
Adenosine Inosine Blood Plasma Clinical analysis Fluorescence

"Bipolar Pulse Conductometric Detection Of Enzyme Reactions In Flow Injection Systems. Urea In Serum And Urine"
Anal. Chim. Acta 1986 Volume 186, Issue 1 Pages 91-100
Douglas Taylor and Timothy A. Nieman

Abstract: Urea(I) is determined by enzymatic hydrolysis on a column (5.7 cm x 3.2 mm) of urease bound to controlled-pore glass, with phosphate buffer (pH 7.3) as the carrier stream (20 mM for urine and 2 mM for serum samples) containing NaCl; the reaction is monitored by measuring conductivity before and after hydrolysis, with automatic processing to give a differential signal. The calibration graph is rectilinear for 10 mM I, with detection limits of 0.1 mM I in urine and 0.01 mM I in serum; the coefficient of variation is 3%. The sampling rate is 20 h-1. The accuracy compares well with that of existing methods.
Urea Blood Serum Urine Clinical analysis Conductometry

"Fundamental And Practical Considerations In The Design Of Online Column Preconcentration For Flow Injection Atomic Spectrometric Systems"
Anal. Chim. Acta 1987 Volume 200, Issue 1 Pages 35-49
Zhaolun Fang, Shukun Xu and Suchun Zhang

Abstract: The terms concentration. efficiency (expressed as the enrichment factor per min), retention efficiency, recovery data and elution efficiency are recommended as criteria for the critical evaluation of an online column pre-concentration. system. Efficiency is improved with time-based sampling and double-column systems without intermediate column washing. Columns (4.5 cm x 3 mm) with sample loading rates of 9.5 mL min-1 are proposed for achieving high efficiency and accuracy with AAS detection. More careful optimization of column dimensions is needed for detection by ICP-AES. A procedure for the online pre-concentration. of Co in water, involving columns of CPG-8Q exchanger (Pierce Chemicals) and a mobile phase of 25 mM ammonium acetate (pH 8), is presented. An enrichment factor of 48 was achieved at a sampling rate of 60 h-1. The coefficient of variation was 1.7% at the 40 µg L-1 level (n = 11) and the detection limit was 0.2 µg l-1.
Cobalt Environmental Spectrophotometry

"Deoxygenation Of Supporting Electrolytes In Stripping Voltammetry By Glucose Oxidase And Catalase In A Flow System"
Anal. Chim. Acta 1987 Volume 200, Issue 1 Pages 313-318
Lars Risinger, Xiurong Yang and Gillis Johansson

Abstract: Carrier solution for flow injection anodic-stripping voltammetry were deoxygenated by reaction with glucose added to the carrier, 0.1 M Na acetate - 0.15 M NaCl (pH 6.0) containing 2.5 mM glucose, before sample injection. The reaction was catalyzed by using an enzyme reactor comprising a Plexiglas tube (2.7 mm i.d.) containing glucose oxidase and catalase, co-immobilized on glutaraldehyde-activated silica gel (SP-500) or porous glass (CPG-10). The removal of O was at least as efficient as with N purging, and allowed a more rapid start-up. The voltammetric behavior of Cd(II), Pb(II) and Zn(II) was unaffected by the presence of glucose and gluconate.
Cadmium(2+) Lead(2+) Zinc(II) Voltammetry

"Flow Injection Determination Of Sugars With Immobilized Enzyme Reactors And Chemiluminescence Detection"
Anal. Chim. Acta 1988 Volume 205, Issue 1-2 Pages 195-205
Cathy A. (Koerner) Swindlehurst and Timothy A. Nieman

Abstract: In the determination of glucose, the sample (80 µL) was injected into a carrier stream of 1 mM phosphate buffer (pH 6.5), which was passed through a reactor (5.6 cm x 3 mm) containing glucose oxidase and aldose 1-epimerase, immobilized on controlled-pore glass, and the H2O2 produced was determined via its chemiluminescence reaction with luminol in the presence of peroxidase at pH 11.6. Sucrose, maltose, lactose and fructose were determined similarly after their enzymatic conversion into glucose (with β-fructofuranosidase and aldose 1-epimerase, glucan 1,4-α-glucosidase, β-galactosidase, and glucose isomerase, respectively). Calibration graphs were rectilinear from 0.2 µM to 1 mM glucose, -sucrose and -maltose (limit of detection 0.1 µM) and 3 µM to 1 mM lactose and -fructose (limit of detection 1 µM). Analysis time was ~2 min.
Sugars Sucrose Maltose Glucose Lactose Fructose Carbohydrates Pharmaceutical Chemiluminescence

"Determination Of Myo-inositol In A Flow Injection System With Immobilized Enzyme Reactors And Amperometric Detection"
Anal. Chim. Acta 1988 Volume 206, Issue 1-2 Pages 49-55
Bo Olsson, György Marko-Varga, Lo Gorton, Roger Appelqvist and Gillis Johansson

Abstract: myo-Inositol 2-dehydrogenase, lactate dehydrogenase and lactate oxidase are co-immobilized on porous glass and used in a packed-bed enzyme reactor. The myoinositol(I) in the biological fluid sample reacts to produce an equivalent amount of H2O2, which oxidizes ferrocyanide to ferrocyanide in a second reactor containing immobilized peroxidase. The ferrocyanide is then detected amperometrically at O mV vs. the SCE in a flow-through detector. The system responds rectilinearly to I concentration. from 1 to 300 µM. The max. throughput was 90 samples h-1, and the enzyme reactor was stable for 5 weeks.
Myo-inositol Biological fluid Amperometry

"Flow Injection System For The Amperometric Determination Of Xylose And Xylulose With Co-immobilized Enzymes And A Modified Electrode"
Anal. Chim. Acta 1988 Volume 213, Issue 1-2 Pages 139-150
E. Dominguez and B. Hahn-Hägerdal, G. Marko-Varga and L. Gorton

Abstract: A purpose-built flow injection analysis system was constructed that incorporated a 25 µL reactor containing xylose isomerase (omitted in determination of xylose alone), aldose 1-epimerase and glucose dehydrogenase immobilized on CPG-10 glass beads (37 to 74 µm diameter; 51.5 nm pore size). The NADH produced from NAD+ incorporated in the carrier solution [0.1 M phosphate buffer (pH 7.0) containing Mg] was detected electrochemically with use of a dye-modified graphite electrode in a wall-jet configuration (cf. Appleqvist et al., Anal. Abstr., 1985, 47, 12D151). Up to 30 determinations per hour were possible and calibration graphs were rectilinear for up to 2 mM xylose or -xylulose.
Xylose Xylulose Feed Industrial Amperometry Electrode Sample preparation

"Immobilized Enzymes In Clinical And Biochemical Analysis. Applications To The Simultaneous Determination Of Acetylcholine And Choline And To The Determination Of Lipids"
Anal. Chim. Acta 1988 Volume 214, Issue 1-2 Pages 173-186
M. Masoom

Abstract: A flow injection analysis system was used with a 10 µL sample-injection loop, one or more immobilized-enzyme reactor columns and an amperometric (for choline or acetylcholine) or a commercial (HPLC) potentiometric detector (for lipids). In the former system, acetylcholinesterase and choline oxidase were immobilized by glutaraldehyde cross-linking to controlled-pore glass and packed into columns (3 cm x 2.5 mm) that were operated at 25°C. The amperometric detector consisted of two Pt electrodes (6 mm x 3 mm) sandwiched between Perspex sheets and separated by a 1-mm-thick sheet of silicone rubber, and the carrier stream (0.5 mL min-1) was 0.1 M phosphate buffer adjusted to pH 8.2. Rectilinear calibration graphs for 10 to 100 µM-choline and -acetylcholine were obtained. For determination of phospholipids, the appropriate phospholipase and glycerol-3-phosphate oxidase were used in the included reactor columns with a carrier stream (1.5 mL min-1) of pH 7.0 containing 20 mM barbitone sodium(I), or a column of co-immobilized phosphatase - choline oxidase was used with a buffered carrier stream of 0.1 M Tris (pH 7.5) or 20 mM I (pH 6.5), Triton X-100 (0.2 or 0.3%) and 0.4 mM ZnCl2 or 30 mM CaCl2. Calibration graphs for phosphatidylcholine were rectilinear for 1 to 10 mM or 50 to 800 mg l-1.
Choline Acetylcholine Phospholipids Blood Amperometry Biochemical analysis Clinical analysis Ion exchange Potentiometry Electrode

"Determination Of Creatinine In Undiluted Blood Serum By Enzymatic Flow Injection Analysis With Optosensing"
Anal. Chim. Acta 1988 Volume 214, Issue 1-2 Pages 147-159
Mette Tranholm Jeppesen and Elo Harald Hansen

Abstract: For the determination of creatinine in serum, a flow injection system was used that included a packed-bed enzyme reactor containing creatinine deiminase bonded to CPG-10 controlled-pore glass to liberate NH4+, which was converted into NH3 and transferred via a gas-permeable membrane (Celgard 2500 polypropylene; 0.04 µm pores) to an indicator stream containing 0.04% bromothymol blue at pH 6.8 for spectrophotometric detection at 620 nm. Endogenous NH3 was either removed by inclusion of a pre-reactor containing CPG-10-bonded glutamate dehydrogenase [NAD(P)+] or was monitored in a parallel system in which the creatinine deiminase reactor was omitted to obtain a reagent blank. The carrier stream in the pre-reactor system was 50 mM Tris containing 5 mM 2-oxoglutaric acid, 2 mM NADH, 20 mM EDTA and 0.5 mM ADP, with a reagent stream of 0.2 M NaOH to provide pH of 11.9. In the reagent-blank system, the carrier stream contained 20 mM Na2B4O7 to give pH of 9.2. The operating range was up to 1 mM for NH3 and for creatinine. Enzymic conversion efficiency was 91 ± 2%, and within-run reproducibility was ±4%.
Creatinine Blood Serum Clinical analysis Spectrophotometry

"Flow Injection Determination Of Inorganic Pyrophosphate With Use Of An Enzyme Thermistor Containing Immobilized Inorganic Pyrophosphate"
Anal. Chim. Acta 1988 Volume 214, Issue 1-2 Pages 409-413
Ikuo Satoh and Taro Ishi

Abstract: Inorganic pyrophosphatase was immobilized on to controlled-pore glass which was then loaded into a plastic column (0.28 mL packed volume; i.d. 7 mm) mounted in a poly(methyl methacrylate) holder. The holder consisted of two channels, one of which contained a glass-encapsulated thermistor fixed on a gold capillary at the outlet of the column; the other channel contained the carrier stream. The assembly was placed in a thermostatted aluminum cylinder containing two heat exchangers. A reference column containing only washed glass beads was also incorporated. Sample solution (500 µL) was injected via a loop-injection rotary value and the carrier solution (1 mL min-1) was 0.1 M Tris - HCl buffer (pH 7.2) that was 1 mM in MgCl2. The calibration graph was rectilinear for 0.5 to 20 mM pyrophosphate and the coefficient of variation (n = 30) was 2%. Calcium and Sr interfered.
Pyrophosphate Enthalpimetry Thermistor

"Enzymatic Methods For The Determination Of Ethanol Based On Linear And Cyclic Flow Injection Systems"
Anal. Chim. Acta 1988 Volume 214, Issue 1-2 Pages 161-172
A. M. Almuaibed and Alan Townshend

Abstract: Mini-columns (2.5 cm x 2.5 mm) containing(I) alcohol dehydrogenase bonded to controlled-pore glass and(II) aldehyde dehydrogenase bonded to CNBr-activated Sepharose 4B were used in a linear or a closed-loop flow injection system for ethanol oxidation with formation and fluorimetric detection of NADH at 460 nm (excitation at 360 nm) or spectrophotometric detection at 340 nm. In the linear system the carrier stream contained 1.8 mM NAD+, 0.15 M KCl, 1 mM 2-mercaptoethanol and 0.05 M Na4P2O7 adjusted to pH 9.0 with 0.1 M NaOH. The cyclic system also contained a column (5 cm x 2.5 mm) of glutamate dehydrogenase for regeneration of NAD+, and the carrier stream (adjusted to pH 8.5 instead of 9.0) contained additionally 0.02 M NH4Cl and 0.01 M 2-oxoglutarate. The linear system provided the more sensitive conditions, with a rectilinear calibration range of 0.17 to 4 mM ethanol.
Ethanol Fluorescence Spectrophotometry

"Flow Injection Amperometric Determination Of Oxalate With Immobilized Oxalate Oxidase"
Anal. Chim. Acta 1989 Volume 218, Issue 1 Pages 1-6
Ala'ddin M. Almuaibed and Alan Townshend

Abstract: Oxalate oxidase (7.2 mg) was immobilized on 0.2 g of aminopropylsilanized controlled-pore glass as described by Masoom and Townshend (Anal. Chim. Acta, 1984, 166, 111) and the glass was packed into a column (2.5 cm x 2.5 mm) which was incorporated into a flow injection system for determination of oxalate. Sample solution (40 µL) was injected into the carrier stream (0.05 M Na succinate buffer of pH 3.5; 1 mL min-1) before passing, via a 15-cm mixing coil, to the enzyme-immobilized column where oxlate was oxidized to CO2 with the production of H2O2. The H2O2 was determined amperometrically at 0.6 V. Interference from Cu was masked by addition of Na2EDTA to the carrier stream or by incorporating a Chelex-100 column before the immobilized-enzyme column. The calibration graph was rectilinear from 6 µM to 0.9 mM oxalate and the detection limit was 5.7 µM. The coefficient of variation (n = 10) was 1% at 0.45 mM. Sample throughput was 40 h-1. The effects of ascorbic and uric acids were studied. Oxalate oxidase (7.2 mg) was immobilized on 0.2 g of aminopropylsilanized controlled-pore glass as described by Masoom and Townshend (Anal. Chim. Acta, 1984, 166, 111) and the glass was packed into a column (2.5 cm x 2.5 mm) which was incorporated into a flow injection system for determination of oxalate. Sample solution (40 µL) was injected into the carrier stream (0.05 M Na succinate buffer of pH 3.5; 1 mL min-1) before passing, via a 15-cm mixing coil, to the enzyme-immobilized column where oxlate was oxidized to CO2 with the production of H2O2. The H2O2 was determined amperometrically at 0.6 V. Interference from Cu was masked by addition of Na2EDTA to the carrier stream or by incorporating a Chelex-100 column before the immobilized-enzyme column. The calibration graph was rectilinear from 6 µM to 0.9 mM oxalate and the detection limit was 5.7 µM. The coefficient of variation (n = 10) was 1% at 0.45 mM. Sample throughput was 40 h-1. The effects of ascorbic and uric acids were studied.
Oxalate Amperometry

"Spectrophotometric Flow Injection Determination Of Urea In Body Fluids By Using An Immobilized Urease Reactor"
Anal. Chim. Acta 1989 Volume 218, Issue 1 Pages 151-155
Petr Solich, Miroslav Poláek and Rolf Karlíek, Olga Valentová and Miroslav Marek

Abstract: Urease was immobilized on poly(glycidyl methacrylate)-coated controlled-porosity glass which was then packed into PTFE tubing. The reactor was incorporated into a flow system for determination of urea. Serum was diluted 1:50 and urine 1:1000 or 1:2000 with water before injection into 0.2 M phosphate buffer (pH 6.9) containing 2 mM Na2 EDTA, 1 mM NaN3 and 5 µM-2-mercaptoethanol. The solution passed to the enzyme reactor before being mixed with 0.37 M Na salicylate - 4 mM Na nitroprusside - 0.25 M NaOH in aqueous 30% ethanol, and then with 30 mM NaClO and passing to a reaction coil (1.5 m x 0.5 mm) at 50°C. The absorbance of the solution was measured at 700 nm. The calibration graph was rectilinear for 25 to 500 µM-urea, and the coefficient of variation (n = 10) for 300 µM-urea was 1.1%.
Urea Blood Serum Urine Spectrophotometry

"Simultaneous Determination Of Ammonia And Urea With An Asymmetric Merging Zones Flow Injection Configuration"
Anal. Chim. Acta 1989 Volume 221, Issue 1 Pages 173-177
J. S. Cosano, J. L. Calle, J. L. Pinillos, P. Linares and M. D. Luque de Castro

Abstract: The system described includes a dual injection valve and parallel reaction loops for the reaction of NH3 with Nessler`s reagent (1.2 mL min-1) in the presence of 0.1 M NaCl (0.5 mL min-) and of urea with immobilized urease (bound to controlled pore glass). The reaction streams were merged and the urea reaction was terminated by the alkalinity of the medium (0.1 M KH2PO4 at pH 8). Total and free NH3 was determined by absorbance measurement at 380 nm. The coefficient of variation at 2 µg mL-1 were 2.3 and 1.9% for NH3 and urea, respectively, with corresponding determination ranges of 1.0 to 5.0 and 1.0 to 6.0 µg mL-1. The method was applied to the determination of NH3 and urea in agricultural irrigation water.
Ammonia Urea Irrigation Solution Spectrophotometry

"Flow Injection Determination Of Malate With Immobilized Malate Dehydrogenase"
Anal. Chim. Acta 1989 Volume 221, Issue 2 Pages 337-340
Ala'ddin M. Almuaibed and Alan Townshend

Abstract: Malate dehydrogenase was immobilized on controlled-pore glass and packed in a glass column (2.5 cm x 2.5 mm); the column was used in a flow injection system. The carrier stream (2 mL min-1) consisted of 0.1 M phosphate buffer (pH 11.5) - 3.6 mM NAD+ (1:1); detection was at 340 nm. The detection limit was 7 µM and calibration graphs were rectilinear for 0.9 mM malate. The coefficient of variation (n = 10) was 1.5% for 0.44 mM. The column was stable for 1 month without loss in activity. The sampling rate was 50 h-1. Malate dehydrogenase was immobilized on controlled-pore glass and packed in a glass column (2.5 cm x 2.5 mm); the column was used in a flow injection system. The carrier stream (2 mL min-1) consisted of 0.1 M phosphate buffer (pH 11.5) - 3.6 mM NAD+ (1:1); detection was at 340 nm. The detection limit was 7 µM and calibration graphs were rectilinear for 0.9 mM malate. The coefficient of variation (n = 10) was 1.5% for 0.44 mM. The column was stable for 1 month without loss in activity. The sampling rate was 50 h-1.
l-Malate

"Flow Injection Determination Of L-tyrosine In Serum With An Immobilized Tyrosinase Reactor And Fluorescence Detection"
Anal. Chim. Acta 1989 Volume 224, Issue 1 Pages 133-138
Nobutoshi Kiba, Masae Ogi and Motohisa Furusawa

Abstract: Serum (100 µL) was deproteinized with Na2WO4 - H2SO4 and filtered, and 50 µL of the filtrate was injected into a carrier stream of 0.3 M phosphate buffer of pH 7.2 (0.6 mL min-1), which passed through a separation column (5 cm x 4 mm) of Capcell 120 C18 (5 µm) and a reactor column (5 cm x 4 mm) containing the cited enzyme immobilized on controlled-pore glass beads and was then mixed with a stream of 5 M KOH (1 mL min-1). The fluorescence was measured at 490 nm (excitation at 375 nm). The calibration graph was rectilinear in the range 0.1 µM to 0.1 mM tyrosine, the detection limit was 50 nM, and the coefficient of variation (n = 10) for the determination of 5.0 µM-tyrosine was 2.0%.
Tyrosine Blood Serum Fluorescence

"Rapid Flow Injection Sandwich-type Immunoassays Of Proteins Using An Immobilized Antibody Reactor And Adenosine Deaminase-antibody Conjugates"
Anal. Chim. Acta 1990 Volume 229, Issue 1 Pages 47-55
I. H. Lee and M. E. Meyerhoff

Abstract: Human IgG (I) and α1-acid glycoprotein (II) were determined in biological samples in a flow injection assay cycle involving sequential injection of sample and adenosine deaminase - antibody conjugate (goat anti-I or rabbit anti-II antibodies for I and II, respectively) into a 0.025 M Tris - HCl (pH 7.5) carrier buffer. Bound antibody - analyte was retained on a column (2.5 cm x 1.54 mm) of goat anti-I or rabbit anti-II antibodies immobilized on controlled-pore glass beads. Substrate (0.25 mM adenosine in 0.5 M Tris - HCl of pH 7.5) was then fed into the reactor and the eluate was monitored by NH4+-selective potentiometry. Between steps a wash stream of 0.1 M glycine - HCl (pH 2.2) was used. Working ranges were from 5 to 400 ng mL-1 of I and 3 to 15 µg mL-1 of II. Results for I agreed well with those by several commercially available methods and recoveries were 102 to 107%. Analysis time was ~12 min.
Protein Biological Immunoassay Potentiometry

"Integrated Reaction And Photometric Detection With Enzymes Immobilized In The Flow Cell Of A Flow Injection System"
Anal. Chim. Acta 1990 Volume 230, Issue 1 Pages 199-202
Pilar Linares, M. D. Luque de Castro and M. Valcárcel

Abstract: Alcohol dehydrogenase was immobilized on the walls of the detector cell by use of controlled-pore glass as support (cf. Masoom and Townshend, Ibid., 1986, 179, 399). Simultaneous dual-valve injections were made of sample and NAD+ into separate streams of 0.8% semicarbazide hydrochloride solution in 0.075 M K4P2O7 buffer (pH 7.5), and the merged streams passed through a delay coil (35 to 65 s) before stopped-flow absorbance measurement at 340 nm. Regression parameters are tabulated for various combinations of optical pathlength (0.5 to 5 mm) and stop-time (1 to 5 s). The calibration graph was rectilinear typically for 2 to 500 µg mL-1 of ethanol.

"Flow Injection Analysis For L-glutamate Using Immobilized L-glutamate Oxidase: Comparison Of An Enzyme Reactor And Enzyme Electrode"
Anal. Chim. Acta 1990 Volume 231, Issue 1 Pages 121-124
Toshio Yao, Naokazu Kobayashi and Tamotsu Wasa

Abstract: The enzyme was covalently immobilized by the use of aminopropyl-controlled-pore glass and glutaraldehyde, and by cross-linking the enzyme and bovine serum albumin with glutaraldehyde on one side of a Pt sheet silanized with 3-aminopropyltriethoxysilane. The former was used in a packed-bed reactor and the latter as a flow-through enzyme electrode in a flow injection system. Both systems were compared in the determination of L-glutamate (I), the H2O2 evolved being monitored amperometrically. The carrier solution was 0.1 M phosphate buffer (pH 7.5) pumped at 1.5 mL min-1. The speed of analysis was 60 samples h-1 with the enzyme electrode compared with 90 to 120 samples h-1 with the enzyme reactor. The detection limits were 2 and 4 µM for the reactor and electrode, respectively. For both systems the peak current was rectilinearly related to the I concentration. from 5 µM to 1 mM. The selectivity and stability of both systems were similar. The methods were applied to the determination of I in Japanese seasonings; the coefficient of variation was 0.7% (n = 12).
l-Glutamate Amperometry Electrode

"Spectrophotometric Determination Of Ethanol In Blood Using A Flow Injection System With An Immobilized Enzyme (alcohol Dehydrogenase) Reactor Coupled To An Online Dialyser"
Anal. Chim. Acta 1990 Volume 231, Issue 1 Pages 115-119
Gabrielle Maeder, Jean-Luc Veuthey, Michel Pelletier and Werner Haerdi

Abstract: Whole blood (110 µL) which was not pre-treated was introduced into the carrier stream of sodium pyrophosphate decahydrate - semicarbazide hydrochloride - glycine - NaCl - NAD+ - water (pH 9.0) by means of a rotary valve. The mixture was passed at 650 µL min-1 to a dialyser comprising a Cuprophan membrane between two Plexiglas plates, then to a controlled-pore glass enzyme reactor with a nylon filter fabric (mesh size 100 µm) at each end, kept at 25°C. The absorbance of NADH was measured at 340 nm. The calibration graph was rectilinear from 3 to 40 µg mL-1 of ethanol, corresponding to 0.3 to 4.0 g of ethanol per 1000 g of whole blood prior to dilution. The results agreed with those from direct-injection GC.
Ethanol Whole Spectrophotometry

"Flow Injection Determination Of Paraoxon By Inhibition Of Immobilized Acetylcholinesterase"
Anal. Chim. Acta 1990 Volume 236, Issue 2 Pages 267-272
M. E. Leon-Gonzalez and Alan Townshend

Abstract: A system incorporating two injection valves and a spectrophotometric detector (operated at 500 nm) is described and illustrated. The sample solution is injected into a stream of 0.05 M phosphate buffer (pH 8.0) containing 45 mM NaCl and 12 µM-MgCl2. A solution (0.3 mM) of 1-naphthyl acetate, as substrate, is injected via the second valve into the middle of the paraoxon zone. The resulting stream is passed through a glass column, maintained at 29°C, containing acetylcholinesterase immobilized (method described) on controlled-porosity glass (80 to 120 mesh; mean pore diameter 22.6 nm) and is then merged with a solution (5 mM) of p-nitrobenzenediazonium fluoroborate to react with the liberated naphthol before reaching the detector. Continuous-flow and stopped-flow versions of the method are described; in the latter, the sample and substrate are stopped for 35 s in the column. Rectilinear ranges of the continuous- and stopped-flow procedures are 0.2 to 15 µM and 10 to 400 nM, respectively; corresponding detection limits and sample throughput are 0.4 µM and 60 h-1, and 8 nM and 30 h-1. The coefficient of variation (n = 6) for the respective procedures were 1.4% at 8 µM and 0.9% at 0.25 µM. Optimization of the method is described.
Paraoxon Spectrophotometry

"Membrane-type Immobilized-enzyme Reactors For Use In Flow Injection Analysis"
Anal. Chim. Acta 1992 Volume 256, Issue 1 Pages 53-57
S. Cliffe*, C. Filippini, M. Schneider and M. Fawer

Abstract: A simple membrane reactor housing with negligible dead volume was constructed to take commercially available membranes. Three immobilized-enzyme systems were investigated: glucose oxidase, glutaminase and laccase for the determination of glucose, glutamine and phenolics, respectively. The response of reactors with Zetaffinity AM membranes was comparable to that of packed bed reactors containing controlled-pore glass or Biosynth VA Epoxy, but the membrane reactors had less pressure build-up and thus increased lifetime compared with the packed beds (which were prone to clogging) in continuous monitoring. Their lifetime was determined by the loss of enzyme activity. Membrane-type reactors were prepared using a commercially available support. In three test systems, involving glucose oxidase, glutaminase and laccase, they compared favorably with packed-bed reactors in a flow injection set-up. Although similar responses were observed for all reactor types, the membranes are easier to handle than microparticles. When making continuous inline measurements of bioprocesses at high sampling frequencies, the lifetimes of packed beds of controlled pore glass were determined by pressure build-up within the reactor. When replaced with membrane reactors, no clogging was observed, the lifetimes being determined by loss of enzyme activity.
Glucose

"Selective Determination Of Dextrins By Liquid Chromatography With Post-column Enzymic Reaction, Using Co-immobilized Enzymes"
Anal. Chim. Acta 1992 Volume 257, Issue 1 Pages 79-87
F. Ortega

Abstract: The CPG-10 support (Serva; pore diameter 51.5 nm, paricle size 37 to 74 µm) was treated with 10% (3-aminopropyl)triethoxysilane solution in toluene, the product was activated with glutaraldehyde, and glucan 1,4-α-glucosidase, glucose dehydrogenase and aldose 1-epimerase were co-immobilized on its surface from 0.1 M phosphate buffer medium (pH 6) at ambient temperature and then at 4°C. Parameters influencing enzyme behavior were optimized by means of three flow injection systems in which reactors containing the immobilized enzymes were incorporated (diagrams given). For the determination of dextrins in wort and beer, diluted samples were cleaned up by membrane filtration (0.22 µm) and passage through Sep-Pak cartridges, and portions (20 µL) were then injected into a column of Aminex HPX-42-A (at 65°C) preceded by a Micro-Guard column (Bio-Rad) for HPLC with water as mobile phase. The eluate passed via a T-piece into a flow of 3 mM NAD+ in 0.3 M phosphate buffer of pH 6 and thence through the enzyme reactor (at 40°), and the final product NADH was detected at 340 nm. Rectilinear calibration graphs based on peak area were obtained for the various dextrins, and detection limits ranged from 200 to 500 ng. The co-immobilized enzymes remained stable for 55 days when stored in 1 M NaCl at 4°.
Dextrins Beer Beer Wort HPLC Amperometry Spectrophotometry

"Sequential Determination Of Glucose, Fructose And Sucrose By Flow Injection Analysis With Immobilized Enzyme Reactors And Spectrophotometric Detection"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 137-143
Cándido García de María and Alan Townsend*

Abstract: The flow system described and illustrated incorporates two injection valves, one preceding and one after a reactor (R3) containing β-fructofuranosidase, reactors containing hexokinase (R2), phosphoglucose isomerase - glucose-6-phosphate dehydrogenase (R4) and glucose-6-phosphate dehydrogenase alone (R1 and R5), and a flow cell monitored by a spectrophotometer. All enzymes are immobilized on controlled porosity glass (Masoom and Townshend, Ibid., 1985, 171, 185). A solution of ATP, NADP+ and Mg2+ in phosphate buffer (pH 8.0) is used as carrier. Three sequential injections are carried out for each sample; the flow passes through R2, R5 and then R1 for glucose; R3, R2, R5 and R1 for glucose plus sucrose; and R2, R4 and R1 for glucose plus fructose. The method shows good selectivity, and covers the range 0.01 to 1 mM. A flow injection spectrophotometric procedure is described for the sequential determination of glucose, fructose, and sucrose in a sample solution by using single and dual immobilized enzyme reactors in a single manifold. Concentrations in the range 10^-5-10-3 M are determined with anal. rates of 30-40 determinations h-1. A relative standard deviation of 0.8% was obtained for glucose and sucrose and of 1.1% for fructose (10 samples of 4 x 10^-4 M in all cases).
Glucose Fructose Sucrose Spectrophotometry

"Amperometric Flow Injection Method For Determination Of Biogenic Diamines And Hypoxanthine By Combined Use Of Immobilized Enzyme Reactors And A Peroxidase Electrode"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 161-165
Toshio Yao*, Masahiro Satomura and Tamotsu Wasa

Abstract: The sample solution (50 µL) was injected into a carrier solution (0.1 M pyrophosphate buffer of pH 8.7), then the flow was split between two reactors containing putrescine oxidase and xanthine oxidase, respectively, immobilized on controlled-pore glass. A delay coil was incorporated in the latter flow line so that two peaks would be obtained for each sample. The flows were reunited before introduction of a mediator solution [1.0 mM Fe(CN)64- in 0.1 M phosphate buffer of pH 7.0] and flow-through a mixing coil to a cell equipped with a vitreous carbon electrode on which peroxidase had been cross-linked by glutaraldehyde for amperometric measurement of the H2O2 formed in the two enzymatic reactions. The first and second signals corresponded to total polyamines (cadaverine, spermine and putrescine) and to hypoxanthine, respectively. The calibration graphs were rectilinear from 1 to 500 µM and the detection limit was 0.5 µM for polyamines or for hypoxanthine. Both reactors maintained adequate activity during repetitive use for 40 days. The method was applied to a deproteinized aqueous extract of pork loin meat undergoing storage at 5°C. A flow injection system is proposed for the determination of meat freshness, based on the simultaneous determination of polyamines and hypoxanthine. A putrescine oxidase reactor and xanthine oxidase reactor were incorporated at fixed positions in a flow system, which was based on the splitting of the flow after sample injection and subsequent confluence before reaching the peroxidase electrode. Because each channel has a different residence time, two peaks were obtained. The first peak corresponded to the total polyamine concentration. (putrescine, cadaverine and spermidine); the second peak to hypoxanthine. The calibration graphs were linear in the range 1 x 10^-6-5 x 10^-4M. The detection limit was 0.5 x 10^-6 M for polyamines and hypoxanthine. The measurement of polyamines and hypoxanthine contents in porcine loin meat could be performed at a rate of 25 samples per h with satisfactory precision (<1.5% RSD).
Amines, biogenic Hypoxanthine Cadaverine Spermine Putrescine Meat Amperometry Electrode

"Total And Individual Determination Of Creatine Kinase Isoenzyme Activities By Flow Injection And Liquid Chromatography"
Anal. Chim. Acta 1992 Volume 263, Issue 1-2 Pages 43-52
M. D. Luque de Castro* and J. M. Fernandez-Romero

Abstract: A flow manifold was devised for online coupling of a cyclic flow injection system to a LC system. Sample was injected simultaneously into the loops of the flow injection and LC systems. By appropriate switching of the selective valve, the combined stream is trapped in a closed circuit consisting of an enzyme reactor containing two auxiliary enzymes co-immobilized on controlled-pore glass (120 to 200 mesh); spectrophotometric or fluorimetric detection was used. The optimum values of the experimental parameters were evaluated. The method was applied to the determination of the total and individual activity of creatine kinase isoenzymes in serum. Recoveries were >95%. Calibration graphs were rectilinear from 0.01 to 2.00 iu L-1 and the coefficient of variation were 2%. A flow injection liquid chromatography method for the simultaneous determination of total and individual creatine kinase isoenzyme activities is proposed. The flow manifold coupled to the liquid chromatograph is a cyclic circuit that allows each eluted isoenzyme to be trapped within. This circuit includes a spectrophotometric detector and a reactor containing co-immobilized auxiliary enzymes that take part in the post-column derivatization reaction. The iterative passage of the reacting zones through the enzymatic reactor provides a series of multiple-peak recordings that can be used to determine total and individual isoenzymes; such recordings supply a wealth of information and enhanced sensitivity for the determinations Linear calibration graphs between 0.01 and 2.00 U L-1 and relative standard deviations <2.0% were obtained in all instances. The application of the method to serum samples provided results consistent with those obtained by the standard method and recoveries within the range 95-105%.
Enzyme, creatine kinase, isoenzymes Blood Serum HPLC

"Determination Of Sub-nanomole Amounts Of Hydrogen Peroxide Using An Immobilized Enzyme Flow Cell: Application To The Determination Of Ethanol"
Anal. Chim. Acta 1992 Volume 266, Issue 2 Pages 309-315
Robert W. Marshall and Timothy D. Gibson

Abstract: A flow injection system was developed to measure hydrogen peroxide using the luminol-4-iodophenol reaction and an immobilized horseradish peroxidase flow cell. The immobilization procedure adopted allowed 17.06 mg of peroxidase to be attached covalently to 1 g of controlled-pore glass. Hydrogen peroxide could be quantitatively detected down to a limiting concentration of 1 times 10^-8 M, which corresponded to 1 pmol. The incorporation of immobilized alcohol oxidase into the flow system allowed the determination of ethanol down to the same low levels.
Hydrogen peroxide Ethanol Spectrophotometry

"Effects Of PH, Temperature And Reaction Products On Performance Of An Immobilized Creatininase - Creatinase - Sarcosine Oxidase Enzyme System For Creatinine Determination"
Anal. Chim. Acta 1992 Volume 268, Issue 2 Pages 331-345
Henning Sakslund and Ole Hammerich*

Abstract: The three cited enzymes (I, II and III, respectively) were immobilized on controlled pore glass CPG-10 (average pore size 70 nm, particle size 75 to 125 µm) by a modification of the method of Weetall (Methods in Enzymology, 1976, 44, 140). Two flow injection systems (illustrated) were used; one had the enzymes separately immobilized in series, the other comprised co-immobilized I, II and III, and II and III in parallel flow paths. After passing the sample through either system, the H2O2 resulting from the coupled reactions was detected amperometrically in an electrochemical cell of wall-jet type equipped with a Pt working electrode, a Pt wire auxiliary electrode and a SCE as reference. The working potential was +650 mV vs. the SCE and the optimal temperature was 25°C at pH 7.7. Glycine increased the activity of I and decreased that of III but had no effect on II. Formaldehyde and urea had no effect on the enzymes. Calibration data for creatinine for 400 µM, with and without the presence of creatine (5 µM), are tabulated. The unused immobilized enzymes maintained their activity for at least 6 months, but with heavy daily use the activities of I and III, but not of II, decreased by ~25% after 20 to 30 days.
Creatine Creatinine Sarcosine Amperometry Electrode Electrode

"Flow Injection Determination Of Glucose In Serum With An Immobilized Pyranose Oxidase Reactor"
Anal. Chim. Acta 1992 Volume 269, Issue 2 Pages 187-191
Nobutoshi Kiba*, Fumito Ueda and Motohisa Furusawa, Takeshi Yamane

Abstract: Pyranose oxidase (I) or glucose oxidase (II) were immobilized on controlled-pore glass beads and packed into a stainless-steel column (5 cm x 4 mm) by the slurry-packing method. Glutaraldehyde solution (2%) in 0.1 M phosphate buffer (pH 7.0) was pumped through the column for 2 h at 0.2 mol min-1 and the column was washed with water. Enzyme solution (1000 iu of I or II in 0.05 M phosphate buffer (pH 7.0) was circulated through the column for 3 h at 0.2 mL min-1. Serum samples (10 µL) were injected into the carrier stream of the flow injection system (diagram given) and the H2O2 produced in the reactor at 40°C was detected by measuring the chemiluminescence emitted on mixing with luminol and K3Fe(CN)6. The calibration graph was rectilinear from 0.2 to 500 and 0.5 to 1000 µM of glucose for immobilized I and II, respectively; the corresponding detection limits were 0.05 and 0.1 µM.
Glucose Blood Serum Chemiluminescence

"Flow Injection Determination Of 1,5-anhydroglucitol In Serum With An Immobilized Pyranose Oxidase Reactor And Chemiluminescence Detection"
Anal. Chim. Acta 1993 Volume 271, Issue 1 Pages 47-51
Nobutoshi Kiba*, Fumito Ueda, Kazuya Saegusa, Yuusuke Toto and Motohisa Furusawa, Takeshi Yamane

Abstract: A stainless-steel enzyme reactor column (5 cm x 4 mm) containing pyranose oxidase bonded via glutaraldehyde onto aminopropyl-bonded controlled-pore glass (200 to 400 mesh; pore diameter 59 nm) was used at 50°C with a pre-column (4 cm x 4 mm) of TSKgel SAX anion-exchange resin (10 µm; Cl- form) for sample cleanup and a flow stream of 0.1 M NaH2PO4 - 0.05 M citric acid (pH 4.5). Post-column reaction with 0.7 mM luminol and 0.2 M K3Fe(CN)6 in a 0.3 M carbonate buffer (pH 10.5) was followed by luminescence detection of H2O2. The rectilinear calibration range was 0.4 µM to 0.2 mM 1,5-anhydroglucitol (I) with a detection limit of 0.2 µM. Human serum was analyzed and recoveries of I were 97 to 104% with coefficient of variation of 2.0% within-day or 2.4% between-day for 144 µM-I.
1,5-Anhydroglucitol Blood Serum Chemiluminescence

"Determination Of L-glutamate And L-glutamine By Flow Injection Analysis And Chemiluminescence Detection: Comparison Of An Enzyme Column And Enzyme Membrane Sensor"
Anal. Chim. Acta 1993 Volume 271, Issue 2 Pages 231-237
Gert Blankenstein, Frank Preuschoff, Uwe Spohn and Karl-Heinz Mohr, Maria-Regina Kula*

Abstract: L-Glutamate and L-glutamine were determined by luminol chemiluminescence with flow injection analysis. Glutamate oxidase and glutaminase were co-immobilized on controlled-poreglass (CPG) for the determination of glutamate and glutamine. The hydrogen peroxide produced by enzymatic degradation was detected by luminol chemiluminescence catalyzed by peroxidase either immobilized on CPG or on a pre-activated membrane. Arthromyces ramosus peroxidase produced a much stronger luminescence signal than horseradish peroxidase. Immobilization of the microbial peroxidase on a membrane inside the flow cell simplified the technique. The membrane sensor had a detection limit of 0.1 µM-L-glutamate and 1 µM-L-glutamine. The calibration graph was rectilinear for 0.1 to 60 µM-L-glutamate and 1 µM to 2.5 mM L-glutamine.
l-Glutamate l-Glutamine Chemiluminescence Sensor

"Flow Injection Analysis For The Measurement Of Penicillin V In Fermentation Media"
Anal. Chim. Acta 1993 Volume 274, Issue 1 Pages 117-123
Morten Carlsen, Lars H. Christensen and Jens Nielsen*

Abstract: Iodimetric, potentiometric and molybdenum blue flow injection analysis systems for the determination of penicillin (I) in fermentation media were evaluated. All three methods were based on the enzymatic hydrolysis of I to penicilloic acid (II) using β-lactamase, followed by the detection of II. β-Lactamase was immobilized on aminopropyl controlled-pore glass beads (120 to 200 mesh) and the beads were packed in a column and used as a packed-bed reactor. Conversion of I to II was 100%, therefore total I and II present in the sample was determined. The iodometric method was the best for measurements in fermentation media. By replacing the enzyme reactor with an enzyme-free dummy reactor, II alone was determined, and hence the concentration. of I was calculated. The limit of detection was 2.5 mg L-1 and calibration graphs were rectilinear from 2.5 to 150 mg l-1. There was no interference from other components in the fermentation medium. The iodometric analyzer. was applied to the monitoring of a fed-batch I fermentation; results correlated well with those obtained by LC.
Penicillin V Fermentation broth Potentiometry Spectrophotometry

"Comparison Between Different Inorganic Supports For The Immobilization Of Amyloglucosidase And α-amylase To Be Used In Enzyme Reactors In Flow Injection Systems. 2. Hydrolysis Of Glycogen"
Anal. Chim. Acta 1993 Volume 276, Issue 2 Pages 319-328
Jenny Emn&eacute;us* and Lo Gorton

Abstract: As a continuation of the work described in Part I, the immobilization procedure for glucan 1,4-α-glucosidase was carried out with four different inorganic supports, and heat-stable α-amylase (Termamyl) was also immobilized on three supports. The highest immobilization efficiency and optimum activity (for the entry of glycogen) were obtained with controlled-pore glass (CPG) supports of pore size 170 and 729 Å, respectively, and for Termamyl with Micropil A and a CPG support of 1489 Å, respectively.
Glycogen

"Comparison Between Different Inorganic Supports For The Immobilization Of Amyloglucosidase And α-amylase To Be Used In Enzyme Reactors In Flow Injection Systems. 1. Hydrolysis Of Maltose, Malto-oligosaccharides And Soluble Starch"
Anal. Chim. Acta 1993 Volume 276, Issue 2 Pages 303-318
Jenny Emn&eacute;us* and Lo Gorton

Abstract: Glucan 1,4-α-glucosidase was immobilized on 12 different inorganic supports by silanization with aminopropyltriethoxysilane (I) followed by glutaraldehyde activation; also, two of the alumina-based supports were treated with PEI before glutaraldehyde activation. The various packings were then packed in identical reactors (1 cm x 2 mm; 30 µL) and compared in respect of optimum pore size, surface area, immobilization efficiency and enzyme activity for the cited substrates. The highest immobilization efficiency was obtained with a controlled-pore glass with a pore size of 170 .angstrom., and the highest enzyme activity with Micropil A (pore size 300 .angstrom.; surface area 100 to 150 m2 g-1). Treatment of the alumina supports with I gave better results than did treatment with PEI.
Maltooligosaccharides Maltose Starch

"Determination Of Xylose And Glucose In A Flow Injection System With PQQ [pyrroloquinoline Quinone-]dependent Aldose Dehydrogenase"
Anal. Chim. Acta 1993 Volume 280, Issue 1 Pages 119-127
Maria Smolander*, Julia Cooper, Wolfgang Schuhmann, Martin H&auml;mmerle and Hanns-Ludwig Schmidt

Abstract: PQQ-dependent aldose dehydrogenase was immobilized on (i) glutaraldehyde-activated controlled-pore glass (1400 .angstrom.), (ii) a heat-treated carbodi-imide-activated graphite electrode or (iii) an Os-containing redox polymer. Different flow injection systems were investigated to optimize the measurements; the highest current densities were obtained with (ii) and the best stability with (i). For the analysis of fermentation samples containing xylose as the only C source the (i) system was used, with 500 µm-phenazine methosulfate added as a mediator in a carrier stream of 50 mM potassium phosphate of pH 6.5. Results for xylose agreed well with those obtained by HPLC. For samples containing both xylose and glucose, the glucose was determined separately by incorporating a glucose oxidase column in the flow system, by use of a test kit or by HPLC; the xylose concentration. could then be calculated by compensating for the effect of glucose on the xylose calibration graph.
Xylose Glucose HPLC

"Selective Flow Injection Determination Of Methanol In The Presence Of Ethanol Based On A Multi-enzyme System With Chemiluminescence Detection"
Anal. Chim. Acta 1993 Volume 280, Issue 2 Pages 179-184
Yoshiie Sekine*, Masayasu Suzuki, Toshifumi Takeuchi, Eiichi Tamiya and Isao Karube

Abstract: Alcohol oxidase, formaldehyde dehydrogenase and catalase were covalently immobilized onto aminopropyl controlled-pore glass beads (200-400 mesh) using glutaraldehyde (details given) and packed sequentially into a stainless-steel column (3 cm x 2 mm i.d.) incorporated into a flow injection system (diagram given). Samples (20 µL) containing methanol (I) were injected into a stream (1 ml/min) of 50 mM phosphate buffer of pH 7 containing 1-methoxy-5-methylphenazinium methylsulfate (II; 30 mg/l), 5 mM EDTA and 200 µM-NAD+. I was oxidized to formaldehyde (III) by alcohol oxidase; the H2O2 formed was destroyed by catalase. III was oxidized by formaldehyde dehydrogenase producing NADH, which was oxidized by II. The reduced II re-oxidized spontaneously producing H2O2 which entered a carrier stream (1.5 ml/min) of 200 mM borate buffer of pH 9.5 containing luminol (1 mg/l) and 5 mM EDTA for chemiluminescence detection. The calibration graph was linear from 0.1 (detection limit) to 50 mg/l of I; the RSD were 3%. The response time was 2 min. I could be measured at 5 mg/l in the presence of 100-20 000 mg/l of ethanol.
Methanol Chemiluminescence

"Glucose Quantitation Using An Immobilized Glucose Dehydrogenase Enzyme Reactor And A Tris-(2,2'-bipyridyl)ruthenium(II) Chemiluminescent Sensor"
Anal. Chim. Acta 1993 Volume 281, Issue 3 Pages 475-481
Alice F. Martin and Timothy A. Nieman

Abstract: A flow injection method for the cited analysis is described (diagram given). Test solution were injected into a 0.1 M potassium phosphate buffer carrier stream (pH 6.5; 2 ml/min) which passed through a reactor column containing glucose dehydrogenase immobilized (using glutaraldehyde) onto controlled-pore glass (method described), and then to a flow cell incorporating a dual Pt electrode coated (described) with Nafion containing immobilized tris-(2,2'-bipyridyl)ruthenium(II) (I), and a Plexiglas window for chemiluminescence detection via a photomultiplier tube. The cell potential was maintained at 1300 mV vs. Ag/AgCl, and a stainless-steel counter electrode was used. The system was used to analyze glucose solution containing 1 mM NAD+; the NADH produced reduced the oxidized-I generating chemiluminescence and thus recycling I. The calibration graph was linear from 10^-2500 µM-glucose and the RSD (for 300 µM-glucose) was 1.1%. Gluconic acid and NAD+ did not interfere, but uric acid and ascorbic acid did. The sensor also responded to NADH, oxalate, proline and tripropylamine. Catechol permanently impaired the response.
Chemiluminescence Sensor

"Industrial Online Monitoring Of Penicillin V, Glucose And Ethanol Using A Split-flow Modified Thermal Biosensor"
Anal. Chim. Acta 1993 Volume 281, Issue 3 Pages 521-526
M. Rank, J. Gram, B. Danielsson

Abstract: Bioreactor fermentation broth was sampled online with a hydrophilized polypropylene filtration probe (Advanced Biotechnology, Puchheim, Germany) and pumped into an enzyme thermistor with a split-flow biosensor comprising a column of β-lactamase, penicillin V acylase, glucose oxidase/catalase or alcohol oxidase immobilized onto controlled-pore glass using γ-aminopropyltriethoxysilane and glutaraldehyde (details given), and a parallel reference column without enzyme. Columns were eluted (0.9 ml/min) with 0.1 M phosphate buffer of pH 7 containing 4 mM NaN3 or 5 g/l of benzoic acid (for the glucose oxidase/catalase column). The difference between the enthalpy changes on the two columns was monitored, amplified and processed by computer. The calibration graph for the penicillin V acylase column was linear from 0.5-150 mM penicillin V (20-500 µL samples); the determinations of penicillin V, penicilloic acid and p-hydroxypenicillin V agreed well with those obtained by LC. The β-lactamase column values were 10% higher than those obtained by LC. Results for ethanol (alcohol oxidase column) agreed with those obtained by GC.
Penicillin V Glucose Ethanol Fermentation broth Industrial Enthalpimetry Sensor Thermistor Biotechnology

"Enhanced Selectivity In Flow Injection Analysis For L-amino Acids Using Electrodialysis With Amino-acid Oxidation"
Anal. Chim. Acta 1993 Volume 282, Issue 2 Pages 369-378
J. C. Cooper*, J. Danzer and H. -L. Schmidt

Abstract: A 30 cm electrodialysis cell with a central channel separated from an upper cathode-containing channel and a lower anode-containing channel by horizontal cation- and anion-exchange membranes, respectively, was used. Electrodialysis was performed at 20 V with the sample solution passing through the central channel and 0.2 M pyrophosphate buffer solution of pH 8 passing through the other channels (each at 0.8 ml/min); arginine migrated into the cathode channel. After adjustment of the pH to 7.5 with 0.2 M citrate buffer, the solution leaving the cathode channel was injected into a carrier stream of 0.2 M phosphate buffer of pH 7.6 (2 ml/min) and passed through two glass columns (2.3 cm x 5 mm i.d.) containing catalase and L-amino-acid oxidase immobilized on controlled-pore glass beads (details given). The resulting H2O2 produced by the enzymatic oxidation of arginine was determined in a flow cell at a Pt electrode at 0.7 V vs. SCE. No details of detection limits are given. Other amino-acids, e.g., lysine, interfered with the method, which should also be applicable to the determination of other L-amino-acids in such mixtures under appropriate electrodialysis conditions.
Amino acids, L Electrochemical analysis

"Enzyme-based Chemically Amplified Flow Injection Determination Of Catechol And Catecholamines Using An Immobilized Tyrosinase Reactor And L-ascorbic Acid"
Anal. Chim. Acta 1993 Volume 282, Issue 2 Pages 363-367
Yasushi Hasebe, Kenichiro Takamori and Shunichi Uchiyama

Abstract: The method exploited a cycle involving the tyrosinase-catalyzed oxidation of the sample molecule by dissolved O2 to a quinone compound which was reduced by L-ascorbic acid to the sample molecule which underwent further oxidation-reduction cycles while passing through the enzyme reactor. This resulted in an increase in the amount of dissolved O2 consumed by each sample molecule which was determined with use of an O2 electrode detector. Dopamine solution (100 µL), e.g., was injected into a stream (2.5 ml/min) of 1 mM L-ascorbic acid in 10 mM phosphate buffer of pH 6.5 saturated with dissolved O2 which was passed via a 0.5 mm diameter PTFE tube to a reactor containing tyrosinase immobilized by diazo-coupling onto controlled-pore glass beads (80-120 mesh). The dissolved O2 content of the stream was monitored by an O2 electrode at -0.7 V vs. an Ag/AgCl reference electrode. Calibration graphs were linear (ranges tabulated). The detection limits ranged from 1 nM-dopamine to 0.5 µM-adrenaline. The RSD (n = 8) was 5%.
Catechol Catecholamines Electrode

"Flow Injection Study Of Inhibition And Reactivation Of Immobilized Acetylcholinesterase: Determination Of The Pesticides Paraoxon And Carbamoylcholine"
Anal. Chim. Acta 1993 Volume 282, Issue 2 Pages 307-312
I. A. Takruni, Ala'ddin M. Almuaibed and Alan Townshend

Abstract: To determine paraoxon (I), the sample solution (60 µL) was injected into a carrier stream of 50 mM potassium phosphate buffer of pH 8 (1 ml/min) flowing through a PTFE manifold (50 µm i.d.). A solution of 7 mM acetylthiocholine iodide (60 µL) was injected into the centre of the I plug 10 cm downstream of the first injection point and the stream was passed through a glass column (2 cm x 2.5 mm i.d.) of acetylcholinesterase immobilized on controlled-pore-glass beads. The presence of I in the column inhibited the catalytic activity of the enzyme in the hydrolysis of acetylthiocholine iodide to thiocholine determined spectrophotometrically according to Quintero et al. (Talanta, 1991, 38, 1273) after mixing the column effluent with 1 mM 5,5'-dithiobis(2-nitrobenzoic acid). The total uninhibited catalytic activity of the acetylcholinesterase was determined similarly by injecting acetylthiocholine iodide after reactivation of the enzyme by pyridine-2-aldoxime methochloride. Calibration graphs were linear up to 0.8 mM I and the detection limit was 50 µM-I. The RSD (n = 5) was 5% for 0.6 mM I. A procedure for determining up to 0.6 mM carbamoylcholine was also described.
Pesticides Paraoxon Carbamoylcholine Spectrophotometry

"Spectrophotometric Determination Of Magnesium In Serum By Using A Flow Injection System With An Immobilized Enzyme Reactor"
Anal. Chim. Acta 1993 Volume 283, Issue 1 Pages 447-452
J. M. Fern&aacute;ndez-Romero, M. D. Luque de Castro and M. Valc&aacute;rcel*, R. Quiles-Zafra

Abstract: The method was based on the activating effect of Mg(II) on the hydrolysis of 2-nitrophenyl β-D-galactopyranoside (I) by β-galactosidase with all other reactants present in excess. In a merging-zones configuration (illustrated), portions of the sample and a solution of 4.7 g/l of NaCl and 60.3 mg/l of I in reagent A (aqueous 0.1 M Tris/4 mM dithiothreitol/0.4 mM EGTA of pH 7.5) were simultaneously injected into a stream of reagent A. In a region maintained at 37°C, the solution were mixed in a single-bead string reactor and passed through a reactor containing β-galactosidase immobilized on controlled-pore glass. A flow of 1 M NaOH was introduced and merged with the stream in a reactor coil, and the absorbance was monitored at 405 nm. Optimized operating parameters were established. Forty samples could be analyzed in 1 h, and response was linear for 5-20 µM-Mg. Within-run and between-run RSD (n = 11) were 0.78-2.91% and 1.7-3.21%, respectively. The method showed good selectivity, with no interference from a 20-fold amount of calcium. Recovery of added Mg was 96-113% and results were well correlated with those of AAS.
Magnesium Blood Serum Spectrophotometry

"Flow Injection Analysis - Wall-jet Electrode System For Monitoring Glucose And Lactate In Fermentation Broths"
Anal. Chim. Acta 1993 Volume 283, Issue 2 Pages 763-771
Yu Liang Huang, Soo Beng Khoo, Miranda G. S. Yap

Abstract: The flow injection manifold consisted of two peristaltic pumps, an injection valve with a 20 µL sample loop, an enzyme reactor column (10 x 1 mm i.d.) containing either glucose oxidase or lactate oxidase immobilized onto controlled-pore glass beads, and a large volume wall-jet detector cell. The detector contained a Pt disc working electrode modified with Fe(II), a Ag/AgCl reference electrode and a vitreous C counter electrode. Fermentation broth was centrifuged and the supernatant was diluted prior to analysis. The resulting solution was injected into the 0.1 M potassium phosphate buffer of pH 7 carrier stream (0.5 ml/min). The H2O2 produced by the enzymatic reaction of both glucose (I) and lactose (II) was detected at 0.65 V vs. Ag/AgCl. The calibration graphs were linear up to 0.5 and 0.04 g/l of I and II, respectively. The RSD (n = 54) was 3% over a 10 h measurement period. The procedure was used to monitor I and II concentrations in various fermentation broths and the results agreed well with those obtained using a commercial I-II analyzer..
Glucose Lactate Fermentation broth Electrode

"Flow Injection Analysis For Total Cholesterol With Photometric Detection"
Anal. Chim. Acta 1994 Volume 287, Issue 1-2 Pages 59-64
Achim Krug*, Roman G&ouml;bel and Robert Kellner

Abstract: Serum (70 µL) was injected into a carrier stream (3 ml/min) of 0.1 M phosphate buffer of pH 7/2-propanol/Triton X-100 (83:13:4) and the stream was passed through a glass column enzyme reactor (10.5 cm x 1.5 mm i.d.) containing a 45 mm section filled with cholesterol esterase immobilized on to controlled pore glass (CPG) beads (120-200 mesh; 127 nm pore diameter) and a 60 mm section filled with cholesterol oxidase immobilized on to CPG. The sample stream passed through the cholesterol esterase section first. Hydrogen peroxide generated was determined by post-column reaction with 3.5 mM 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate) catalyzed by horse-radish peroxidase (5 U/ml) and with detection at 423 nm. The calibration graph was linear for 0.1-0.86 mM cholesterol and the RSD (n = 8) for 5.1 mM cholesterol was 2.5%. The sample throughput was 50 samples/h. Reducing species do not interfere at concentrations >10 times their normal levels in serum.
Cholesterol, total Blood Serum Spectrophotometry LC

"Selective Enzyme Amplification Of NAD+/NADH Using Co-immobilized Glycerol Dehydrogenase And Diaphorase With Amperometric Detection"
Anal. Chim. Acta 1994 Volume 290, Issue 3 Pages 335-342
Karin Kronkvist, Katarina Wallentin and Gillis Johansson*

Abstract: A FIA system for substrate recycling of NAD+/NADH was constructed by connecting an enzyme reactor containing diaphorase and glycerol dehydrogenase (1110 and 40 iu, respectively) immobilized onto controlled pore glass beads (40-80 µm, pore size 38 nm) with an amperometric detector (details given). Test solution (100 µL) containing NADH were injected into a stream of 0.1 M potassium phosphate buffer of pH 8 containing 50 mM glycerol and 30 mM ammonium sulfate (buffer A) which merged with a stream of 2 mM hexacyanoferrate(III) in buffer A before passing through the enzyme reactor. The product, hexacyanoferrate(II), was detected at a vitreous C electrode at 450 mV vs. SCE and graphite electrodes were modified with a phenoxazine mediator for detection of NADH at 0 V vs. SCE; Pt wires were used as auxillary electrodes. The amplification increased linearly with residence time (150-fold for a 100 µL reactor at a flow rate of 0.5 ml/min). Under these conditions the calibration graph was linear for 0.1-5 µM-NAD+ and the detection limit was 0.09 µM. For a stopped-flow of 4 min the detection limit was 0.01 µM (13 300-fold amplification).
Nicotinamide adenine dinucleotide oxidized Nicotinamide adenine dinucleotide reduced Biological Amperometry Electrode Electrode

"Continuous-flow Assay Of Ammonia In Plasma Using Immobilized Enzymes"
Anal. Chim. Acta 1994 Volume 294, Issue 1 Pages 43-47
R. Quilesa, J. M. Fern&aacute;ndez-Romerob, E. Fern&aacute;ndeza and M. D. Luque de Castrob,*

Abstract: Heparinized plasma containing NH3 were diluted sixfold with carrier-buffer solution before injection of a 0.3 mL portion for FIA into the 0.15 M triethanolamine carrier, adjusted to pH 8 with 5 M HCl, (0.8 ml/min) containing 25 mM 2-oxoglutarate, 1 mM ADP and 0.2 mM NADH. The carrier was fed through a reaction tube (5 cm x 1 mm i.d.) of glutamate dehydrogenase [NAD(P)+] immobilized on controlled-pore glass (120-200 mesh) at 37°C to the flow-cell detector of a spectrofluorimeter for measurement of the decrease in fluorescence due to NADH consumption. The calibration graph was linear for 5 µM to 0.5 mM NH3 with RSD 2% and sampling frequency of up to 40/h. Results correlated well with those obtained using a Kodak Ektachem analyzer. and bromophenol blue.
Ammonia Blood Plasma Fluorescence

"Flow Analysis With Membrane Separation And Time Based Sampling For Ethanol Determination In Beer And Wine"
Anal. Chim. Acta 1995 Volume 305, Issue 1-3 Pages 241-247
Jochen Mohns and Wolfgang K&uuml;nnecke*

Abstract: The flow analyzer. consisted of a dual-channel peristaltic pump, a gas diffusion unit incorporating a PTFE membrane (channel; 0.5 mm depth x 1.4 mm width x 21 mm length), an enzyme reactor containing 10 mg of alcohol oxidase immobilized on to controlled pore glass beads and an electrochemical detection cell operated at 700 mV. One channel of the pump propelled the acceptor stream (0.1 M potassium phosphate buffer at pH 7.5) through the gas diffusion unit and, after the switching of the appropriate valves, through the enzyme reactor and the detector cell. The other channel of the pump propelled samples and standard solutions through the donor channel of the gas diffusion unit. All flow rates were 1.5 ml/min. The flow of both donor and acceptor solutions was stopped to allow ethanol to diffuse across the PTFE membrane. The accumulated ethanol plug was then carried to the enzyme reactor where it was converted to acetaldehyde and H2O2. H2O2 was detected electrochemically. The thickness and pore size of the PTFE membrane controlled the diffusion of ethanol. With 550, 500 and 400 µm thick membranes, the calibration graphs were linear up to 0.6, 7 and 15% ethanol, respectively. The detection limit was 0.0001% ethanol. The RSD (n = 3) for the analysis of samples within the linear calibration range was 2%. The ethanol content of beer and wine samples were determined without sample pretreatment apart from manual degassing. The sampling frequency was 30 samples/h.
Ethanol Beer Wine Amperometry

"Fluorimetric Flow Injection Determination Of Theophylline Based On Its Inhibitory Effect On Immobilized Alkaline Phosphatase"
Anal. Chim. Acta 1995 Volume 308, Issue 1-3 Pages 159-163
M. S&aacute;nchez-Cabezudo, J. M. Fern&aacute;ndez-Romero and M. D. Luque de Castro*

Abstract: The dual channel flow injection manifold for the determination of theophylline consisted of a dual injection valve, a reactor at 40°C containing bovine alkaline phosphatase immobilized on controlled pore glass and a fluorimetric detector. Sample (500 µL) and 225 µL 2 mM 4-methylumbelliferone phosphate solution (substrate) were simultaneously injected into two carrier streams (both at 1.2 ml/min) of 0.5 M Tris hydrochloride buffer of pH 10. The streams were merged and the flow was passed through the reactor (50 cm x 0.5 mm i.d.) to the detector. The released 4-methylumbelliferone was detected at 445 nm (excitation at 365 nm). The presence of theophylline inhibited the enzymatic reaction and reduced the fluorimetric signal. The calibration graph was linear for 10^-200 µM-theophylline and the RSD (n = 3) at the 20 µM level was 3.5%. The sampling frequency was 40 samples/h. The recovery of 20-60 µM-theophylline from spiked blood serum was >93%.
Theophylline Blood Serum Fluorescence

"Dynamic Analysis Of The Binding Process Of Bovine Serum Albumin On Glutaraldehyde-activated Controlled Pore Glass"
Anal. Chim. Acta 1995 Volume 308, Issue 1-3 Pages 261-268
Hiroyuki Ukeda*, Tohru Ishii, Masayoshi Sawamura and Hirozo Kusunose

Abstract: Twenty microlitre volumes of BSA solution (5 mg/ml) were repeatedly injected into a column (5 cm x 1.68 mm i.d.) packed with aminopropyl-controlled pore glass activated with glutaraldehyde (GA-CPG). The elution profile of BSA was recorded using phosphate buffer eluents (0.5 ml/min) and detection at 280 nm. The results were analyzed using a model based on the assumption that two modes are involved in binding BSA to GA-CPG. Binding process parameters such as bound amounts and binding rate constants were estimated by a curve fitting method. An increase in ionic strength of the carrier solution resulted in a reduction in the total amount of BSA bound. The maximum bound amount occurred at pH 6 with low ionic strength carriers and at pH 7 for higher ionic strength carriers. The reduction of GA-CPG with sodium borohydride reduced the bound amount while blocking treatment with amine had no effect.
Albumin Cow Serum Spectrophotometry

"Selective Flow Injection Determination Of Methanol Using Immobilized Enzyme Reactors"
Anal. Chim. Acta 1995 Volume 309, Issue 1-3 Pages 241-250
C&aacute;ndido Garc&iacute;a de Mar&iacute;a*, Teresa Manzano, Roc&iacute;o Duarte and Angel Alonso

Abstract: Portions of 50 iu of formaldehyde dehydrogenase (I) were immobilized, and 250 iu of alcohol oxidase (II) and 70 000 iu of catalase (III) coimmobilized onto 100 mg of 80-120 mesh, 240 µm controlled-pore glass as described by Masoom and Townshend (Ibid., 1985, 171, 185), and the immobilized enzymes packed into variable length PTFE columns. Portions of 40 µL 100 mM sodium pyrophosphate buffer of pH 8 and containing methanol were injected into a carrier stream (0.6 ml/min) of buffer and through a II-III-column (2 cm x 0.8 mm i.d.). The eluate was then mixed with a carrier stream (0.2 ml/min) of 1.5 mM NAD+ in buffer and passed through a I-column (3 cm x 0.8 mm i.d.) and the NADH produced monitored by fluorimetric detection at 460 nm (excitation at 340 nm). Calibration graphs were linear for 4-80 µM-methanol with a detection limit of 1.2 µM. At 24 µM-methanol a RSD (n = 10) of 1.8% was obtained. The selectivity of the method in the presence of glycerine, lower alcohols and aldehydes, carboxylic acids, nitrite and sulfite was evaluated. When stored in buffer of pH 6 and at 4°C, the I- and II-III enzyme reactors were stable for 8 and 4 months, respectively.
Methanol Fluorescence

"Fluoride Determination By Its Inhibitory Effect On Immobilized Liver Esterase"
Anal. Chim. Acta 1995 Volume 310, Issue 1 Pages 173-180
J. Marcos and A. Townshend*

Abstract: A 30 µL portion of 16 mM ethyl butyrate (EBYR) and a 0.5 mL portion of 0.1 mM KF were injected sequentially into 2 mM CaCl2 of pH 4 (adjusted with HCl). The solution was pumped (1.1 ml/min) through a 1 mm reactor that contained liver esterase immobilized on controlled pure glass (CPG; 120-200 mesh; preparation described) and mixed with 20 mM glycine buffer of pH 10 (1.1 ml/min) that contained 0.9 g/l of NAD+, prior to passage through a 10 mm reactor that contained alcohol dehydrogenase immobilized on CPQ (120-200 mesh; preparation described). The NADH released was detected at 340 nm. The quantity of ethanol released following hydrolysis of EBYR, and hence the quantity of NADH detected was inversely proportional to the fluoride concentration. Fluoride inhibition of the immobilized liver esterase was fast and reversible, and there was linear relationship between percentage inhibition and fluoride concentration for 15-150 µg/l. The limit of detection was 0.16 µM and the RSD (n = 5) were 4%.
Fluoride Spectrophotometry

"Development Of An Optical Flow-through Biosensor For The Determination Of Sulfite In Environmental Samples"
Anal. Chim. Acta 1995 Volume 311, Issue 3 Pages 281-287
M. D. Luque de Castro* and J. M. Fern&aacute;ndez-Romero

Abstract: The method was based on the enzymatic oxidation of sulfite in the presence of sulfite oxidase (SOD) to yield sulfate and H2O2. The H2O2 reacted with 4-aminophenazone (4-AAF) and 3-methyl-N-ethyl-N'-(β-hydroxyethyl)aniline (MEHA) in the presence of peroxidase (HPOX) to yield an alkyl aniline cationic derivative (4-AMHA) which was detected spectrophotometrically following retention on an ion exchanger. Water sample (2 ml) was injected into a stream (0.6 ml/min) of 10 mM Tris hydrochloride buffer of pH 8.4 and passed through a column containing SOD immobilized onto controlled pore glass beads. The eluate was merged with a stream (0.6 ml/min) containing 10 µM-4-AAF and 200 µM-MEHA in 50 mM citrate buffer of pH 5 and passed through a column containing an upper layer of HPOX immobilized on to controlled pore glass beads and a lower layer of Sephadex carboxymethyl cation ion-exchange resin. The absorption of the retained 4-AMHA was monitored at 565 nm and the retained 4-AMHA was then eluted with 15 mM NaCl. The calibration graph for sulfite was linear for 10^-1000 ng/ml, the detection limit was 3 ng/ml and the RSD (n = 11) were 0.7-2.6%. The recoveries of 30-100 ng/ml sulfite from spiked water were >94%. The sampling frequency was 16/h.
Sulfite Environmental Spectrophotometry Sensor

"Flow Injection Column Preconcentration Directly Coupled With Electrothermal Atomization Atomic Absorption Spectrometry For The Determination Of Aluminum. Comparison Of Column Packing Materials"
Anal. Chim. Acta 1995 Volume 316, Issue 3 Pages 313-322
Dongxing Yuan and Ian L. Shuttler*

Abstract: A method has been developed for the determination of endogenous levels of aluminum ( gt 1 µg l-1) in water samples using an automated online pre-concentration system with flow injection coupled directly to an electrothermal atomic absorption spectrometer. Two pre-concentration materials, 8-quinolinol immobilized on controlled-pore glass (8-Q-CPG) and Amberlite XAD-2, poly(styrene/divinyl benzene) copolymer (XAD-2) were investigated and compared. Both systems were found to be suitable for pre-concentration. However, the sampling flow-rate for the 8-Q-CPG system was found to be much lower than that of the XAD-2 system, relative to the same magnitude of pre-concentration. The chelating kinetics of the 8-Q-CPG system were less favourable than the adsorption kinetics of the XAD-2 system. The detection limits (3SD) varied from 15 to 40 ng l-1, depending on the pre-concentration time, with RSDs of the order of 4% for a 1 µg L-1 concentration of aluminum. The optimum concentration range for the application of the method developed was 50 ng L-1 to 3 µg l-1. Recoveries for drinking water were in the range 100-115% for the 8-Q-CPG system and 90-100% for the XAD-2 system. The method developed was applied with varying results to the analysis of potable, fresh, river and seawater samples. (23 References)
Aluminum Water River Sea Spectrophotometry

"Characterization And Application Of An Online Flow Injection Analysis/wall-jet Electrode System For Glucose Monitoring During Fermentation"
Anal. Chim. Acta 1995 Volume 317, Issue 1-3 Pages 223-232
Yu Liang Huanga, Titus J. Foellmera, Koon Chye Anga, Soo Beng Khoob,* and Miranda G. S. Yapa

Abstract: An online system for glucose monitoring during fermentation was developed. The system employed flow injection analysis coupled with a wall-jet electrode. Glucose was converted enzymatically in a tubular reactor packed with controlled pore glass beads containing immobilized glucose oxidase. The hydrogen peroxide produced was detected amperometrically. A filtration/sampling module operated by a peristaltic pump enabled sample withdrawal from the fermenter. The system was interfaced to a programmable language controller which was in turn linked to a microcomputer. The dual injection valve of the flow system was operated in a novel configuration to achieve sample dilution, by dispersion, and standardization simultaneously. The developed system was characterized for dilution factor, stability and linearity of response. After conditioning of the reactor, sample peak heights were found to have a precision of 1.07% (RSD, 25 determinations) when the glucose concentration was 1.32 g l-1. Under the conditions studied, linear response to glucose concentration from 0.062 to 4.0 g L-1 was obtained. Sample throughput was about 7 per hour. Interferences from the fermentation media were negligible after filtration through the sampling module and dilution. The online system was applied to glucose monitoring during E. coli fermentation. Good results were obtained as shown by the high cell density (OD6-00 nm = 168 in 9 h fermentation), constant specific growth rate (µ= 0.66) and lower acetate accumulation (less than 2 g L-1 when OD-600 nm was over 50). (18 References)
Glucose Fermentation broth Electrode

"Pervaporation: An Interface Between Fermentors And Monitoring"
Anal. Chim. Acta 1996 Volume 330, Issue 2-3 Pages 265-272
I. Papaefstathioua, U. Bilitewskib and M. D. Luque de Castroa,*

Abstract: An enzymatic spectrophotometric method is described for determining acetaldehyde in fermentation broths. A 1 mL sample of the fermentation broth is injected into a carrier stream of water and propelled at 0.49 ml/ml through a pervaporation module (at 60°C) fitted with a PTFE membrane (1.5 mm thickness, 5 µm pore size). The vaporized acetaldehyde diffuses through the membrane and is collected in an acceptor stream (0.49 ml/min) containing 1 mM NAD+, 0.2 M KCl and 6 mM 2-mercaptoethanol in 0.1 M Na2HPO4/NaH2PO4 buffer at pH 8. The acceptor stream passes through an enzyme reactor (at 30°C) containing aldehyde dehydrogenase immobilized on controlled-pore glass beads and a spectrophotometric detector equipped with a 1 cm detector cell; the NADH formed by the enzymatic oxidation of acetaldehyde is detected at 340 nm. The calibration graph was linear for 1-30 µg/ml of acetaldehyde, the detection liwas 0.9 µg/ml and the RSD (n = 8) at 20 µg/ml was 2.7%. The sampling frequency was 8 per h. The recoveries of 6.5 µg/ml of acetaldehyde added to fermentation broths containing 2-15 µg/ml of acetaldehyde were >92.7%.
Acetaldehyde Fermentation broth Spectrophotometry

"Development Of An Automated Controlled-pore Glass Flow-through Immunosensor For Carbaryl"
Anal. Chim. Acta 1997 Volume 347, Issue 1-2 Pages 199-205
M. A. Gonz&aacute;lez-Mart&iacute;nez, S. Morais, R. Puchades*, A. Maquieira, A. Abad and A. Montoya

Abstract: The application of controlled-pore glass (CPG) as solid support for immobilization of immunoreagents in order to develop flow-through immunosensors is described. Monoclonal antibodies (MAbs) to carbaryl were site-directed immobilized on CPG through covalent attachment of their oxidized carbohydrate moieties to amine groups generated on the surface of silanyzed CPG. The automated immunosensor system is based on the LIB-CNA36 MAb in a direct competitive assay format, with horseradish peroxidase as enzyme label and fluorimetric detection. The dynamic range of the sensor is 0.05-1 µg l-1, with a detection limit of 0.029 µg l-1, being sensitive enough to be applied to drinking water samples without pre-concentration. The immobilized antibody reactor is able to run a whole assay in 20 min, and is reusable for more than 100 of consecutive assay cycles without significant loss of performance. The recognition of l-naphthol - the main metabolite of carbaryl - and other N-methylcarbamate insecticides are also studied, none of these compounds showing cross-reactivity higher than 7%. A preliminary validation of the immunosensor, carried out by analyzing real samples spiked with carbaryl, shows good results for bottled water and for commercial honey diluted with PBS (1 gl-1) as the only sample pretreatment.
Carbaryl Sensor

"Comparison Of Flow Injection Analytical Biosystems Based On Open-tube And Packed-bed Enzyme Reactors"
Anal. Chim. Acta 1998 Volume 370, Issue 1 Pages 47-58
M. Jurkiewicz, S. Alegret and E. F&agrave;bregas*

Abstract: A comparison between the most widely used enzyme reactors in flow injection analysis (FIA) systems was realized. These reactors were the open-tube reactors and the packed-bed reactors. The comparison was realized with three different enzymes (urease, creatinine, iminohydrolase and creatine amidinohydrolase). The enzymes were covalently immobilized on controlled-pore glass beads (CPG). These beads were used as the filling for the packed-bed reactors. The enzymes were also immobilized covalently on the inner wall of nylon tube reactors. All three enzymes were used in clinysis analysis and they have clearly distinguished activities. All of them produced ammonium ions as a consequence of their interaction with their respectively. substrates. The single channel FIA system used for the evaluation and the comparison of the reactors worked automatically and included a flow bioreactor and an ammonium-ion sensor. The FIA systems used with the different bioreactors were optimized to obtain the largest signal in the shortest time. Once optimized, the performance of the reactors were evaluated and compared while working with each enzyme. The parameters to evaluate were: calibration parameters (y-ordinate, slope and linear), reproducibility, conversion ratio (yield) of the enzyme reaction in the reactor, dynamic parameters (response time and anal. time) and lifetime. The comparison of the bioreactors working under optimal conditions showed that the packed reactors had a higher conversion ratio, a higher sensitivity (slope of the calibration curve), a smaller lower detection limit and shorter response times. Open-tube reactors showed a higher reproducibility, a higher upper detection limit and a diminished amt. of reagent at the optimal flow rate. Anal. time was lower in open-tube reactors since the packed-bed reactors are difficult to wash after sample injection and the signal took longer to return to the baseline after an FIA peak. In some cases, the baseline value was not fully reached again.
Uric acid Creatine Creatinine Electrode Potentiometry

"Microwave-assisted Saponification Of Animal Greases For Cholesterol Determination"
Anal. Chim. Acta 1998 Volume 371, Issue 2-3 Pages 297-303
Gerardo Pi&ntilde;eiro-Avila, Amparo Salvador and Miguel de la Guardia

Abstract: An alternative method for the hydrolysis of cholesterol esters in animal grease samples has been developed. The method consists of the microwave-assisted treatment of 4 g of animal fat or oil with 40 mL of an ethanolic KOH solution inside a 115 mL closed reactor in which samples were irradiated for 2.5 min at 50% power level of an exit power of 700 W. After extraction with petroleum ether and dissolution of the unsaponifiables in toluene, total cholesterol was determined spectrophotometrically by using p-anisidine as a test reagent of the enzymatic reaction of cholesterol through a bienzymic reactor in which cholesterol oxidase and horseradish peroxidase were noncovalently immobilized in controlled pore,glass beads. The method provided a quantitative recovery of spiked free cholesterol (100% recovery for an added concentration level of 36 mg g-1) and cholesteryl palmitate (98.7% recovery for an added concentration of 37 mg g-1) and results comparable to those obtained after saponification with ethanolic KOH at 90°C under reflux conditions for 1 h, saving a considerable time with a comparable figures of merit which involve a limit of detection of 1 x 10^-6 M total cholesterol an average interday relative standard deviation of 3.5% for three independent analysis of a series of different samples.
Cholesterol Fat Sample preparation Spectrophotometry

"Flow Injection Determination Of Neostigmine And Galanthamine By Immobilized Acetylcholinesterase Inhibition"
Anal. Chim. Acta 1998 Volume 372, Issue 3 Pages 379-386
T. Ghous and A. Townshend*

Abstract: Neostigmine (1 x 10^-7 - 1 x 10^-6 M) and galanthamine (5 x 10^-7 - 6 x 10^-6 M) are determined by measuring their inhibition of acetylcholinesterase immobilized on controlled pore glass. The determination is carried out by a flow injection procedure with spectrophotometric detection. The 3s limits of detection were 0.5 x 10^-7 M neostigmine and 5 x 10^-7 M galanthamine. The relative standard deviations were 1.3% for five determinations of 5 x 10^-7 M neostigmine and 2.0% for six determinations of 2 x 10^-6 M galanthamine. Several reagents for achieving online reactivation of inhibited enzyme were studied. As a result, 2 x 10^-3 M NaF was recommended for reactivation after neostigmine inhibition, and 2 x 10^-4 M substrate solution for reactivation after galanthamine inhibition. The efficiency of the reactivation process was interpreted in terms of the inhibitory mechanisms relating to the above drugs.
Neostigmine Galanthamine Spectrophotometry

"Flow Injection Determination Of L-tyrosine In Serum With Immobilized Tyrosinase"
Talanta 1993 Volume 40, Issue 7 Pages 995-998
Nobutoshi Kiba, Hiroshi Suzuki and Motohisa Furusawa

Abstract: Serum was deproteinized by 5% sodium tungstate solution and 0.15 M H2SO4, filtered and a portion was injected into 0.2 M phosphate buffer solution of pH 7.2 (0.4 ml/min). The mixture passed through a separation column (4 cm x 4 mm) of Capcell 120 C18 and then through the enzymatic reactor (5 cm x 4 mm) containing monophenol monooxygenase immobilized on controlled pore glass (cf. Kiba et al., Anal. Chim. Acta, 1989, 224, 133). The effluent from the reactor was mixed with 30 mM 1,2-diphenylethylenediamine in 0.6 M HCl/ethanol (3:7) and the mixture passed through a reaction coil (1 m) held at 105°C and then through a cooling coil (10 cm) at 15°C, before measurement of the fluorescence at 480 nm (excitation at 350 nm) in a flow-through cell. The calibration graph was linear for 1-200 µM-L-tyrosine with a detection limit of 0.2 µM. Interference from cysteine or ascorbate was avoided. Recoveries of 70-985 µM-L-tyrosine from serum were 98-105%. Within- and between-day RSD were 1 and 1.8%, respectively, for 64 µM-L-tyrosine. Results agreed well with those obtained by an amino-acid analyzer..
Tyrosine Blood Serum Fluorescence

"Biosensing Based On NADH Detection Coupled To Electrogenerated Chemiluminescence From Ruthenium Tris(2,2'-bipyridine)"
Talanta 1994 Volume 41, Issue 6 Pages 1035-1040
Kenji Yokoyamaa, Satoshi Sasakib, Kazunori Ikebukurob, Toshifumi Takeuchic, Isao Karubeb,*, Yumi Tokitsud and Yuzo Masudad,

Abstract: For the FIA of ethanol, phosphate buffer solution containing 1 mM Tris-(2,2'-bipyridyl)ruthenium(III) dichloride was used as carrier flowing at 2 ml/min, into which a solution of ethanol containing 1 mM NAD+ was injected. The resulting solution was passed through a column containing alcohol dehydrogenase immobilized on glass beads to a flow cell equipped with a vitreous carbon electrode and a stainless steel tube as counter electrode. The chemiluminescence generated by applying a potential of 1.6 V was measured. A curvilinear calibration graph was obtained, from which 10 ppm to 5% of ethanol could be determined. The detection limit could be lowered to 0.1 ppm by immobilizing the enzyme on controlled-pore aminopropyl glass beads. NADH could be determined in an analogous system without the enzyme reactor. The linear range was 10^-100 µM, or 10^-250 µM with a log./log. plot and the RSD for 1 mM NADH at a flow rate of 2 ml/min was 5.5% (n = 28).
Ethanol Nicotinamide adenine dinucleotide reduced Chemiluminescence Electrode

"Amperometric Detection Of Uric Acid And Hypoxanthine With Xanthine Oxidase Immobilized And Carbon Based Screen-printed Electrode"
Talanta 1997 Volume 44, Issue 11 Pages 2151-2159
M-A. Carsol, G. Volpe and M. Mascini*

Abstract: Carbon-based screen-printed electrodes are suitable for uric acid detection. Xanthine oxidase (XO) was immobilized either directly on the surface of the electrode or in a reactor with CPG aminopropylsilane in a FIA assembly. Higher reproducibility and lifetime was obtained with the reactor. Optimum conditions were found for the determination of Hypoxanthine (Hx), Inosine (HxR) and Inosine monophosphate (IMP). Calibration curves for IMP, HxR and Hx are linear up to 50 µM with detection limit of 1 µM for 50 µl injection. One assay is completed within 30 s. The reproducibility of 20 µM of Hx was obtained with CV 2%.
Uric acid Hypoxanthine Food Marine Electrode Electrode Amperometry

"Flow Injection Analysis Use Of Immobilized Enzymes For The Determination Of Ethanol In Serum"
Analyst 1987 Volume 112, Issue 3 Pages 259-261
Juan Ruz, Mar&iacute;a Dolores Luque de Castro and Miguel Valc&aacute;rcel

Abstract: Alcohol dehydrogenase was immobilized on silanized controlled-pore glass with glutaraldehyde as the coupling agent (details given). A single-channel system included a reactor (20 cm x 1.2 mm o.d.) containing the immobilized enzyme. Normal-flow and stopped-flow methods were used. Serum (50 µL) was diluted to 5 mL with carrier solution, aliquots (30 to 280 µL) were injected into the carrier stream (1 mL min-1) and the NADH formed from NAD+ was detected at 340 nm. For normal flow injection analysis the calibration graphs were rectilinear for 1 to 24 µg mL-1 with a detection limit of 0.3 µg mL-1 and a sampling frequency of 40 hr-1. For stopped-flow analysis the calibration graph was rectilinear for 0.5 to 28 µg mL-1, with a detection limit of 0.1 µg mL-1 and a sampling frequency of 30 hr-1.
Ethanol Blood Serum Spectrophotometry

"Determination Of Creatine Kinase Activity Using A Co-immobilized Auxiliary Enzyme Reactor Coupled Online With A Flow Injection System"
Analyst 1991 Volume 116, Issue 2 Pages 167-169
J. M. Fern&aacute;ndez-Romero and M. D. Luque de Castro

Abstract: Two flow injection methods (based on spectrophotometric and spectrofluorimetric detection) were developed for the determination of over-all creatine kinase activity. Despite the complexity of the reactions involved (both include three enzyme-catalyzed steps), the manifold is very simple because the two auxiliary enzymes which catalyse the two-step indicator reaction are co-immobilized on controlled-pore glass. The features of the proposed methods (calibration ranges between 0.1 and 2.0 and 0.01 and 1.0 U l-1, relative standard deviation 0.93 and 0.53% for the spectrophotometric and spectrofluorimetric methods, respectively) allow the successful determination of the analyte activity in serum samples (recoveries better than 95-105% for both methods). In the flow injection method described, a sample of serum is injected into a stream of 100 mM Tris - acetate buffer (pH 7.00) and mixed with the reagent stream containing 20 mM Mg acetate, 1 mM EDTA, 10 µM-p'p5-di(adenosine-5')pentaphosphate, 1 or 0.5 mM AMP, 10 mM N-acetyl-L-cysteine, 2 or 1.5 mM ADP, 15 mM creatine phoshate, 1.5 mM D-glucose and 1.5 or 2 mM NADP in the same buffer. The mixture was passed through a reaction coil and an enzymatic reactor containing hexokinase and glucose-6-phosphate dehydrogenase immobilized on controlled pore glass (cf. Masoom and Townshend, Anal. Chim. Acta., 1984, 166, 111). Detection was by spectrophotometry or fluorimetry (where two concentration. are given, they refer to the two methods, respectively). Calibration graphs were rectilinear for 0.1 to 2 and 0.01 to 1 iu l-1, respectively, and coefficient of variation were 1% (n = 11). Recoveries were 95 to 105%.
Enzyme, creatine kinase Blood Serum Fluorescence

"Flow Injection Electrochemical Enzyme Immunoassay For Theophylline Using A Protein A Immunoreceptor And P-aminophenyl Phosphate P-aminophenol As The Detection System"
Analyst 1992 Volume 117, Issue 11 Pages 1679-1682
Derek A. Palmer, Tony E. Edmonds and Nichola J. Seare

Abstract: An online immobilization of the antibody - antigen complex on controlled-pore glass - protein. A competitive electrochemical enzyme immunoassay was developed (details given). The p-aminophenol phosphate hydrolysis product was determined at ± 0.2 V vs. SCE. In the analysis of serum, the coefficient of variation were 6.8 to 8.7%; the detection limit was 25 ng mL-1 of theophylline. Recoveries were 95.5%. A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass-protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V vs. the SCE. For each sample the antibody-protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.
Theophylline Blood Serum Electrode Immunoassay

"Selective Continuous-flow - Stopped-flow - Continuous-flow Determination Of Sulfite In White Wines Using Immobilized Sulfite Oxidase On A Rotating Reactor"
Analyst 1994 Volume 119, Issue 9 Pages 2093-2096
Maria Olimpia Rezende and Horacio A. Mottola

Abstract: White wine was adjusted to pH 8 with 1 M NaOH and kept at room temperature for 10 min. A 5 mL portion was then diluted to 25 mL with 0.1 M phosphate buffer of pH 7.5 and the resulting solution (53 µL) was injected into a carrier stream (1.38 ml/min) of 0.1 M phosphate buffer of pH 7.5 and merged with a reagent stream (1.38 ml/min) of 0.05 M hexacyanoferrate(III). The mixture was pumped through a column packed with ascorbate oxidase immobilized on controlled-pore glass (CPG) and then into a flow-through cell, where contact was made with a rotating (840 rpm) disc containing sulfite oxidase immobilized on CPG (preparation described). The flow was stopped and the hexacyanoferrate(II) produced was detected amperometrically at a stationary Pt ring electrode at a potential of +0.38 V vs. Ag/AgCl. A diagram of the flow system used is given. The analysis could be performed without use of the ascorbate oxidase column by employing the standard additions method. The calibration graph was linear from 0.1-0.8 mM sulfite. Recoveries were quantitative. The results obtained agreed with those obtained by the official AOAC modified Monier-Williams method.
Sulfite Wine White Amperometry Electrode Electrode

"Simultaneous Spectrofluorimetric Determination Of Glycerol And Ethanol In Wine By Flow Injection Using Immobilized Enzymes"
Analyst 1995 Volume 120, Issue 1 Pages 179-182
I. L. Mattos, J. M. Fernandez-Romero, M. D. Luque De Castro and M. Valc&aacute;rcel

Abstract: Wine was diluted 2500-fold in 100 mM KHCO3/Na2CO3 buffer of pH 10 (buffer A). Two volumes of the sample solution (150 and 50 µL for glycerol and ethanol determination, respectively) were injected via a dual injection valve into two symmetrical channels containing a reagent stream (0.6 and 1 ml/min) for glycerol and ethanol determination, respectively) of 4.5 mM β-NAD in buffer A. Each plug passed through a 1.5 cm long enzyme reactor (glycerol dehydrogenase or alcohol dehydrogenase immobilized on controlled-pore glass) maintained at 35°C. A coiled reactor (300 cm long) was inserted in the ethanol channel to produce a delay between the two streams subsequently merged and passed through a 50 cm long mixing coil before fluorimetric detection at 460 nm (excitation at 340 nm). A diagram of the manifold used is given. Calibration graphs were linear from 1-10 and from 10^-100 µg/ml of glycerol and ethanol, respectively. The RSD (n = 11) were 1%. Recoveries were 97-105%. The throughput was 60 samples/h.
Ethanol Glycerol Wine Fluorescence

"Development Of A Flow Fluoriimmunosensor For Determination Of Theophylline"
Analyst 1995 Volume 120, Issue 10 Pages 2589-2591
Carlos M. Rico, M. del Pilar Fernandez, Ana M. Gutierrez, M. Concepcion Perez Conde and Carmen Camara

Abstract: A 30 µL portion of theophylline (I) polyclonal antibody diluted 100-fold was injected into a carrier stream (0.18 ml/min) of 50 mM Tris buffer of pH 8.8 in 0.5 M NaCl (buffer A). The antibodies were carried to a flow cell packed with protein A immobilized on controlled-pore glass. After allowing 80 s for binding to occur, a portion (30 µL) of a mixture of fluorescein isothiocyanate-labelled I (200 µg/l) and standard I solution was injected into a carrier stream (0.18 ml/min) of buffer A. The two antigens were carried to the flow cell containing the bound antibodies and, after 200 s had elapsed, the fluorescence intensity was measured at 520 nm (excitation at 495 nm). The calibration graph was linear up to 10 µg/l of I and the detection limit was 0.3 µg/l. The within-day RSD (n = 6) was 3.3-4.4%. The through-put was 10^-12 samples/h. The method was applied to the analysis of I in serum. Results ageed well with those obtained by an HPLC reference method.
Theophylline Blood Serum Immunoassay Fluorescence Sensor

"Flow-through Spectrofluorimetric Sensor For The Determination Of Glycerol In Wine"
Analyst 1995 Volume 120, Issue 12 Pages 2837-2840
Pilar Ca&ntilde;izares and M. D. Luque de Castro

Abstract: A flow-through sensor based on transient immobilization of NADH, the product of the reaction between glycerol and NAD(+) catalyzed by glycerol dehydrogenase, is proposed, The biocatalyst is immobilized on controlled-pore glass, where the enzymatic reaction takes place, the product of the reaction being transiently retained and spectrofluorimetrically monitored on passage through a flow cell packed with a solid support, The proposed method affords a linear range between 0.3 and 5.0 µg mL-1 of glycerol, and a detection limit of 0.1 µg mL-1, with a relative standard deviation of less than 2%, The performance of the sensor was checked by applying it to the determination of glycerol in wine. The results obtained agree well with those provided by a conventional method based on HPLC. (14 References)
Glycerol Wine Fluorescence Sensor

"Amperometric Determination Of L-malic Acid In A Flow Injection Analysis Manifold Using Packed-bed Enzyme Reactors"
Analyst 1996 Volume 121, Issue 4 Pages 435-439
Mamas I. Prodromidis, Stella M. Tzouwara-Karayanni, Miltiades I. Karayannis, Pankaj Vadgama and Andrew Maines

Abstract: The sample (130 µL) was injected into a carrier stream (0.19 ml/min) of 0.05 M glycylglycine buffer of pH 9.5 (buffer A) containing 1.75 mM hexacyanoferrate(III) and merged with a reagent stream (0.14 ml/min) of 9.5 mM NAD+ in buffer A. The mixture was passed through a packed-bed reactor (3 cm x 2 mm i.d.) containing malate dehydrogenase and diaphorase co-immobilized on isothiocyanate-modified controlled-pore glass (preparation described). The hexacyanoferrate(II) produced was monitored amperometrically at a graphite electrode held at +0.3 V vs. Ag/AgCl. The calibration graph was linear for 20-400 µM malate, the detection limit was 1 µM and the RSD (n = 5) at the 100 µM malate level was 1.2%. The throughput was 30 samples/h. The method was applied to fruits and vegetables. Recoveries of malate were 95-108%. The results obtained agreed with those obtained by an enzymatic test-kit method.
l-Malic acid Fruit Vegetable Amperometry

"Thermometric Determination Of Copper(II) Using Acid Urease"
Analyst 1996 Volume 121, Issue 11 Pages 1717-1720
Claudia Preininger and Bengt Danielsson

Abstract: A flow injection enzymatic-thermometric method for Cu determination is described. A portion (0.5 ml) of 0.5 M urea was injected into a carrier stream (0.9 ml/min) of 0.1 M maleate buffer of pH 6 in a flow injection manifold (schematic shown). The mixture was passed through a column of acid urease immobilized on controlled-pore glass (preparation described) and the heat produced was measured with a Wheatstone bridge. Then, 0.5 mL Cu solution was injected into the system for 0.5 min to inhibit the enzyme. After 5 min, a further portion (0.5 ml) of 0.5 M urea was injected into the system and the heat produced by the urea/urease reaction was measured. The percentage inhibition was calculated from the peak heights before and after Cu injection, and was used for quantification of Cu(II). The calibration graph was linear up to 0.1 mM Cu(II), the detection liwas 0.32 ppm and the RSD (n = 5) was 4%.
Copper Thermometry

"Technique And Support For Micro-organism Immobilization. Application To Trace Metals Enrichment By Flow Injection Atomic Absorption Spectrometry"
Analyst 1996 Volume 121, Issue 11 Pages 1633-1640
Angel Maquieira, Hayat A. M. Elmahadi and Rosa Puchades

Abstract: The fungus Penicillium notatum was covalently immobilized on glutaraldehyde-treated controlled-pore glass and sand. Two methods for dissolution of the fungus before immobilization were employed, viz., (i) treatment NaOH and (ii) ultrasonic agitation. The immobilized fungus was incorporated in a flow injection AAS system to assess effectiveness in pre-concentrating various metals, viz., Cu, Cd, Fe, Pb and Zn, from aqueous solution. Detection limits in the ng/ml range were achieved, with enhancement factors of 14-625 depending on the metal and on the method and support used to immobilize the fungus. The pre-concentration method was used to determine Cu and Zn in tap and mineral water. The results obtained agreed with those obtained by voltammetry.
Copper Trace elements Mineral Water Spectrophotometry

"Non-aqueous Enzymic Flow Injection Determination Of Cholestanol In Sediments"
Analyst 1998 Volume 123, Issue 11 Pages 2291-2295
Gerardo Pi&ntilde;eiro-Avila, Amparo Salvador and Miguel de la Guardia

Abstract: A procedure was developed for the spectrophotometric determination of cholestanol in sediments based on its extraction with chloroform-methanol (2+1 v/v), dissolution of the extracts, after pre-concentration, in pH 7.0 buffer-saturated toluene containing 10^-3 M p-anisidine and enzymatic determination in non-aqueous media using a bienzymatic reactor consisting of 1 mg of cholesterol oxidase and 1 mg of horseradish peroxidase non-covalently co-immobilized on controlled pore glass beads. A limit of detection of 2.0 x 10^-6 M was obtained under the optimum experimental conditions and recoveries of 95-118% were obtained in the anal. of water and beach sediment samples spiked with cholestanol at concentrations 0.4-1.0 mg/g. This method can also be used for the determination of cholesterol in sediment extracts, the analytical sensitivity for cholestanol being half that for cholesterol, studies were carried out in order to determine accurately both compounds in the same sample.
Cholestanol Cholesterol Beach Water Spectrophotometry

"Immobilized Alga As A Reagent For Preconcentration In Trace Element Atomic Absorption Spectrometry"
J. Anal. At. Spectrom. 1991 Volume 6, Issue 8 Pages 643-646
Hayat A. M. Elmahadi and Gillian M. Greenway

Abstract: Metal ions in buffer solution were injected into a carrier stream (2 mL min-1) of water in a flow injection system (diagram given). The mixture was passed through a column (5 cm x 2.5 mm) packed with the alga Selenstrum capricornutum immobilized on controlled pore glass (details given). After washing with water, the accumulated ions were released by injection of HNO3 and passed to the flame for detection by AAS. Under optimized conditions (details given), the calibration graphs were rectilinear for 5 to 45, 10 to 100, 55 to 300, 650 to 5000, 15 to 80 and 60 to 450 ng mL-1 of Cu2+, Zn2+, Co2+, Hg2+, Cd2+ and Pb2+, respectively; corresponding detection limits were 0.05, 0.2, 8, 30, 2 and 2.5 ng mL-1. The sampling rate was 20 h-1.
Trace elements Spectrophotometry

"Immobilized Cysteine As A Reagent For Preconcentration Of Trace Metals Prior To Determination By Atomic Absorption Spectrometry"
J. Anal. At. Spectrom. 1993 Volume 8, Issue 7 Pages 1011-1014
Hayat A. M. Elmahadi and Gillian M. Greenway

Abstract: Trace metals were determined by AAS in a flow injection system (manifold illustrated) that provided online pre-concentration on glutaraldehyde-immobilized cysteine on controlled-pore glass. A Varian model AA75 atomic absorption spectrophotometer was used with an air-acetylene flame, and the immobilized cysteine was contained in a 5 cm x 2.5 mm column. Cu, Zn, Cd, Pb, Co and Hg were pre-concentrated with capacities of 7.86, 7.88, 12.48, 11.66, 5.50 and 6.06 mmol/g. The presence of other metals at 1 and 5 µg/ml produced no interference, but at 20 µg/ml there was some negative interference. Co was only poorly retained, and was displaced by the other cited metals, but not by Mg.
Metals, trace Copper Zinc Cadmium Lead Mercury Spectrophotometry

"Speciation And Preconcentration Of Trace Elements With Immobilized Algae For Atomic Absorption Spectrophotometric Detection"
J. Anal. At. Spectrom. 1994 Volume 9, Issue 4 Pages 547-551
H. A. M. Elmahadi and Gillian M. Greenway

Abstract: A previously described procedure for the immobilization of Selenestrum capricornutum on controlled-pore glass and its use for the pre-concentration and determination of metal ions by flow injection AAS (Elmahadi and Greenway, Ibid., 1991, 6, 643) was used to immobilize a different alga, Chlamydomonus reinhartii, and to compare its performance with that of the previously immobilized alga. The metal ion solution was passed through a column of the immobilized alga incorporated in a flow injection manifold. The accumulated ions were eluted by injecting the appropriate eluent reagent into a water carrier stream, followed by measurement by AAS. Optimum conditions for the pre-concentration and elution of Cu(II), Ag(I), Cr(III) and Cr(VI) were established (details given). The method was used for the speciation of Cr(III) and Cr(VI) by employing two flow injection manifolds connected together and to determine Cu(II) and Zn(II) in a certified sediment reference material.
Copper(II) Chromium(III) Chromium(VI) Silver(I) Zinc(II) Environmental Spectrophotometry

"Application Of Multielement Time-resolved Analysis To A Rapid Online Matrix Separation System For Inductively-coupled Plasma-mass Spectrometry"
J. Anal. At. Spectrom. 1995 Volume 10, Issue 11 Pages 929-933
Simon M. Nelms, Gillian M. Greenway and Robert C. Hutton

Abstract: A rapid online matrix separation system for ICP-MS, using multi-element time-resolved analysis, was developed for the determination of several trace elements in complex matrix samples, A flow injection manifold was constructed consisting of a mini-column of 8-hydroxyquinoline covalently immobilized on to controlled pore glass, Analytes retained on the column were eluted using 0.1 mi of 2.0 mol L-1 nitric acid, Sample volumes of 0.5 mi were analyzed, yielding a pre-concentration factor of 5 in addition to matrix separation, The system was optimized with respect to the variables of buffer concentration, buffer pH and eluent acid volume and concentration, Calibrations from both pure water and synthetic seawater compared well and showed good linearity, with correlation coefficients of 0.988-0.999 for a range of analytes, The method showed good within-run reproducibility with precisions (s(r)) at the 1 ng mL-1 level of typically <3%, In general, recoveries between 89 and 104% were obtained, with the exception of Ni, which showed a recovery of 78% under the compromise conditions used. The method was validated by the analysis of estuarine (SLEW-1) and coastal (CASS-2) certified reference materials, Good agreement with the certified values was obtained for both of these materials. (17 references)
Metals, trace Sea Estuarine NRCC CASS-2 NRCC SLEW-1 Mass spectrometry Sample preparation

"Preconcentration And Determination Of Some Lanthanide Elements With Immobilized Bacteria By Flow Injection Inductively Coupled Plasma Atomic-emission Spectrometry"
J. Anal. At. Spectrom. 1996 Volume 11, Issue 2 Pages 99-106
Angel Maquieira, Hayat Elmahadi and Rosa Puchades

Abstract: Lanthanides were determined using a previously described FIA method (Anal. Chem., 1994, 66, 3632). The analytes were pre-concentrated by Spirulina plantensis bacteria dissolved in aqueous NaOH, immobilized on controlled-pore glass and packed into a methyl methacrylate mini-column (2.5 cm x 2 mm i.d.). The lanthanides were detected by ICP-AES with an Ar plasma and a cross-flow nebulizer at 3.2 bar using wavelengths of 394.91, 408.672 and 401.225 nm for La3+, Ce3+ and Nd3+, respectively. The effects of flow rate and pH on the pre-concentration is discussed. Detection limits were 9, 0.21 and 0.54 ng/ml for La(III), Ce(III) and Nd(III), respectively, with corresponding recoveries of 100.2±0.04, 105.2±0.18 and 100.1±0.2%. The method was applied to the determination of La(III) and Nd(III) in high-purity Ce(III).
Lanthanum Neodymium High purity Spectrophotometry

"Evaluation Of Controlled-pore Glass-immobilized Iminodiacetate As A Reagent For Automated Online Matrix Separation For Inductively Coupled Plasma Mass Spectrometry"
J. Anal. At. Spectrom. 1996 Volume 11, Issue 10 Pages 907-912
Simon M. Nelms, Gillian M. Greenway and Dagmar Koller

Abstract: A 0.04 g portion of PROSEP Chelating-1 iminodiacetate reagent (Bioprocessing, Consett, Co. Durham, UK) was packed as dry powder into a glass column (2.5 cm x 3 mm) that was incorporated in a flow injection manifold (diagram presented). A 3 mL sample was injected into a stream of H2O; transition-metal, U and Pb cations were retained on the column and subsequently eluted in an elution volume of 0.3 mL with 0.5 M HNO3. Calibration graphs prepared on the basis of water and seawater as matrices agreed, and both sets showed good linearity. RSD (n = 5) at 5 ng/ml were 5%, and recoveries were 62-113% for all the metals except Mn (35%).
Metals, heavy Environmental Environmental Sea Mass spectrometry

"Determination Of Conjugated Glucuronic Acid By Combining Enzymic Hydrolysis With Lucigenin Chemiluminescence"
Anal. Chem. 1985 Volume 57, Issue 1 Pages 46-51
Lori L. Klopf and Timothy A. Nieman

Abstract: The samples pass in a continuous-flow system through a column packed with controlled-porosity glass on which β-glucuronidase is immobilized, and the resulting glucuronic acid is mixed with streams containing NaOH, lucigenin and Na dodecyl sulfate, the chemiluminescent emission intensity being proportional to the free glucuronic acid and therefore to the original glucuronide concentration. Detection limits for phenyl, nitrophenyl, methylumbelliferyl, bromonaphthyl, oestradiol and androsterone glucuronides are from 5 to 10 µM, calibration graphs being rectilinear to 2 mM. The placement of a HPLC Partisil SAX column after the enzyme reactor removes interfering compounds. Results are given for the determination of total glucuronides in urine, and the application of the method to blood is envisaged.
Glucuronic acid Glucuronides, phenyl Glucuronides, nitrophenyl Glucuronides, methylumbelliferyl Glucuronides, bromonaphthyl Glucuronides, oestradiol Glucuronides, androsterone Urine Chemiluminescence

"Immobilized Fluorophores In Dynamic Chemiluminescence Detection Of Hydrogen Peroxide"
Anal. Chem. 1985 Volume 57, Issue 11 Pages 2071-2074
Gerald Gubitz, Piet Van Zoonen, Cees Gooijer, Nel H. Velthorst, and Roland W. Frei

Abstract: Various procedures are described for the immobilization of 3-aminofluoranthene(I) on solid carriers such as cellulose, silica gel and glass beads for use in the chemiluminescence detection of H2O2 in aqueous solution. Controlled pore glass, the most suitable carrier, with immobilized I, was packed in a silica cell and used in a flow injection system also containing a bed reactor of solid bis-(2,4,6-trichlorophenyl) oxalate. A detection limit of 10 nM-H2O2 was obtained, and the calibration graph was rectilinear for 100 µM.
Hydrogen peroxide Chemiluminescence

"Construction And Performance Of Plastic-embedded Controlled Pore Glass Open Tubular Reactors For Use In Continuous-flow Systems"
Anal. Chem. 1986 Volume 58, Issue 7 Pages 1585-1587
Matthew C. Gosnell, Ricky E. Snelling, and Horacio A. Mottola

Abstract: The surface area of open-tubular reactors is increased by embedding controlled-pore glass (CPG) particles in the walls of the glass tubing. The tubes are used in the construction of immobilized-enzyme reactors for continuous-flow systems. Typical flow injection signal profiles of benzylpenicillin with borosilicate glass and CPG - Tygon reactors are presented. The latter reactors give peak heights 15 to 18 times greater than the former type.
Benzylpenicillin

"Chemiluminescence Analysis In Flowing Streams With Luminol Immobilized On Silica And Controlled-pore Glass"
Anal. Chem. 1987 Volume 59, Issue 6 Pages 869-872
Kevin Hool and Timothy A. Nieman

Abstract: Luminol(I) and isoluminol(II) were immobilized on silica (180 to 250 µm) and on controlled-pore glass (125 to 180 µm; pore diameter 522 .ANG.) by silanization of the substrate with 3-aminopropyltriethoxysilane, treatment with glutaraldehyde, and then treatment with a solution of I or II in ethanol - DMSO (1:1). Loadings of I were 29 µmol g-1 on glass and 86 µmol g-1 on silica. Determination of peroxide was performed with these materials in a flow injection system with water as mobile phase and 5 µM-haemin as catalyst, and with a photomultiplier for detection. Calibration graphs were rectilinear in the range 20 to 600 µM-H2O2 with use of luminol as reagent; coefficient of variation were 3% (silica) and 4% (glass) from 1 µM to 1 mM H2O2. Inclusion of 25 mM NaOH in the water stream gave the best compromise between sensitivity and preservation of the reagent. It was estimated that >500 assays could be done with 1 g of silica containing I.
Hydrogen peroxide Chemiluminescence

"General Approach To The Development Of Luminescent Cation Detectors"
Anal. Chem. 1989 Volume 61, Issue 3 Pages 211-215
T. C. Werner, John G. Cummings, and W. Rudolf Seitz

Abstract: A cation-selective ionophore is immobilized on controlled-pore glass, which is then packed into a glass capillary tube and placed in a fluorimeter. If an aqueous phase containing the cation of interest and 8-anilinonaphthalene-1-sulfonic acid (I) is passed through the tube, the cation binds reversibly to the ionophore and the complex forms an ion-pair with I on the glass surface. The fluorescence of I increases strongly when bound to a surface, the increase being related to the concentration. of the cation. Several ion - ionophore systems are presented, including K+ - dibenzo-18-crown-6, K+ - valinomycin and NH4+ - nonactin, for which the detection limits were 0.3 mM K+, 50 µM-K+ and 60 µM-NH4+, respectively. The detectors have applications in flow injection analysis.
Cations Fluorescence Luminescence

"Flow System For Starch Determination Based On Consecutive Enzyme Steps And Amperometric Detection At A Chemically Modified Electrode"
Anal. Chem. 1990 Volume 62, Issue 3 Pages 263-268
J. Emneus and L. Gorton

Abstract: A flow injection system is described for the determination of the total glucose content of starch. The system comprises three reactors, in each the required enzyme is immobilized on glutaraldehyde-activated, aminopropyl-silanized controlled-pore glass (CPG) of specific diameter and pore size, these being, respectively, (i) Termamyl 120L (Novo Industri, Denmark), used at 60°C on CPG (75 to 125 µ diameter), (ii) amyloglucosidase, from Aspergillus niger, on CPG (125 to 180 µm) and (iii) porcine mutarotase co-immobilized with glucose oxidase, on CPG (37 to 74 µm). The H2O2 released in (iii) is detected at a graphite electrode with a catalytic layer of Au - Pd (3:2), operated at +0.6 V vs. Ag - AgCl. The carrier flow (0.5 mL min-1) is 0.1 M acetate buffer of pH 5.0 for (i) and (ii) adjusted to pH 7.0 for (iii) by addition of 0.2 M phosphate (pH 7.6). Calibration graphs are rectilinear from 10 µM- to 0.6 mM of glucose with an injection volume of 160 µL and 15 samples h-1 throughput.
Glucose Organic compound Amperometry Electrode

"Flow Injection Analysis Of L-lactate With Enzyme Amplification And Amperometric Detection"
Anal. Chem. 1990 Volume 62, Issue 7 Pages 708-712
Moore U. Asouzu, William K. Nonidez, and Mat H. Ho

Abstract: A flow injection analysis method for the determination of the lactate anion with enzyme amplification and amperometric detection is described. The system utilizes an oxygen electrode for measurement of changes in the oxygen concentration in the flow stream. Two enzymes, lactate oxidase and lactate dehydrogenase, were randomly coimmobilized on aminopropyl controlled-pore glass (AMP-CPG) and packed into a reactor. β-NADH was used as a coenzyme for the regeneration of lactate from pyruvate. The experimental conditions for the determination of the lactate anion were studied for this system by the simplex and the univariant methods. The results obtained under these two conditions were compared. The simplex experimental condition yielded a calibration curve whose linear portion had a slope that was 1.2 times greater than that of the linear portion of the curve obtained under univariant conditions. The limit of detection under simplex condition was 1.19 x 10^-7 M vs 3.29 x 10^-7 M lactate under univariant conditions. The relative standard deviation obtained for this system at 6 x 10^-6 M lactate (n = 10) was about 2.5% under simplex conditions and 3.6% under univariant maximization conditions.
l-Lactate Amperometry Electrode

"Determination Of Monosaccharides In Cellulosic Hydrolysates Using Immobilized Pyranose Oxidase In A Continuous Amperometric Analyser"
Anal. Chem. 1990 Volume 62, Issue 24 Pages 2688-2691
Lisbeth Olsson, Carl Fredrik Mandenius, and Jindrich Volc

Abstract: Purified pyranose oxidase (details given) was immobilized on controlled pore glass by using the glutaraldehyde activation method and stabilized by co-immobilization with catalase. For the determination of glucose, xylose and galactose, the immobilized enzyme reactor was installed in a pseudo flow injection system and oxygen consumption was measured with an amperometric electrode (Clark-type). The electrode response after partial transfer of the sample through a dialysis membrane was rectilinear from 0.6 to 30, 1.0 to 50 and 2.0 to 100 mM, respectively. The analytical system was tested for bioreactor monitoring on laboratory scale by interfacing with a 10-l fermenter containing spent sulfite liquor, and no adverse effects were observed with regard to pyranose response; after 2000 injections into the enzyme reactor the decay of enzyme activity was 17%. This analytical system has also been applied to the continuous monitoring of ethanolic fermentation.
Monosaccharides Glucose Galactose Xylose Amperometry

"Operation Of Ion-selective Electrode Detectors In The Sub-Nernstian/linear Response Range: Application To Flow Injection/enzymatic Determination Of L-glutamine In Bioreactor Media"
Anal. Chem. 1991 Volume 63, Issue 18 Pages 1906-1909
Wojciech Matuszewski, Sara A. Rosario, and Mark E. Meyerhoff

Abstract: A novel approach for eliminating positive errors from endogenous ionic interferences when using ion-selective electrodes as detectors in flow injection enzyme-based biosensing configurations is described. The method involves using a high background level of interfering ions in the sample diluent/carrier stream to convert the normally logarithmic potentiometric sensor into a linear detector over a given concentration range of primary ions. A split-stream single-detector arrangement provides a convenient means to compensate for varying levels of background interferent ions in the injected samples. One portion of the split stream passes directly to the ion-electrode detector, yielding a signal linearly related to the concentration of endogenous primary ions in the sample. The second portion of the split sample is delayed while passing through an immobilized enzyme that generates electrode detectable primary ions in proportion to the concentration of the substrate analyte in the sample. Two linear equations with two unknowns describe the twin potentiometric responses observed. The concept is demonstrated by the accurate determination of L-glutamine in hybridoma bioreactor media via the use of an ammonium-ion-selective membrane electrode detector and immobilized glutaminase enzyme. A system is described for elimination of positive errors from endogenous ionic substance in flow injection enzymatic determinations with ion-selective electrodes as detectors. For determination of glutamine (I), the injected sample is split into two streams, one flowing directly to the ion-selective-electrode detector via a short delay loop and the second passes through a packed controlled pore glass reactor containing immobilized glutaminase and then through a delay loop to the detector. Two detector responses are obtained, the first to endogenous ammonium-N and the second to total NH4+ present after passing through the enzyme reactor. If both responses are rectilinear, simultaneous equations (derived) can be solved to obtain the I concentration. of the sample.
l-Glutamine Electrode Potentiometry Sensor

"Enzymic Flow Injection Analysis In Non-aqueous Media"
Anal. Chem. 1992 Volume 64, Issue 2 Pages 129-133
Lorenzo Braco, Jose A. Daros, and Miguel De la Guardia

Abstract: The use of non-covalently immobilized enzyme reactors operated in non-aqueous media is described, with a co-immobilized cholesterol oxidase - horse-radish peroxidase reactor for flow injection spectrophotometric determination of cholesterol (I) in toluene as a model system. Reactors were prepared by adsorption of the biocatalyst on to controlled-pore glass beads and incorporated into a flow injection system. For determination of I, the carrier solution was H2O-saturated toluene containing 1 mM p-anisidine. Spectrophotometric detection was at 458 nm. Calibration graphs were rectilinear from 20 µM to 0.18 mM I. The detection limit was 1 µM I. A novel enzymatic flow injection approach is proposed, involving the use of non-covalently immobilized enzyme reactors operating in non-aqueous media. The feasibility of this approach was tested and successfully demonstrated by using as a model system a co-immobilized cholesterol oxidase-peroxidase reactor for the determination of cholesterol in toluene. The response was linear up to 1.8 x 10^-4 M cholesterol, with a detection limit of 10^-6 M. The throughput was 60 samples/h. The reactor was stable for >4 months with a half-life of 81 days. The strategy developed combines the remarkable benefits offered by enzymology in organic solvents and the advantages inherent in flow injection methods. Among them, the biocatalyst can be immobilized in a rapid, complete, and irreversible manner by simple adsorption to the solid support; poorly water-soluble compounds can be readily dissolved in the mobile phase and directly analyzed; and most importantly, the enzymatic reactor exhibits an enhanced stability in the water-restricted environment.
Cholesterol Organic compound Spectrophotometry

"Continuous-flow Sensor Strategy Comprising A Rotating Bioreactor And A Stationary Ring Amperometric Detector"
Anal. Chem. 1993 Volume 65, Issue 5 Pages 636-639
Kiyoshi Matsumoto, J. J. Baeza Baeza, and Horacio A. Mottola

Abstract: Details are given of a Plexiglas cell incorporating a stationary Pt-ring electrode and a rotating enzyme reactor comprising a PTFE disc coated with glucose oxidase immobilized on aminopropyl-modified controlled-pore glass. This cell was used in conjunction with a stainless-steel auxiliary electrode and a Ag - AgCl reference electrode for the continuous-flow amperometric measurement of glucose via the H2O2 produced enzymatically; the carrier electrolyte was 0.1 M phosphate buffer of pH 7.00, the Pt-ring electrode was maintained at +0.60 V, and the signal was measured under stopped-flow conditions. With rotation of the reactor at 847 rpm, response was rectilinearly related to glucose concentration. up to 1 mM; the detection limit was 2.3 µM-glucose and the coefficient of variation (n = 10) at 0.5 mM was 1.6%. The sampling rate was ~30 h-1; the lower the rotation rate the wider the dynamic range.
Amperometry Sensor

"Use Of Saccharomyces Cerevisiae In Flow Injection Atomic Absorption Spectrometry For Trace Metal Preconcentration"
Anal. Chem. 1994 Volume 66, Issue 9 Pages 1462-1467
Angel Maquieira, Hayat A. M. Elmahadi, and Rosa Puchades

Abstract: Freeze-dried yeast was washed with CHCl3 and hot water, digested with 0.1 M HNO3/HClO4 (1:1) on a steam bath for 30 min. Ethanol was added dropwise and the resulting solution was diluted with water and adjusted to pH 7 with 2 M NaOH. The solution was treated with phosphate buffer solution of pH 5.5, 6.5, 7.5 or 8.5 before the addition of glutaraldehyde-treated controlled pore glass. Immobilization proceeded for 24 h at 4°C under N2, the glass was filtered off and air-dried. Immobilization was unsatisfactory at pH 8.5 and slightly less effective at pH 5.5 than at pH 6.5 or 7.5. Methacrylate columns (4 cm x 2.5 mm i.d.) of the yeast-treated glass were evaluated for the concentration of trace Cd, Zn, Cu(II), Pb(II) and Fe(III) ions from 0.1 M phosphate buffer of pH 6.5. The solution was applied to the column; elution was effected with acid and the metal ions were determined by AAS (details given). Zn and Cu(II) were eluted by 0.1 M HNO3, Pb(II) and Cd by 0.5 M HNO3 and Fe(II) by 0.5 M HClO4/HNO3 (1:1). Linear ranges extended up to 1500 ng/ml (Pb) and the detection limits were 0.1 (Zn) to 8.0 ng/ml (Pb). RSD (n = 10) were 2.6%. The method was used to determine Cu and Cd in sediment.
Cadmium Zinc Copper(II) Lead(2+) Iron(III) Environmental Spectrophotometry

"Immobilized Cyanobacteria For Online Trace Metal Enrichment By Flow Injection Atomic Absorption Spectrometry"
Anal. Chem. 1994 Volume 66, Issue 21 Pages 3632-3638
Angel Maquieira, Hayat A. M. Elmahadi, and Rosa Puchades

Abstract: Cyanobacteria (Spirulina platensis) immobilized on controlled pore glass pre-concentrate Cu(II), Zn(II), Cd(II), Pb(II), and Fe(III) from aqueous solution with high efficiency as ascertained using an online flow injection atomic absorption spectrometry system. The degree of metal binding depends on the pH of the solution. Quantitative retention of copper, zinc, and cadmium occurred at a wide range of pH values, while the retention for lead and iron was pH-dependent. The latter metals were adsorbed strongly only at pH 6 and 7, respectively. The breakthrough capacity was determined from the breakthrough curve, with values of 0.0035, 0.0008, 0.0011, 0.0028, and 0.0017 ng/mL for Cu, Zn, Cd, Pb, and Fe, respectively, being obtained. The analysis of a certified reference sample, sewage sludge of domestic origin (BCR No. 144), for cadmium and copper with a high accuracy ensures the feasibility of this technique for environmental analysis. Copyright 1994, American Chemical Society.
Copper Cadmium Lead Iron Sludge BCR 144 Spectrophotometry

"Immobilized Amino Aromatics For Solid-phase Detection Using Imidazole-mediated Bis(trichlorophenyl) Oxalate Chemiluminescence"
Anal. Chem. 1995 Volume 67, Issue 23 Pages 4302-4308
Einar Ponten, Malin Stigbrand, and Knut Irgum

Abstract: This investigation examines six amino polycyclic aromatic hydrocarbons, covalently immobilized onto non-porous and porous methacrylate, controlled-pore glass, and Merrifield (S-DVB) beads, for their suitability in solid phase peroxyoxalate chemiluminescence detection, The solid phase luminophores were packed in a now cell mounted adjacent to a photomultiplier tube, and the properties of the different reagents were evaluated by injecting aqueous hydrogen peroxide into a stream of premixed bis(trichlorophenyl) oxalate and imidazole in acetonitrile, The highest sensitivity was obtained with 3-aminoperylene and 3-aminofluoranthene, A high degree of functionalization as well as a large surface area of the support increased the efficiency, The porous methacrylate material was found to give better sensitivity and precision than a non-porous material of similar composition, In this respect, the porous methacrylate support was also superior to controlled-pore glass, although these materials gave similar detection limits. The microporous S-DVB copolymer with the same overall degree of functionalization as the porous methacrylate support showed a lower sensitivity, since its subsurface functionalization did not contribute to the efficiency. (49 references)
Hydrogen peroxide Chemiluminescence

"Immobilized Enzyme Reactors. Diffusion/convection, Kinetics, And A Comparison Of Packed-column And Rotating Bioreactors For Use In Continuous-flow Systems"
Anal. Chem. 1996 Volume 68, Issue 10 Pages 1701-1705
Pablo Richter, Beatriz L&oacute;pez Ruiz, Mercedes S&aacute;nchez-Cabezudo, and Horacio A. Mottola

Abstract: The advantages of rotating disk bioreactors over conventional packed-column reactors used in continuous-flow analysis are demonstrated. Such reactors give minimum dispersion and maximum utilization of immobilized active sites and small amounts of relatively expensive biocatalysts can be utilized effectively. The oxidation of glucose by dissolved oxygen, catalyzed by immobilized glucose oxidase, was used as a model system. The enzyme was immobilized by glutaraldehyde binding to aminopropyl-modified controlled pore glass. The H2SO4 generated was detected at a Pt working electrode using a Ag/AgCl (3-M-NaCl) reference electrode. The system was described previously (Matsumoto et al., Ibid., 1993, 65, 636).
Electrode

"Flow Injection - Based Renewable Electrochemical Sensor System"
Anal. Chem. 1996 Volume 68, Issue 21 Pages 3808-3814
Michael Mayer and Jaromir Ruzicka

Abstract: A renewable electrochemical sensor was developed in which electrically conducting beads formed the disposable electrode and enzymes immobilized on to non-conducting beads formed renewable enzymatic layers. The sensor was based on a sequential injection system equipped with planar concentric Pt electrodes mounted at the base of a stainless steel tube. The central electrode (5.5 mm2) was the working electrode and the beads were piled on to this electrode to form a packed-bed column. The outer Pt electrode (2.8 mm2) was the quasi-reference electrode and the steel tube was the counterelectrode. Each measurement was carried out by injecting a suspension of beads into the stainless steel column to form a packed bed. The analyte was then injected into the carrier stream and transported through the beads. Detection was by measuring the oxidation current. The approach was tested using different immobilized oxidases (galactose, lactate, alcohol or glucose) and various conducting and non-conducting beads (controlled pore glass, oxirane acrylic, glassy C and graphite). Detection limits were 0.085 and 0.005 mM alcohol, and galactose, glucose and lactate, respectively. The approach was applied to determine glucose and alcohol in beer and wine samples.
Alcohol Glucose Beer Wine Sensor

"Flow Injection Ion-exchange Preconcentration For The Determination Of Iron(II) With Chemiluminescence Detection"
Fresenius J. Anal. Chem. 1990 Volume 337, Issue 7 Pages 848-851
Abdulrahman A. Alwarthan, Khalil A. J. Habib and Alan Townshend

Abstract: Solutions of 10 µM-luminol and 10 µM-H2O2, each in 0.1 M carbonate buffer, were continuously pumped, each at 2 mL min-1, through the manifold of the flow injection system; the sample (3 ml; adjusted with 0.1 M phosphate buffer to pH 6.0) was pumped at 1.5 mL min-1 through a chelating column (2 cm x 2.5 mm i.d.) containing 8-hydroxyquinoline-5-sulfonic acid immobilized by azo-coupling on controlled-pore glass (pore diameter 22.6 nm; 80 to 120 mesh). The sample flow was then replaced by water to wash the column for 3 min. For elution of Fe, 52 µL of 0.15 M HCl was injected via the injection valve into the column, and the eluate was mixed with the reagent stream before measurement of the emitted chemiluminescence. A log - log calibration graph was rectilinear for Fe(II) pre-concentrated from triple 52 µL injections of Fe(II) solution (10 to 70 ng mL-1), and the detection limit was ~2 pg mL-1. Of the foreign ions tested, Mo(VI), VV, Ti(IV) and AgI increased and Cu(II) slightly decreased the chemiluminescence signal; Co did not interfere. One analysis took 5 min.
Iron(2+) Chemiluminescence

"Immobilized 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole As Solid-phase Luminophore In Peroxyoxalate Chemiluminescence"
Fresenius J. Anal. Chem. 1996 Volume 356, Issue 1 Pages 84-89
Einar Pont&eacute;n, Patrik Appelblad, Malin Stigbrand, K. Irgum and Kenichiro Nakashima

Abstract: A new luminophore for application in peroxyoxalate chemiluminescence is presented. An analogue of the well-known chemiluminescence compound lophine, i.e. 2-(4-hydrazinocarbonylphenyl)-4,5-diphenyl-imidazole (HCPI), has been covalently immobilized to controlled pore glass and a porous methacrylate resin. By using this reagent in a solid phase detection reactor, sensitive determinations of hydrogen peroxide have been demonstrated. In homogeneous solution HCPI emits poorly as a result of 1,1'-oxalyldiimidazole excitation, but when immobilized its efficiency is almost comparable to highly efficient luminophores such as 3-aminofluoranthene. Linearity extends in the single stream flow system over several orders of magnitude with both materials. The limit of detection was 1 nmol/l (10 fmol injected), when using the porous methacrylate support.
Hydrogen peroxide Chemiluminescence HPLC

"Flow Injection System Using Immobilized Peroxidase And Chromogenic Reagents For Possible Determination Of Hydrogen Peroxide"
Microchim. Acta 1985 Volume 86, Issue 3-4 Pages 211-221
Bo Olsson

Abstract: Horse-radish peroxidase (I) was immobilized on to glycerylpropyl-derivatized controlled pore glass (Pierce CPG/460 Glucophase G, particle size 37 to 73 µm) with 2,2,2-trifluoroethanesulfonyl chloride (II) or diazo-coupling. In combination with these immobilization methods, several chromogenic reagents were studied, in particular with regard to the adsorption of the colored product on the enzyme support. With the use of a reactor containing I - II, and the reagents 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate), 4-amino-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one, 3,5-dichloro-2-hydroxybenzenesulfonate and chromotropic acid, H2O2 was determined with use of a calibration graph which was rectilinear for 1000 µM with a detection limit of 0.1 µM. The system incorporated a Gilson Minipuls 2 pump, carrier- and reagent-stream flow rates were 0.9 and 0.45 mL min-1, and the absorbance was monitored in a 10 µL flow-through cell. The sample throughput was 210 h-1.
Hydrogen peroxide Spectrophotometry

"Enzyme-linked Flow Injection Immunoassay Using Immobilized Secondary Antibodies"
Microchim. Acta 1988 Volume 96, Issue 1-6 Pages 207-221
Ing Huei Lee and Mark E. Meyerhoff

Abstract: A system is described in which immobilized secondary antibodies are used to separate antibody-bound from free enzyme-labelled analyte in a flowing sample solution Theophylline(I), with adenosine deaminase as labelling enzyme and sheep anti-I as first antibody, and insulin(II), with horse-radish peroxidase as labelling enzyme and guinea-pig anti-II as first antibody, are used as model analytes. The secondary antibody (a fraction of rabbit anti-sheep whole serum antiserum for I and of sheep anti-guinea-pig whole serum antiserum for II) was contained in a silicone tube (2.7 cm x 2.54 mm) and was immobilized on carbonyldi-imidazole-activated glycerol-coated controlled-pore glass beads. The buffer carrier stream was 0.05 M Tris - HCl (pH 7.5) for I and 0.05 M sodium phosphate (pH 7.0) for II. After treating the reactor with substrate (details given), the enzymatic reaction products were detected with a NH4+-selective electrode for I or spectrophotometrically at 405 nm for II. Working ranges were 0.025 µM for I and 1 to 250 µg mL-1 for II in the injected solution.
Theophylline Insulin Pharmaceutical Immunoassay Electrode Spectrophotometry

"Fructose Determination Using Immobilized Enzymes In A Flow System With Special Emphasis On The Effect Of Isomerism"
Microchim. Acta 1988 Volume 96, Issue 1-6 Pages 131-142
Mikael Skoog, Gillis Johansson, Bo Olsson, and Roger Appelqvist

Abstract: The sample (25 µL) was injected into a water carrier stream (0.5 mL min-1) which was merged with a buffer stream (0.5 mL min-1) of 0.2 M Tris containing 6 mM Mg2+, 2 mM ATP and 0.7 mM NAD+ (pH 7.6) and thence through three successive tubes packed with CPG-10 controlled-pore glass (40 to 80 µm, silanized and activated with glutaraldehyde) with coatings of (i) glucose oxidase - catalase - aldose-1-epimerase (for glucose removal), (ii) hexokinase and (iii) glucose-6-phosphate isomerase - glucose-6-phosphate dehydrogenase. The NADH formed was detected amperometrically with a graphite-rod working electrode impregnated with Mirak's blue [bis(benzophenoxazinyl)terephthaloyl chloride] and operated at +0.05 V vs. a SCE (Anal.Abstr., 1985, 47, 12D151). Mean recovery of fructose was 91%, the detection limit was~1 µM, and at 100 µM the repeatability was ±0.3%.
Fructose Amperometry Electrode

"Determination Of Sucrose By Flow Injection Analysis With Fourier Transform Infrared Detection"
Microchim. Acta 1995 Volume 119, Issue 1-2 Pages 73-79
Bernhard Lendl and Robert Kellner

Abstract: The cited analysis was based on the invertase-catalyzed cleavage of sucrose (I) to α-D-glucose and β-D-fructose in a reactor (3 cm x 3 mm i.d.) packed with invertase immobilized on aminopropylated controlled pore glass using glutaraldehyde (details given). A manifold (diagram given) incorporating two internally-coupled injection valves enabled FTIR spectra of the unreacted and reacted sample to be obtained. Sample (75 µL) was injected into 0.2 M acetate buffer of pH 4.2 (2.1 ml/min) and FTIR spectra were recorded continuously at 8 cm-1 resolution with use of Brucker OPUS GC-IR software and the difference spectrum of the unreacted and reacted sample was obtained. The difference in absorption at 998 and 1038 cm-1 was used to determine I. The calibration graph was linear for 10^-100 mM I. No detection limit or RSD is given. Sample throughput was 45/h. The method was applied to soft drinks.
Sucrose Soft drink Spectrophotometry

"Preconcentration Of Metal Ions Using An Immobilized Dialkyldithiocarbamate In A Flow System"
Microchem. J. 1992 Volume 45, Issue 2 Pages 210-218
Alan Townshend* and Khalil A. J. Habib

Abstract: Controlled-pore glass (22.6 nm pore diameter, 80 to 100 mesh) was silylated and treated with CS2 (details given) to form the NN-dialkyldithiocarbamate. The treated glass was then packed into a glass tube (4 cm x 2.5 mm) that formed part of a flow injection system. Optimum conditions were established for the extraction of metal ions and for their elution with HNO3, which was followed by direct nebulizer injection for determination by AAS. The capacity of the collector varied from 0.9 mmol g-1 for Rh(III) to ~4 mmol g-1 for Co2+, Cu2+ and Hg2+. The calibration graphs for Rh(III), Co2+, Cu2+, Hg2+ and Hg22+ were rectilinear up to 0.6, 1, 0.14, 2.5 and 5 µg mL-1, respectively, and the corresponding detection limits were 1, 2, 0.05, 50 and 25 ng mL-1 for 5- or 10 mL samples; the coefficient of variation were 2.2 to 2.7%. Little or no interference occurred with various other ions up to 50 µg mL-1. Flow injection analysis with online pre-concentration using a minicolumn loaded with dialkyldithiocarbamate immobilized on controlled pore glass is described for the determination of Rh(III), Co2+, Cu2+, Hg2+, and Hg22+. The detection limits range from 0.05 ng mL-1 for Cu2+ to 50 ng mL-1 for Hg2+ for 5- or 10 mL samples, improvement of 2-3 orders of magnitude compared with direct injection. The operating conditions are optimized and the effects of interferents are studied. The capacity of the collector varied from 0.9 mmol g-1 for Rh(III) to ~4 mmol g-1 (Co2+, Cu2+, Hg2+).
Rhenium(III) Cobalt(II) Copper(II) Mercury(II) Mercury(1+) Spectrophotometry

"Immobilized Chloroxine As A Preconcentration Reagent For Atomic Absorption Spectrometry"
Microchem. J. 1996 Volume 53, Issue 2 Pages 188-194
H. A. M. Elmahadi and G. M. Greenway

Abstract: Portions (5 ml) of standard metal solutions in 0.1 M phosphate or 0.1 M Tris buffer solutions (pH/buffer listed/each metal) were injected into a water carrier solution (2 ml/min) of a flow injection manifold (schematic shown) and the metal ions were trapped on a column (5 cm x 2.5 mm i.d.) packed with chloroxine (5-chloroquinolin-8-ol) immobilized on controlled pore glass (preparation details given). The metals were eluted from the column with 40 µL 1 M HNO3 [0.5 M for Co(II)] injected into the water carrier stream prior to the column. The absorbance was measured by AAS at 324.8, 213.9, 228.8, 240.7 and 217 nm, respectively, for Cu(II), Zn(II), Cd(II), Co(II) and Pb(II). The corresponding calibration graphs were linear from 10^-100, 25-100, 20- 130, 50-300 and 60-500 ng/ml with detection limits of 0.5, 0.2, 0.4, 0.6 and 4 ng/ml. RSD (n = 5) were 1.4, 1.3, 1.1, 1.2 and 1.2%, respectively, for 50, 100, 40, 250 and 200 ng/ml of Cu(II), Zn(II), Cd(II), Co(II) and Pb(II), respectively. The corresponding average recoveries were 98.2, 98.8, 89.2, 87.2 and 72%. The enhancement in sensitivity (enrichment factor) was of the order 49-136 better than that for direct injection, using a 5 mL sample with a sampling rate of 20/h. The effect of the other cations on the absorbance signal of each metal ion was also investigated.
Cadmium(2+) Cobalt(II) Copper(II) Lead(2+) Zinc(II) Spectrophotometry

"Determination Of Acetylcholine And Choline By Flow Injection With Immobilized Enzymes And Fluorimetric Or Luminometric Detection"
Anal. Biochem. 1989 Volume 176, Issue 2 Pages 221-227
Jan R&iacute;cn&yacute;, Jil&iacute; Coupek and Stanislav Tucek

Abstract: Acetylcholine and choline were determined in solution and tissue extracts by a continuous-flow system with a sequence of enzyme reactors containing immobilized acetylcholinesterase, choline oxidase and peroxidase. Additional reactors with immobilized choline oxidase and catalase were used to remove choline and choline-generated H2O2 from samples when acetylcholine was being determined. Acetylcholinesterase, choline oxidase and catalase were immobilized on Separon HEMA E and peroxidase was immobilized on Separon HEMA (for fluorimetric detection) or on a coiled glass capillary or on controlled-pore glass beads (for luminometric detection). Detection was by measuring fluorescence generated from the reaction of H2O2 with 3-(4-hydroxyphenyl)propionic acid in presence of peroxidase, or measuring luminescence generated from the reaction of H2O2 with luminol in presence of peroxidase. Calibration graphs were rectilinear up to 100 pmol of acetylcholine with fluorescence detection. Detection limits were 10 or 25 pmol of acetylcholine with the coiled-capillary peroxidase or controlled-pore-glass peroxidase columns, respectively, with luminometric detection, and 0.2 pmol with fluorimetric detection.
Acetylcholine Choline Biological tissue Luminescence Fluorescence

"Determination Of Inorganic Phosphate By Flow Injection Method With Immobilized Enzymes And Chemiluminescence Detection"
Anal. Biochem. 1989 Volume 182, Issue 2 Pages 366-370
Hideki Kawasaki, Katsumi Sato, Jyunko Ogawa, Yukio Hasegawa and Hidetaka Yuki

Abstract: Inorganic phosphate (I) was determined by flow injection analysis with use of two glass columns (0.9 cm x 3 mm), one packed with purine-nucleoside phosphorylase-coated controlled-pore glass beads (AMP-500; 120 to 200 mesh), and the second packed with xanthine oxidase and urate oxidase-coated beads (preparation described). The carrier solution (1 mL min-1) was 25 µM inosine in 10 mM HEPES - NaOH buffer (pH 7.5) and the H2O2 produced was determined with use of 10 µM luminol and 0.8 µM peroxidase in 50 mM carbonate buffer (pH 10.5) and chemiluminescence detection. The calibration graph was rectilinear for 5 to 250 pmol of K2HPO4 and the detection limit was 500 fmol. The within-assay coefficient of variation was 1.03 to 9.36%. The method was applied in determination of I in DNA.
Phosphate DNA Chemiluminescence

"Determination Of Phosphatidylcholine In A Flow Injection System Using Immobilized Enzyme Reactors"
Anal. Biochem. 1990 Volume 187, Issue 2 Pages 240-245
Mohammed Masoom, Rita Roberti and Luciano Binaglia

Abstract: Two alternative procedures are described for the quantitative determination of phosphatidylcholine in a flow injection system utilizing immobilized enzymes. Phospholipase C from Bacillus cereus and phospholipase D from cabbage were covalently bound to the surface of controlled-pore glass beads and the enzyme-derivatized beads were packed in small columns. In the first procedure, the phospholipase C column was connected with a second column containing coimmobilized alkaline phosphatase and choline oxidase. In the alternative procedure, the column packed with immobilized phospholipase D was connected with a column packed with immobilized choline oxidase. The hydrogen peroxide produced through the action of choline oxidase in both flow injection systems was detected amperometrically. Both procedures are suitable for an accurate and rapid quantitation of phosphatidylcholine. The sensitivity of the method based on phospholipase C and alkaline phosphatase is higher than that using phospholipase D. Quantitation of phosphatidylcholine at the nanomole level can be easily obtained using the first methodology. Membrane lipids were extracted from brain by the method of Folch et al. (J. Biol. Chem., 1957, 226, 497) and dissolved in CHCl3 - methanol (2:1). The solution was evaporated in vacuo and the lipid residue was dissolved in 0.1 M Tris - HCl buffer of pH 7.5 containing 0.3% of Triton X-100 and 0.4 mM ZnCl2. The suspension was injected into a stream (pH 6.5) of 30 mM CaCl2 containing 0.3% of Triton X-100 and 20 mM diethylbarbitone and passed through columns of phospholipase C immobilized on glass beads and of alkaline phosphatase and choline oxidase immobilized on glass beads before electrochemical detection with a vitreous-carbon electrode at +0.6 V. The calibration graph was rectilinear for 0.05 to 2 mM phosphatidylcholine. The sensitivity was higher than that of a flow injection method using phospholipase D.
Phosphatidylcholine Amperometry Electrochemical analysis Electrode

"A Flow Injection Biosensor System For The Amperometric Determination Of Creatinine: Simultaneous Compensation Of Endogenous Interferents"
Anal. Biochem. 1993 Volume 210, Issue 1 Pages 163-171
Rui C. S., Sonomoto K., Ogawa H. I. and Kato Y.

Abstract: Creatinine deaminase and L-glutamate oxidase were immobilized separately on propylamine - controlled pore glass with glutaraldehyde through a Schiff base, and glutamate dehydrogenase was coupled to succinate - controlled pore glass activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The two channel flow injection system (illustrated) comprised a peristaltic pump, injection valve, immobilized enzyme packed in a glass tube and a control tube of controlled pore glass with delay coil, a galvanic O electrode with flow cell, a potentiostat and a recorder. Urine samples were diluted (x 20) and portions (30 µL) were introduced into the system at a flow rate of 1.6 mL min-1. Double peak recording was obtained using the delay coil in one channel; one peak being creatinine, endogenous ammonia and glutamate, and the other being endogenous ammonia and glutamate. Calibration graphs were rectilinear from 0.1 (detection limit) to 2 mM creatinine with coefficient of variation of 2.3% (within-day; n = 12) and 3.9% (between-day; n = 8). Lower detection limits (~0.01 mM), suitable for creatinine determination in serum, were obtainable by increasing the injection volume, e.g., to 100 µL. A flow injection biosensor system was developed for the amperometric determination of creatinine based on coupled reactions of three sequentially aligned enzyme reactors, creatinine deiminase, glutamate dehydrogenase, and glutamate oxidase, using an oxygen electrode as the detector. To overcome the problem of endogenous ammonia and glutamate, the flow was split into two channels after the injector and rejoined before the glutamate dehydrogenase reactor. Double peak recording was obtained by setting a delay coil and a reference column in one of the two channels. The first peak gave the sum response of creatinine, endogenous ammonia, and glutamate, and the second that of endogenous ammonia and glutamate. By this method compensation for endogenous ammonia and glutamate, as well as for interfering ascorbic acid, was achieved simultaneously. The system gave linear calibrations up to 2 mM for the first peak and 3 mM for the second one. The lower detection limits were 0.1 and 0.02 mM for 35- and 100 µL injection of sample, respectively. One run was completed within 2 min. The system showed good reproducibility (<3%) and long operational stability (> 1300 runs). The assay results of creatinine in urine showed good correlation with those obtained from the chemical method of Jaffe.
Creatine Urine Amperometry Sensor

"Peroxidase- And Tetracyanoquinodimethane-modified Graphite Paste Electrode For The Measurement Of Glucose/lactate/glutamate Using Enzyme-packed Bed Reactor"
Anal. Biochem. 1995 Volume 224, Issue 1 Pages 428-433
Pandey P. C. and Weetall H. H.

Abstract: A flow injection analysis sensor for the measurement of glucose/lactate/glutamate is reported. The glucose oxidase/glutamate oxidase/lactate oxidase was immobilized on silanized controlled pore glass particles and packed into a Teflon column (i.d., 1.2 mm; length, 40 mm) to give a bed for glucose/lactate/glutamate. The hydrogen peroxide formed by the enzymatic reaction in the packed bed was monitored by a horseradish peroxidase- and tetracyanoquinodimethane (TCNQ)- modified graphite paste electrode at 50 mV vs Ag/AgCl. The glucose oxidase/lactate oxidase/glutamate oxidase were regenerated in the packed bed, whereas peroxidase was regenerated in the TCNQ-mediated graphite paste electrode by the oxidation of TCNQ. The oxidized TCNQ was electrochemically reduced at 50 mV vs Ag/AgCl. The cathodic current obtained by the reduction of TCNQ determined the concentration of the injected analytes in the packed bed. The system showed very rapid response. Response curves for the analysis of peroxide, glucose, lactate, and glutamate are reported.
Glucose Lactate Glutamate Sensor Electrode

"Enzymatic Assay By Flow Injection Analysis With Detection By Chemiluminescence. Determination Of Glucose, Creatinine, Free Cholesterol And Lactic Acid Using An Integrated Flow Injection Analysis Microconduit"
Anal. Lett. 1986 Volume 19, Issue 5&6 Pages 649-665
Petersson, B.A.;Hansen, E.H.;Ruzicka, J.

Abstract: A miniaturized flow injection system is described for determination of the cited compounds by use of the appropriate oxidases which, together with ancillary enzymes (for creatinine assay), are immobilized on controlled-porosity glass in PVC column reactors. The H2O2 generated in the individual reactions is determined by chemiluminescence with alkaline luminol - Fe(CN)63- reagent. The injection valve, flow channels, enzyme reactor and light detector are integrated into a flow injection analysis microconduit (diagram given). The detection limits were 0.03 mg dl-1 for D-glucose or L-lactate, 0.3 mM creatinine and 0.5 mg dl-1 of cholesterol. Good sensitivity, high sampling rate (70 to 120 h-1) and inexpensive instrumentation makes this method attractive for clinical applications.
Glucose Lactic acid Creatinine Cholesterol, free Chemiluminescence Clinical analysis

"Continuous-flow Determination Of Phenol With Chemically Immobilized Polyphenol Oxidase"
Anal. Lett. 1989 Volume 22, Issue 5 Pages 1145-1158
Zachariah, K.;Mottola, H.A.

Abstract: The open tubular reactor was made from Tygon tubing (20 or 60 cm x 1.3 mm), on the inner surface of which controlled-pore glass (200 to 400 mesh) was thermally embedded. The glass surface was modified by reaction with (aminophenyl)trimethoxysilane and the adsorbed aminophenyl groups were converted into diazonium groups by reaction with HNO2. Tyrosinase (I) was then attached via the diazonium groups (cf. Gosnell et al., Anal. Chem., 1986, 58, 1585). The reactor tube was coiled and used to detect phenol in water. Phenol was converted by I to 1,2-benzoquinone; in the presence of ferrocyanide (II), the quinone formed ferricyanide which was detected by absorbance mesurement at 420 nm; 10 to 40 µg mL-1 of phenol could be determined. For amperometric detection, a carbon paste electrode containing immobilized II was used; mesurement was at -0.2 V vs. Ag - AgCl. This method could determine up to 500 ng mL-1 of phenol, with a detection limit of 18 ng mL-1 for a 30-s contact time.
Phenol Environmental Spectrophotometry Electrode

"L-Malate Determination In Wines And Fruit Juices By Flow Injection Analysis. Adaptation Of A Coupled Dehydrogenase - Transferase System"
Anal. Lett. 1989 Volume 22, Issue 15 Pages 2897-2913
Chemnitius, G.C.;Schmid, R.D.

Abstract: Malate dehydrogenase and aspartate aminotransferase were co-immobilized on controlled-pore glass; the carrier stream (0.1 M K phosphate of pH 7.0) contained NAD+ to generate oxalacetate (I) and NADH from L-malate and the first enzyme, and L-glutamate to trap I with the second enzyme. The NADH was detected fluorimetrically. The rectilinear range was 5 to 100 µM-L-malate, but up to 50 mM L-malate could be determined directly by zone sampling, a flow injection configuration which is described. From 12 to 20 samples could be analyzed per hour. Results agreed with those obtained by an official method.
l-Malate Wine Fruit Fluorescence

"Photometric And Fluorimetric Determination Of Creatine Kinase Activity By Using Co-immobilized Auxiliary Enzymes And An Open - Closed Flow Injection Manifold"
Anal. Lett. 1991 Volume 24, Issue 5 Pages 749-765
M. D. Luque de Castro; J. M. Fern&aacute;ndez-Romero

Abstract: The method involves reaction between creatine phosphate (I) and ADP catalyzed by creatine kinase (II), phosphorylation of D-glucose by the produced ATP catalyzed by hexokinase (III), and oxidation of the resulting D-glucose 6-phosphate by NADP+ catalyzed by glucose-6-phosphate dehydrogenase (IV); the NADPH formed is monitored photometrically at 340 nm or fluorimetrically at 470 nm (excitation at 340 nm). The serum sample is diluted with 0.1 M Tris - acetate buffer of pH 7.0 before injection into a stream of the same buffer, which is then merged with a stream containing ADP and I and passed through a reaction coil. Subsequently, the sample plug is passed repeatedly through an enzyme reactor containing III and IV co-immobilized on controlled-pore glass, the flow-through detector and two reaction coils; the series of peaks of increasing height thus obtained is used to provide fixed-time or reaction-rate measurements of II activity. Optimum values of the reaction variables are tabulated. Typical rectilinear ranges of calibration were 0.01 to 1.00 or 2.00 iu L-1 of II, and recoveries of added II (0.1 or 0.2 iu l-1) were quantitative.
Enzyme, creatine kinase Blood Serum Fluorescence

"Flow Injection Immunosensor For Theophylline"
Anal. Lett. 1993 Volume 26, Issue 7 Pages 1425-1439
Palmer, D.A.;Edmonds, T.E.;Seare, N.J.

Abstract: Glass micro-columns (5 cm x 3 mm) were packed with controlled pore glass-protein A and mounted on an FIA system. Theophylline (I) antisera in PBS (pH 7.4) was injected into a Tris buffer (pH 8.8) carrier stream (0.5 ml/min) and immobilized on the column. A mixture of alkaline labelled I and I standard was similarly loaded onto the reactor column. The label and standard competed for the immobilized antibody. A solution of p-aminophenol was measured amperometrically. The calibration range (not linear) was from 60-300 ng/ml of I. The detection limit was 25 ng/ml of I. Recoveries were 85%. Intra- and inter-day RSD ranged from 8.9-7.7% and 10.4-11.7%, respectively.
Theophylline Immunoassay Amperometry Sensor

"Model Online Flow Injection Fluorescence Immunoassay Using A Protein A Immunoreactor And Lucifer Yellow"
Anal. Lett. 1993 Volume 26, Issue 12 Pages 2543-2553
Palmer, D.A.;Ren, X.Z.;Fernandez Hernando, P.;Miller, J.N.

Abstract: A diagram of the system used is given. Transferrin antiserum (1:49 dilution in 0.15 M PBS of pH 7.4), followed by a mixture of Lucifer yellow VS-labelled transferrin (model analyte; prep. described) and transferrin standard were injected on to the immunoreactor (glass microcolumn of 5 cm x 3 mm packed with controlled pore glass protein A) using Tris buffer of pH 8.8 (0.5 ml/min). After 350 s the flow was changed to citrate buffer (0.5 ml/min) of pH 2.5 to elute the bound complex which was detected fluorimetrically at 530 nm (excitation at 430 nm); the flow was switched back to the Tris buffer after 500 s. The calibration graph was linear from 25 (detection limit) to 600 µg/ml of transferrin. The method was applied to human serum diluted 1:9 or 1:19 with 0.15 M PBS of pH 7.4 with RSD of 4.5 and 3.8%, respectively. The immunoreactor needed replacing after ~60 runs. The method could be applied to different analytes.
Immunoassay Fluorescence

"Flow Injection Determinations Of Creatine And Creatinine Using Packed Bed Enzyme Reactors Under Equilibrium Conditions"
Anal. Lett. 1994 Volume 27, Issue 3 Pages 495-510
Moges, G.;Johansson, G.

Abstract: Plexiglas tube reactors were included in series in a flow injection system for determination of creatinine (I) and creatine (II). The tubes contained immobilized enzymes on CPG-10 controlled-pore glass pre-silanized with aminopropylsilane and activated with glutaraldehyde. The immobilized enzymes were creatinine amidohydrolase (E.C. 3.5.2.10.) in reactor A for determination of I and creatine kinase (E.C. 2.7.3.2), pyruvate kinase (E.C. 2.7.1.40.) and lactate dehydrogenase (E.C. 1.1.1.27.) in reactor B for determination of II, pyruvate and ADP. The flow stream buffer contained 13 mM magnesium acetate, 13 mM potassium acetate, 1.1 mM ATP, 1 mM phosphenol pyruvate and 0.25 mM NADH in 70 mM imidazole acetate of pH 7.8 and detection was at 340 nm. Reactor A was bypassed if only II was determined. Linear calibration graphs were obtained for 5-400 µM-I and -II with detection limits of 6.4 µM-I and 3 µM-II. The RSD was 0.7% for 100 µM of both I and II. Reactor working lives were >5 months for A and 3 months for B.
Creatine Creatinine Biological material Spectrophotometry

"Flow Injection Fluoroimmunoassay For Human Transferrin Using A Protein A Immunoreactor"
Anal. Lett. 1994 Volume 27, Issue 6 Pages 1067-1074
Ren, X.Z.;Miller, J.N.

Abstract: Transferrin (I) was determined in serum by flow injection fluoroimmunoassay using Lucifer Yellow VS as the label, with detection at 525 nm (excitation at 433 nm). A controlled-pore glass micro-column (5 cm x 3 mm i.d.) of PROSEP-A with two injection valves was used. Goat anti-human transferrin (50 µL) was injected into binding buffer (PBS, pH 7.4) through one valve and sample (50 µL) through the other. When washing of tracer was complete, the flow was switched to elution buffer (0.1 M citrate, pH 3) for 6 min, then switched back to binding buffer for 4 min. Flow rates were 0.25 ml/min throughout. The calibration graph was linear for up to 500 µg/ml of I. The RSD (n = 8) for 102 and 208 µg/ml of I were 10.6 and 9.4%, respectively.
Transferrin Blood Serum Immunoassay Fluorescence

"New Chemiluminometric Method For The Determination Of Glycerol In Wine By Flow Injection Analysis With Immobilized Glycerol Dehydrogenase In Combination With NADH Oxidase"
Anal. Lett. 1994 Volume 27, Issue 8 Pages 1489-1505
Kondruweit, S.;Dremel, B.A.A.;Schmid, R.D.

Abstract: Wine (100 µL) and 40 µL of aqueous 5 mM NAD+ were introduced into a stream (0.8 ml/min) of 0.1 M potassium phosphate buffer of pH 9 containing 0.02% sodium azide (buffer A) and, for fluorimetric determination, carried to an enzyme reactor (30°C) containing glycerol dehydrogenase (GDH) immobilized onto controlled-pore glass with glutaraldehyde, where glycerol was converted to dihydroxyacetone; the NADH produced was detected at 460 nm (excitation at 340 nm). The calibration graph for glycerol is shown; no detection limit or RSD are given. For chemiluminometric (CL) determination, the sample first passed to an enzyme reactor (30°C) containing immobilized GDH, and then to a reactor (30°C) containing immobilized NADH oxidase, before merging with pre-mixed streams of 0.8 mM luminol and 40 mM potassium hexacyanoferrate(III), both in buffer A, for detection of H2O2. The calibration graphs for H2O2 and glycerol are shown and the detection limits were 1 nM and 25 µM, respectively; the working range was from 0.1-10 mM glycerol (RSD 2%). For both systems, sample throughput was 40/h.
Glycerol Wine Fluorescence Chemiluminescence

"Determination Of Glutamine In Mammalian Cell Cultures With A Flow Injection Analysis - Wall-jet Electrode System"
Anal. Lett. 1995 Volume 28, Issue 4 Pages 593-603
Huang, Y.L.;Khoo, S.B.;Yap, M.G.S.

Abstract: The cited system (diagram given) incorporated a reactor (2 cm x 5 mm i.d.) containing glutamate dehydrogenase (GDH) immobilized on aminopropyl-derivatized controlled-pore glass beads (AMP-CPG; 120-200 mesh) which was situated upstream of a second reactor (3 cm x 1 mm i.d.) packed with glutamate oxidase and glutaminase co-immobilized on AMP-CPG (immobilization methods given). Cell culture supernatants were pumped at 0.42 ml/min into a carrier stream (1 ml/min) of 0.1 M potassium phosphate buffer of pH 5.5. The glutamine was converted to H2O2 in the second reactor; the GDH reactor eliminated interference from up to 0.08 g/l glutamate. H2O2 was determined at the wall-jet electrode (Gunasingham and Tan, Anal. Chim. Acta, 1990, 234, 321) at +0.65 V vs. Ag/AgCl (using 3 mm Pt disc working and 5 mm vitreous C disc counter electrodes). The calibration graph was linear from 0.01-0.2 g/l glutamine and the RSD was 5.1%. No detection limit is given. Recoveries were 98%. After 70 determinations 70% of the initial reactor activity remained. The results generally agreed well with those obtained by HPLC.
Glutamine Cell Amperometry Electrode Electrode

"A Microdialysis Probe Coupled With A Miniaturized Thermal Glucose Sensor For In Vivo Monitoring"
Anal. Lett. 1995 Volume 28, Issue 13 Pages 2275-2286
Amine, A.;Digua, K.;Xie, B.;Danielsson, B.

Abstract: A miniaturized thermal biosensor was coupled with a microdialysis probe (MP) for subcutaneous glucose (I) monitoring. The thermal biosensor measured the heat evolved during enzyme catalyzed reactions and consisted of a stainless steel column (15 mm x 1.5 mm i.d.) filled with controlled pore glass beads (125-140 µm) onto which glucose oxidase and catalase had been co-immobilized. The temperature signal was measured with a Wheatstone bridge. The biosensor was incorporated into a FIA system equipped with a 1 µL sample loop and operated with 0.1 M sodium phosphate buffer carrier stream (60 µL/min). The MP was fitted with a polycarbonate-polyether co-polymeric membrane (1.6 cm x 0.5 mm i.d., 20 000 Da molecular weight cut-off) and 0.1 M PBS at pH 7.2 was used as the perfusion fluid at 3 µL/min. The thermal sensor exhibited a linear response from 1-50 mM I and a response time of ~e;85 s. In vivo experiments were performed by inserting a 1 cm portion of the MP hollow fiber into the skin of a healthy volunteer who had been orally dosed with I and the levels of I in subcutaneous fluid was measured at 5 min intervals over a period of 180 min after dosage and the results were compared with levels of I in blood measured at 20 min intervals. Subcutaneous concentrations were found to follow the blood concentrations with a time lag of 18 min.
Glucose Subcutaneous Fluid Blood Sensor Thermistor

"Simultaneous Enzymatic Determination Of Glucose And Ascorbic Acid Using Flow Injection Amperometry"
Electroanalysis 1990 Volume 2, Issue 2 Pages 147-154
Wojciech Matuszewski, Marek Trojanowicz *, Liliana Ilcheva

Abstract: Sample solution (10 µL) is injected into a carrier stream of water (2 mL min-1) that subsequently merges with a stream of phosphate buffer solution The mixed stream then divides into two; one passes directly to the amperometric detector, whilst the other passes through a reactor containing glucose oxidase immobilized on controlled-pore glass and then through a delay coil before passing to the detector. The resulting signal consists of two peaks, the first corresponding to oxidation of ascorbic acid (I) and the second to the oxidation of I and enzymatically produced H2O2. Results for synthetic mixtures of I and glucose (up to 20 and 100 mM, respectively) agreed well with expected values. Recoveries of I and glucose added to fresh citrus juice were 100 to 101.8% and 100 to 102.5%, respectively. The sampling rate is 20 h-1
Glucose Ascorbic acid Amperometry

"FIA Analysis With Immobilized Oxidase/peroxidase Enzymes And Fluoride Electrode Detection"
Electroanalysis 1990 Volume 2, Issue 7 Pages 525-531
Wojciech Matuszewski, Marek Trojanowicz*, Mark E. Meyerhoff

Abstract: Details are given of flow injection manifold arrangements in which (a) glucose oxidase (I) and horse-radish peroxidase (II) are co-immobilized on nylon net or a polyester membrane, which is then mounted on a F--selective electrode; (b) the two enzymes are immobilized on controlled-pore glass beads in a single reactor preceding the electrode; or (c) I is immobilized on glass beads in a reactor following the sample injection valve in the carrier stream, and II is so immobilized in a reactor following the merge point of the carrier and reagent streams and preceding the electrode. The sample is a substance that is enzymatically (by I) oxidized to yield H2O2, and the H2O2 then reacts with a fluoriaromatic compound as reagent, catalyzed by II, to liberate F- for detection. Arrangements (b) and (c) showed greater catalytic efficiency and better sensitivity than arrangement (a); glucose could be determined at 0.1 to 10 mM at throughputs of >30 h-1 by using arrangement (b). Either 4-fluorianiline or pentafluoriphenol (5 mM) in acetate buffer of pH 5.5 could be used as reagent; incorporation of 10 or 20 µM-NaF in the reagent stream stabilized the baseline potential. The F- electrode was more selective then amperometric detection systems for H2O2.
Hydrogen peroxide Electrode Amperometry

"NADH Electrochemical Sensor For The Enzymatic Determination Of L- And D-lactate And 3-hydroxybutyrate Using A Flow Injection Analysis"
Electroanalysis 1994 Volume 6, Issue 3 Pages 221-226
G. Marrazza, A. Cagnini, M. Mascini

Abstract: The NADH sensor was prepared from spectroscopic graphite (2 cm x 3 mm) assembled in a wall-jet cell using a FIA procedure and was coupled with enzyme reactors. The reactors were prepared by immobilizing L-, D-lactate or 3-hydroxybutyrate dehydrogenases on to aminopropyl-controlled pore glass beads using glutaraldehyde in a packed-bed enzyme reactor. Measurements were performed at an applied potential of +500 mV vs. Ag/AgCl. 3-hydroxybutyrate and L- and D-lactate were oxidized in the presence of NAD+ and the NADH produced was measured. By the appropriate choice of buffer, pH and NAD+ concentration, the analytes could be measured in the range 1 x 10^-6 to 1 x 10^-4 M in a few seconds. Calibration graphs are shown.
d-Lactate l-Lactate 3-hydroxybutyrate Biological Sensor

"Simultaneous Assays Of Glucose, Urate And Cholesterol In Blood Serum By Amperometric Flow Injection Analysis"
Electroanalysis 1995 Volume 7, Issue 2 Pages 143-146
T. Yao, M. Satomura, T. Nakahara

Abstract: The flow system featured a 16-way switching valve and a parallel configuration of three immobilized enzyme reactors. The enzymes; namely glucose oxidase, uricase and cholesterol oxidase were immobilized on controlled-pore glass packed into glass columns (1.8 cm x 3 mm i.d). Nafion film-coated and Nafion/cellulose acetate-coated Pt electrodes were used to selectively detect the H2O2 generated without any interference from electroactive species like L-ascorbate and proteins present in serum. The FIA system was similar to one described earlier (J. Biotechnol., 190, 14, 115). Serum was injected into two carrier streams of 0.1 M phosphate buffer of pH 7.5 and a stream of 0.1 M phosphate buffer of pH 7.5 containing 0.75% Triton X-100 (1 ml/min) which then passed through the enzyme reactors to the electrode detectors. The electrochemical flow cell had an Ag/AgCl reference electrode and a stainless steel tube as auxiliary electrode. Signal current was linear for 5-700 mg/dl glucose, 1-80 mg/dl uric acid and 5-100 mg/dl cholesterol. The RSD was better than 2.8% for the simultaneous assay of the three species in human control serum. Up to 32 samples per hour could be assayed.
Cholesterol Glucose Uric acid Blood Serum Amperometry Electrode Electrode

"Amperometric Flow Injection Determination Of Citric Acid In Food Using Free Citrate Lyase And Co-immobilized Oxalacetate Decaboxylase And Pyruvate Oxidase"
Electroanalysis 1995 Volume 7, Issue 6 Pages 527-530
Kiyoshi Matsumoto, Tadayuki Tsukatani, Yuko Okajima

Abstract: Controlled-pore glass was refluxed with 6 M HCl for 6 h, dried and silanized with 10% (3-aminopropyl)triethoxysilane solution in toluene at 130°C for 10 h. Oxalacetate decarboxylase and pyruvate oxidase were co-immobilized (Matsumoto et al., Anal. Chem., 1988, 60, 147). This support was packed in to a glass tube (10 cm x 2 mm i.d.) and used in a FIA system with a carrier solution of 0.1 M phosphate buffer of pH 7 containing 10 mM MgCl2/80 µM-thiamine pyrophosphate/ 10 µM-flavine adenine dinucleotide. The sample (50 µL) and citrate lyase solution in phosphate buffer of pH 7 (80 µL) were injected in to the carrier stream (0.85 ml/min) and the mixture was pumped through a reactor containing ascorbate oxidase immobilized on controlled-pore glass to remove L-ascorbate, and then through the prepared reactor. H2O2 was determined amperometrically in a flow-through cell equipped with a Pt working electrode set at +0.6 V vs Ag/AgCl. Calibration graphs were linear from 0.1-1 mM citrate. The detection limit was 0.02 mM; RSD was 1.23% at the 0.5 mMlevel (n=10). An interference study was carried out (details given). The frequency was 15 tests/h. The system was applied to determination of citrate in several fruits; results correlated well with those from a kit enzymatic method.
Citric acid Fruit Amperometry Electrode

"New Approaches To Coupling Flow Injection Analysis And High Performance Liquid Chromatography"
J. Chromatogr. A 1992 Volume 600, Issue 2 Pages 183-188
M. D. Luque de Castro* and M. Valc&aacute;rcel

Abstract: In pre-column arrangements (A), the flow injection system is placed before the LC column, whilst in post-column couplings (B), the chromatographic process takes place before the flow injection analysis. In each instance the downstream unit incorporates the detection module. Examples of the use of A include: the determination of Zn based on its activating effect on metal-free carboxypeptidase A immobilized in a controlled-pore-glass reactor; the coupling of a liquid - liquid extractor to a normal-phase chromatograph for the determination of caffeine in beverages and urine; and the coupling of an ultrasonic leaching cell for solid - liquid extraction processes such as in the determination of B in soil. Only those B systems incorporating two valves are considered to be true HPLC - flow injection analysis configurations. Examples of the use of B include: determination of aflatoxins in foodstuffs, based on enhancing their fluorescence by means of a redox reaction with Br; and the determination of bile acids, based on the production of NADH. A review with 15 references. An overview of the advantages gained in coupling a flow injection manifold to a liquid chromatograph is presented. Improvements in the analytical features arising from this association and the peculiar pre- and post-column arrangements are discussed, as are the promising prospects of arrangements to be developed for avoiding the preliminary steps of the analytical process.
Caffeine Environmental Urine Beverage HPLC

"Sensitive Flow Injection Method With Peroxyoxalate Chemiluminescence Detection Combined With Preparative High Performance Liquid Chromatography For Determination Of Choline-containing Phospholipids In Human Serum"
J. Chromatogr. B 1996 Volume 678, Issue 2 Pages 129-136
Mitsuhiro Wada, Kenichiro Nakashima*, Naotaka Kuroda, Shuzo Akiyama and Kazuhiro Imai

Abstract: Serum samples (5 µL) were diluted to 2 mL with 0.1% aqueous Triton X-100. A 20 µL portion was analyzed for total choline-containing phospholipids (PL) on an immobilized enzyme reactor column (7 cm x 2 mm i.d.) containing phospholipase D and choline oxidase on aminopropyl controlled-pore glass, linked with glutaraldehyde. The carrier solution was 10 mM imidazole buffer of pH 8 containing 0.1% Triton X-100 (1 ml/min) and the post-column chemiluminescent reagent contained 0.4 mM bis-(2,4,6-trichlorophenyl)oxalate and 0.5 µM-2,4,6,8-tetrathiomorpholinopyrimido[5,4,-d] pyrimidine in acetonitrile (1.2 ml/min). Separation of choline-containing PL was performed by adding 100 µL of CHCl3/methanol (1:1) to 5 µL of serum, filtering, then analyzing on a 5 µm Daisopak-SP-120-5-APS aminopropyl column (15 cm x 6.0 mm i.d.) with acetonitrile/methanol/10 mM ammonium phosphate buffer of pH 5.8 (615:264:150) as mobile phase and detection at 205 nm. Fractions were analyzed on the FIA system described above. Calibration graphs were linear for up to 1 nmol PL and the detection limits were 1.3-1.6 pmol; RSD were 1.6-3.3%. RSD for serum were 1.7-7.7%.
Phospholipids Blood Serum HPLC Chemiluminescence

"Enzymatic Flow Injection Method For Determination Of Formate"
J. Pharm. Biomed. Anal. 1990 Volume 8, Issue 8-12 Pages 991-994
F. Ortega*, M. Ballesteros, J. I. Centenera and E. Dom&iacute;nguez

Abstract: Formate dehydrogenase was immobilized on controlled pore glass (CPG-10; pore diameter 51.5 nm, particle size 37-74 µm) after silanization of the glass with 2-aminopropyl triethoxy xilane and activation with glutaraldehyde. The immobilized enzyme was packed into Plexiglass reactors with polypropylene nets at each end. The flow injection apparatus (diagram given) consisted of a pump, an injection valve, the immobilized enzyme unit reactor, a UV detector and a recorder. Sample (50 µL) was injected into a carrier solution (0.5 mL min-1) of 2 mM NAD+ in 0.1 M phosphate buffer (pH 7) and the NADH formed was determined from the absorbance at 340 nm. The optimum temperature was 30°C. Response was rectilinear from 5 to 80 and from 50 to 2000 µM-formate with use of 400- and 50 µL reactors, respectively. The coefficient of variation were 5%. The method was applied in the analysis of commercially available carbohydrate solution for parenteral use.
Formate

"Kinetic Determination Of Aspartate Aminotransferase In Human Serum With A Flow Injection/multidetection System"
J. Pharm. Biomed. Anal. 1991 Volume 9, Issue 9 Pages 679-684
J. M. Fern&aacute;ndez-Romero* and M. D. Luque De Castro

Abstract: Photometric-kinetic methods for the determination of activity of aspartate aminotransferase are proposed. The flow injection manifold used for this purpose includes a selecting valve which allows the sample to be trapped in a closed circuit where a solid reactor housing an auxiliary enzyme and a conventional single detector allows a multipeak recording to be obtained for each sample. This record represents a typical kinetic curve from which much information can be obtained to develop fixed-time and reaction-rate methods for the determination of the analyte based on its catalytic action on the L- aspartic acid-2 oxoglutarate system. The linear range is found to be between 1 and 500 U l-1, with relative standard deviation less than 1%. The utility of the methods is illustrated by the determination of the analyte in human serum from healthy and sick individuals. Sample was injected into a stream of 0.1 M Tris - HNO3 buffer of pH 7.5 which merged successively with 6 mM 2-oxoglutarate in Tris buffer and 200 mM aspartic acid and 1 mM NADH also in Tris buffer. The mixture passed through the manifold (illustrated) to a selecting valve which trapped the reacting plug in a closed circuit. The plug passed through an enzymatic reactor (5 cm long) containing malate dehydrogenase immobilized on controlled-pore glass and the decrease in absorbance was monitored at 340 nm. The calibration graph was rectilinear for 1 to 500 U L-1 of aspartate aminotransferase; coefficient of variation was 1% for 30 U l-1.
Enzyme, aspartate aminotransferase Serum Human

"An Online Immunoassay Method For Theophylline Using A Protein A Immunoreactor"
J. Pharm. Biomed. Anal. 1991 Volume 9, Issue 10-12 Pages 1121-1123
Pilar Fernandez-Hernando and James N. Miller

Abstract: A heterogeneous fluorescence immunoassay for theophylline has been automated using a flow injection analysis system containing a protein A solid phase reactor to separate antibody-bound and unbound fluorescein-theophylline. For each sample the antibody-protein A reaction takes place at near neutral pH, and the complexes are eluted at acid pH. The antibody-binding capacity of the reaction greatly exceeds the antibody level in each sample incubation mixture, and a single reactor can be repeatedly cycled between neutral and acid pHs. Experimental variations such as reactor size, flow rate, pH values, and reactant concentrations have been studied. Theophylline could readily be determined at the µg mL-1 level with online incubation with antibodies. Sheep anti-theophylline antiserum, serum sample and fluorescein isothiocyanate-theophylline were injected into a carrier stream of 50 mM Tris - HCl buffer of pH 8.8 containing 0.1 M NaCl. The mixture passed through a column (5 cm x 3 mm) of protein A immobilized on controlled pore glass particles; elution was effected by the injection of 0.5 M citrate buffer of pH 3.5 containing 0.1 M NaCl. The eluate was detected fluorimetrically at 525 nm (excitation at 495 nm). The flow rate was 0.2 mL min-1 throughout. Recoveries of theophylline were 80.5 to 94.8% and the within- and between-day coefficient of variation (n = 10) were both 7.9%.
Theophylline Fluorescence Immunoassay

"Flow Injection Immunoassay Using A Protein A Immunoreactor"
J. Pharm. Biomed. Anal. 1991 Volume 9, Issue 10-12 Pages 1115-1120
J. N. Miller, D. A. Palmer and M. T. French

Abstract: Competitive immunoassay have been developed for the determination of cyclosporin A (I) and theophylline (II) using microcolumns of protein A immobilized on controlled pore glass and flow injection techniques. For cyclosporin, the assay was based on a monoclonal antibody with fluorimetric detection; for theophylline, sheep antiserum and electrochemical detection was used. The limits of detection were 9 ng mL-1 of I and 2.5 µg mL-1 of II.
Cyclosporin A Theophylline Electrochemical analysis Fluorescence Immunoassay

"Selective Catalytic Detection Of Dopamine"
J. Pharm. Biomed. Anal. 1996 Volume 14, Issue 8-10 Pages 1157-1162
Fidel Ortega and Elena Dom&iacute;nguez*

Abstract: The FIA system for the determination of dopamine consisted of an enzyme reactor column (3.2 cm x 1 mm i.d.) packed with tyrosinase immobilized onto controlled-pore glass beads (51.5 nm pore diameter, 37-74 µm particle size) and a spectrophotometric detector at 475 nm in parallel with a electrochemical detector at -50 mV vs. SCE. Samples of 25 µL were injected into a carrier stream (0.9 ml/min) of 0.1 M phosphate buffer of pH 7. The linear calibration ranges, detection limits and RSD (n = 10) at the 0.5 mM level were 0.005-1 mM, 3.5 µM and 1.7%, respectively, for electrochemical detection and 0.05-0.75 mM, 8.4 µM and 0.8%, respectively, for photometric detection. The FIA system can by used for the post-column detection of dopamine in LC.
Dopamine Amperometry Spectrophotometry

"Amperometric Biosensing Of Copper(II) Ions With An Immobilized Apoenzyme Reactor"
Sens. Actuat. B 1990 Volume 1, Issue 1-6 Pages 499-503
Ikuo Satoh*, Teruo Kasahara and Naoto Goi

Abstract: An amperometric flow injection system, incorporating an immobilized-apoenzyme reactor for specific recognition of Cu, is presented (diagram given). Copper ions were selectively determined in the range 0.1 to 10 mM by activation of immobilized metal-free galactose oxidase (I). The I was immobilized on alkylamino controlled pore glass beads (120 to 200 mesh; pore diameter 51.5 nm) and the preparation was loaded into a plastic column which was mounted in a water-jacketed holder. A polarographic O electrode, or a cellulose-acetate-membrane covered Pt - Ag - AgCl electrode pair, housed in a plastic flow-through cell was connected to the outlet of the enzyme reactor to monitor the amount of O consumed, or H2O2 generated, by the activated enzyme. A constant voltage of -0.7 V vs. Ag - AgCl for O, or 0.7 V vs. Ag - AgCl for H2O2, was applied to the Pt electrode. The carrier solution (1 mL min-1) was 0.05 M phosphate buffer (pH 6) containing 0.1 M KCl. D-Galactose solution was added as substrate solution and 10 mM N,N-diethyldithiocarbamate solution (pH 8) was used to regenerate the reactor.
Copper(II) Amperometry Electrode Sensor

"Fast Determination Of Whole Blood Glucose With A Calorimetric Micro-biosensor"
Sens. Actuat. B 1993 Volume 15, Issue 1-3 Pages 141-144
Bin Xie, Ulrika Hedberg, Michael Mecklenburg and Bengt Danielsson

Abstract: A calorimetric flow injection sensor was used to provide rapid determination of whole-blood glucose without pre-treatment. Glucose oxidase was immobilized together with catalase on controlled-pore glass beads in a micro-column (1.5 cm x 0.6 mm i.d.). When the sample was reduced in volume from 20 to 1 µL the linear range for glucose was increased from 0.5-2.5 mM to 0.5-20 mM. Results correlated well with those obtained with a Reflolux-S blood glucose analyzer.. The RSD (n = 100) was 3.7%. The sensor could be used for at least one month if stored at 4°C. The measurement period was 40 s and the device could be used to analyze 90 samples per hour.
Glucose Whole Calorimetry Sensor

"A Comparison Of Preconcentration Reagents For Flow Injection Analysis Flame Atomic Spectrometry"
Spectrochim. Acta B 1996 Volume 51, Issue 14 Pages 1909-1915
G. M. Greenway*, S. M. Nelms, I. Skhosana and S. J. L. Dolman

Abstract: The chelating resins Chelamine and Chelosolve (Fluka), the immobilized-iminodiacetate controlled-pore glass PROSEP Chelating-1 (Bioprocessing Ltd., Consett, UK) and an in-house-prepared immobilized-quinolin-8-ol controlled-pore glass (CPG-8-HQ) were evaluated for rapid online pre-concentration and matrix separation in the flow injection AAS determination of Cd, Cu(II), Mn(II), Ni(II) and Zn in a flow injection manifold designed for rapid matrix separation. With all four materials the matrix-matching of standards for determining the cited heavy metals in seawater was unnecessary. The most suitable material was PROSEP Chelating-1, which, however, showed inferior selectivity to CPG-8-HQ.
Cadmium Sea Spectrophotometry

"Flow Injection Analysis For Hydrogen Peroxide By Using Immobilized Horseradish Peroxidase And Its Fluorogenic Substrate 3-(p-hydroxyphenyl) Propionic Acid"
Anal. Sci. 1985 Volume 1, Issue 1 Pages 65-68
Yohji HAYASHI, Kiyoshi ZAITSU and Yosuke OHKURA

Abstract: The sample solution (20 µL) was injected into a carrier stream (0.5 mL min-1) of water that then merged with a stream (0.5 mL min-1) of 10 mM 3-(4-hydroxyphenyl)propionic acid in 0.2 M Tris - HCl buffer that was 0.3 M in NaCl (pH 8.5). The combined streams passed through a mixing coil and a PTFE tube (8 cm x 0.5 mm) packed with horse-radish peroxidase immobilized on controlled-pore glass beads (prep. described), both at 37°C, and then through a fluorescence detector (excitation at 305 nm, emission at 405 nm), the peak height being recorded. The best-shaped peaks were obtained with flow injection tubing of 0.25 mm i.d. The calibration graph was rectilinear up to 1 nmol of H2O2 and slightly curvilinear up to 1.5 nmol, and the limit of detection was 0.5 pmol. The coefficient of variation (n = 10) for 1, 10 and 100 pmol and 1 nmol of H2O2 were 3.6, 0.68, 0.35 and 0.24%, respectively, and the sampling rate was up to 120 h-1.
Hydrogen peroxide Fluorescence

"Amperometric Flow Injection Biosensor System For The Simultaneous Determination Of Urea And Creatinine"
Anal. Sci. 1992 Volume 8, Issue 6 Pages 845-850
C.-S. RUI, K. SONOMOTO and Y. KATO

Abstract: A single-channel and a dual-channel flow injection system are described and illustrated. The method involved the urease-catalyzed hydrolysis of urea or the creatinine deiminase-catalyzed hydolysis of creatinine to yield NH3, and amperometric detection of the NH3 via coupled enzymatic reactions in which (i) 2-oxoglutarate was reduced to L-glutamate and H2O2 in the presence of NH4+, NAD(P)H and glutamate dehydrogenase, and (ii) the L-glutamate was re-oxidized in the presence of glucose oxidase; the consumption of O was detected with an O electrode. Each of the enzymes was immobilized on modified controlled-pore glass and packed into a glass tube for use in the flow systems. With use of the dual-channel system to determine urea and creatinine simultaneously, the response was rectilinear from 0.1 to 5 mM analyte, and the coefficient of variation (n = 12) were 5%. A flow injection biosensor system was developed for the simultaneous assay of urea and creatinine, with a single injection and one detector. The amperometric detection of urea or creatinine was based on coupled reactions of three sequentially aligned enzyme reactors, urease or creatine deiminase, glutamate dehydrogenase and glutamate oxidase. Ammonia produced by the enzymatic hydrolysis of urea or creatinine was converted to glutamate, and the oxygen consumption due to the oxidation of glutamate by glutamate oxidase was detected with an oxygen electrode. A split and confluence of the flow stream between the injector and the glutamate dehydrogenase reactor resulted in a two-channel system. Double-peak recording was achieved by putting a delay coil at one of the two channels. The system gave linear calibration curves over a range of 0.1-5.0 mM for both urea and creatinine. The assay procedure is simple and one run can be completed within 3 min. The system was reproducible within 5% of the relative standard deviation.
Urea Creatinine Amperometry Sensor

"Applications Of Immobilized Enzymes In Flow Injection Analysis"
Anal. Proc. 1985 Volume 22, Issue 1 Pages 6-8
Stephen, M. Masoom, Alan Townshend

Abstract: Samples of blood serum and soft drinks were injected into a stream of phosphate buffer solution (pH 7) which then passed through a glass column (2.5 cm x 2.5 mm) containing glucose oxidase (immobilized on controlled-pore glass) to a flow-through amperometric detector for the determination of H2O2. Sucrose in soft drinks was determined by use of immobilized β-D-fructofuranosidase, aldose 1-epimerase and glucose oxidase. The limit of detection was 1 µM-H2O2.
Glucose Sucrose Beverage Blood Serum Amperometry

"ISFET-based Enzyme Sensors For Urea: Enzyme-modified ISFET And Column-immobilized Enzyme Flow Injection Analysis"
Anal. Proc. 1989 Volume 26, Issue 5 Pages 154-156
M. J. Eddowes, G. K. Chandler and J. R. Dodgson

Abstract: Enzyme-modified ISFET were prepared by immobilizing urease in either poly-(2-hydroxyethyl methacrylate) hydrogel or in bovine serum albumin - glutaraldehyde cross-linked gel, and placing the gel in contact with matched ISFET - metal oxide semiconductor FET pairs having Ta2O5 pH membranes. A flow injection system was constructed of a column containing urease immobilized on controlled-pore glass and two Ta2O5-membrane ISFET - metal oxide semiconductor FET pairs, one placed before and one after the enzyme column. The gel-immobilized-enzyme ISFET were problematic, but with use of the flow injection system, with 2 mM phosphate buffer (pH ~6.86) as carrier stream, the response time was ~30 s and the calibration graph was rectilinear up to 25 mM urea (30 µL injections).
Urea Field effect transistor Sensor

"Flow Injection Immunoassay For Theophylline Using A Protein A Immunoreactor"
Anal. Proc. 1992 Volume 29, Issue 3 Pages 98-99
Derek A. Palmer, Tony E. Edmonds, Nichola J. Seare

Abstract: Alkaline phosphatase-labelled theophylline (50 µL) and sample in Tris buffer was incubated with anti-theophylline antibody in phosphate-buffered saline (50 µL) for 10 min. The mixture was injected into a controlled pore glass reactor containing protein A with use of Tris buffer at 0.4 mL min-1. After 20 s the reactor was washed, 1 M p-aminophenyl phosphate (substrate solution) was applied to the reactor at 0.6 mL min-1 and the product of the enzyme reaction was measured downstream by an electrochemical cell in a wall jet configuration. The calibration graph was rectilinear up to 500 ng mL-1 of theophylline. Analysis time was 18 minutes; the amino reactor was regenerated by washing with citrate buffer solution for 2 min.
Theophylline Immunoassay Electrode

"Very Near-infrared Dyes As Labels In Immunoassays"
Anal. Proc. 1993 Volume 30, Issue 3 Pages 144-145
Derek A. Palmer, James N. Miller

Abstract: Fluorescence emission and absorption max. are reported for Nile blue (C. I. Basic Blue 12), toluidine blue (C. I. Basic Blue 17), methylene blue (C. I. Basic Blue 9), oxazine 750, oxazine 4, Azure A, Azure B and cresyl violet, each of which was subjected to flow injection analysis with Tris or borate buffer solution of pH 9.0 or phosphate-buffered saline (PBS; pH 7.4) as carrier, both with and without an immunoreactor containing controlled pore glass and Sepharose. The best flow injection profile was obtained by using PBS containing 0.1% of Triton X-100 in conjunction with a Sepharose immunoreactor. The theophylline - Nile blue conjugate was prepared from theophylline-8-butyric acid lactam, and the conjugate and the free dye were subjected to TLC with butanol - acetic acid -H2O (4:1:3) as mobile phase; the respective RF values were 0.71 and 0.64. Incorporation of the conjugate into a flow injection immunoassay using Sepharose protein A is being investigated.
Immunoassay Fluorescence

"Online Immobilized Chelating Agents In Flow Injection Systems"
Anal. Proc. 1993 Volume 30, Issue 11 Pages 438-440
G. M. Greenway, A. Townshend

Abstract: The chelating agents quinolin-8-ol-5-sulfonic acid, dithiocarbamate, dithizone, cysteine and Selenestrum capricornulum were immobilized on to controlled-pore glass particles and packed into columns (4 cm x 2 mm). These columns were incorporated into a flow injection system and sample (5 or 10 ml) was passed through the column. Accumulated metal ions were eluted with 100 µL of dilute HNO3 or HCl, and determined in the eluate by AAS. The flow injection system has a throughput of 20 samples/h. Detection limits and uptake capacities of the chelating agents for Cd, Co, Cu, Hg, Pb and Zn are tabulated.
Metals Cadmium Copper Cobalt Mercury Lead Zinc Spectrophotometry

"Use Of Protein A In A Liposome-enhanced Flow Injection Immunoassay"
Anal. Proc. 1994 Volume 31, Issue 11 Pages 339-340
Geoffrey S. Rule, Derek A. Palmer, Stuart G. Reeves and Richard A. Durst

Abstract: A glass column (10 cm x 6 mm i.d.) containing controlled-pore glass coated with protein A was used in the cited immunoassay for alachlor (I). The column was activated by injecting anti-I antibody (raised in rabbits) onto the protein A matrix. Alachlor (1 mg/ml in methanol) was diluted with TBS of pH 7.4 and injected onto the column. Liposomes (100 mM sulforhodamine B in TBS encapsulated in lumen) were then injected onto the column. A detergent solution (octyl β-L-glucopyranoside) was then passed through the column. The fluorescence generated by the released sulforhodamine B was measured at 572 nm (excitation at 556 nm). The antibody was then removed with 20% acetic acid (pH 2.2) and the column reconditioned for the next analysis by returning the mobile phase to pH 7.4 TBS. Assays were conducted at a flow rate of 0.8 ml/min. Each analysis took 11 min. The detection limit was 10 ng of I injected.
Alachlor Biological Immunoassay Fluorescence

"Studies On Flow Injection Chemiluminescence Immunoassay. 2. Hetermination Of HRP And Its Conjugates With Coupled Reaction"
Acta Chim. Sin. 1997 Volume 55, Issue 6 Pages 590-594
Shao Qian; Ma Wangbai; Feng Manliang; Zhang Zhujun

Abstract: In this paper chitoson, controlled pore glass and silica - gel have been investigated with respect to its potentiality of being used as solid support of flow - injection immunoassay. A new flow - injection immunoassay end - point detection method has also been developed on the basis of coupling the reaction of H2O2 and K4Fe(CN)(6), catalyzed by HRP, with H2O2 and K3Fe(CN)(6) co - oxidized chemiluminescence reaction of luminol. As enzymatic and chemiluminescent reaction were generated at different sites of the detection system. The general shortcomings of such as unable to select a optimum pH for both enzymatic and chemiluminescent reaction, incomplete contact between enzyme and substrate, and scattering of support have been eliminated It has the advantages of high sensitivity and good accuracy. HRP and its conjugates at f mol level can be detected within 1 similar to 2 min. and the relative standard deviation is 3.9%. 2 References
Enzyme, horseradish peroxidase Chemiluminescence Immunoassay

"New Fluorescence Immunoassay For Adrenocorticotropic Hormone Determination Using Flow Injection Analysis"
An. Quim. 1996 Volume 92, Issue 1 Pages 37-40
Martinesteban, A.;Fernandez, P.;Perez Conde, C.;Gutierrez, A.M.;Camara, C.

Abstract: A new competitive fluorescence immunoassay for adrenocorticotropic hormone (ACTH) is described, using online flow injection and protein A immobilized on controlled pore glass packed-bed reactor. The preparation of Lucifer Yellow VS labelled ACTH is described. Variables such as binding/elution flow rates, binding/elution pH effect and the antibody dilutions were optimized. The proposed method allows a simple method to monitore ACTH levels between 0.2-10 mg L-1.
Adrenaline Hormone, adrenocorticotropic Fluorescence Immunoassay

"Using Porous Glass In Enzyme Immobilization"
Bioforum 1998 Volume 21, Issue 3 Pages 108-109
Janasek, D.;Spohn, U.

Abstract: Porous glass (TRISOPERL) was used for enzyme immobilization in enzyme reactors in a flow injection analysis system for the detection of creatine, creatinine, uric acid, and NH4+. Creatine and creatinine were determined using immobilized creatininase and sarcosine oxidase from Pseudomonas sp. and immobilized creatinase from Actinobacillus. Uric acid was determined with immobilized uricase from Arthrobacter globiformis. NH4+ was determined with immobilized glutamate dehydrogenase from liver or immobilized glutamate oxidase from Streptomyces sp. The detection limits were 5 x 10^-5 M for creatinine, and 1-5 x 10^-6 M for the other analytes. The detection range was adequate for clinical purposes.
Uric acid Creatine Creatinine Ammonium

"Porous Glass As Carrier In Enzyme Immobilization For Flow Injection Analysis"
Bioforum Int. 1998 Volume 2, Issue 1 Pages 38-39
Janasek, D.;Spohn, U.

Abstract: This article describes the use of controlled pore glass Trisoperl for the covalent immobilization of hydrolases, dehydrolases and oxidases.

"Evaluation Of Polypyrrole - Glucose Oxidase Electrodes In Flow Injection Systems For Sucrose Determination"
Biosens. Bioelectron. 1991 Volume 6, Issue 3 Pages 263-273
Wolfgang Schuhmann and Ruth Kittsteiner-Eberle

Abstract: Thin films of polypyrrole were deposited on Pt or vitreous-carbon disc electrodes (1 or 3 mm diameter) by electropolymerization of the monomer. The polymer film was nitrated by treatment with acetic anhydride and Cu(NO3)2 and the nitro-groups were reduced electrochemically to amino-groups. The glucose oxidase was immobilized on the electrode surface via the formation of amides or secondary amines (full details given). The electrodes were operated at a working potential of +600 mV (vs. the SCE), and in the flow injection mode, with 0.5 M NaCl - 0.1 M phosphate (pH 7.4) as supporting electrolyte, responses were rapid (~10 s) and rectilinear for 0.05 to 10 mM glucose. The response fell to 60 to 80% of its initial value after 2 days, but then remained fairly constant for a further 20 to 23 days. Attempts to determine simultaneously glucose and sucrose in the same solution, by use of two electrochemical sensors, in combination with immobilized β-fructofuranosidase (I) and aldose 1-epimerase (II), were unsuccessful owing to inhibition of glucose oxidase by the relatively high concentration. of sucrose. However simultaneous determination of glucose and sucrose was possible in a flow injection system containing enzyme columns with glucose dehydrogenase, I and II immobilized on controlled-pore glass, with fluorimetric determination of enzymatically generated NADH.
Glucose Sucrose Electrochemical analysis Electrode Electrode Electrode Electrode Fluorescence

"Online Determination Of Glucose And Lactate Concentrations In Animal Cell Culture Based On Fibre Optic Detection Of Oxygen In Flow Injection Analysis"
Biosens. Bioelectron. 1992 Volume 7, Issue 2 Pages 133-139
B. A. A. Dremel, S. -Y. Li and R. D. Schmid

Abstract: A flow injection (FIA) system based on fiber-optic detection of O consumption using immobilized glucose oxidase and lactate oxidase (Boehringer, Mannheim, Germany) from Pediococcus sp. is described. The enzymes were immobilized on controlled-pore glass in enzyme reactors linked to a specially designed fiber-optic flow-through cell covering the optrode. The enzyme reactors were stable for >1 month in continuous operation and it was possible to analyze 20 samples h-1. The system was applied to the online monitoring of glucose (I) and lactate (II) concentration. of an animal cell culture designed for the production of recombinant antithrombine III. The calibration graphs were rectilinear for 0 to 30 mM I or II and the coefficient of variation (n = 5) were 5%. A flow injection analysis (FIA) system based on fiber optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the online monitoring of glucose and alctate concentrations. in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by mol. oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fiber optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an RSD of 5% at 30 mM (5 measurements) and for 0-30 mM lactate, with an RSD of 5% at 30 mM (5 measurements). The enzyme reactors used were stable for more than 4 wk in continuous operation, and it was possible to analyze up to 20 samples per h. The system has been successfully applied to the online monitoring of glucose and lactate concentrations. of an animal cell culture designed for the prodn. of recombinant human antithrombin III (AT-III). Results of the online measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyzer from Yellow Springs Instrument Company (YSI).
Glucose Lactate Fermentation broth Fluorescence Optrode

"An Amperometric Flow Injection Analysis Enzyme Sensor For Sucrose Using A Tetracyanoquinodimethane Modified Graphite-paste Electrode"
Biosens. Bioelectron. 1996 Volume 11, Issue 8 Pages 719-723
J. L. Lima Filho, P. C. Pandey*, H. H. Weetall

Abstract: FIA systems for the determination of sucrose are described in which (i) invertase and µarotase were immobilized on to controlled pore glass beads in an enzyme reactor and the C-paste detection electrode was modified with glucose oxidase (I) and tetracyanoquinodimethane (II) as electron transfer mediator or (ii) invertase was incorporated into the C-paste electrode together with I and II (an enzyme reactor was not used). Both systems were operated with 0.1 M phosphate buffer of pH 7 as the carrier stream (30 ml/h) and a 250 µL sample loop. The amperometric response was measured at 200 mV vs. Ag/AgCl. The detection range was 0.025-200 mM for i and up to 2 M for ii.
Sucrose Amperometry Electrode Electrode Sensor

"Phosphate Sensing System Using Pyruvate Oxidase And Chemiluminescence Detection"
Biosens. Bioelectron. 1996 Volume 11, Issue 10 Pages 959-965
Kazunori Ikebukuro, Hideaki Wakamura, Isao Karube*, Izumi Kubo, Masako Inagawa, Takako Sugawara, Yoshiko Arikawa, Masayasu Suzuki, Toshifumi Takeuchi

Abstract: Pyruvate oxidase (POP) was immobilized on to aminoalkylated controlled pore glass (80-120 mesh; 500 .angstrom. pore size) by cross-linking with gluteraldehyde. The POP column formed part of a system consisting of two pumps, a sample injector, a mixing joint and a luminometer. The pyruvate and thiamine pyrophosphate chloride (cofactor solution) were pumped on to the POP column along with a phosphate-containing sample solution. The H2O2 generated by the reaction of phosphate and POP was mixed with luminol/10 µm-4-iodophenol/2.8 iu/ml horseradish peroxidase in 0.2 M sodium carbonate buffer of pH 9.5 or Tri- hydrochloride buffer of pH 8.5 (chemiluminescence reagent) which was delivered by the other pump. The resulting luminescence was detected with a photomultiplier tube. Calibration graphs were linear from 4.8-160 µM-phosphate; one measurement took ~3 min.
Phosphate Sensor Chemiluminescence

"Flow Injection Spectrophotometric And Amperometric Determinations Of Ammonia With Glutamate Dehydrogenase Reactor"
Bull. Chem. Soc. Eth. 1993 Volume 7, Issue 2 Pages 99-112
Ghirma Moges, Theodros Solomon, Gillis Johansson

Abstract: A flow injection method for indirect spectrophotometric and amperometric determinations of ammonia and ammonium, based on a 100 µL-glutamate dehydrogenase (GIDH) reactor, is described. GIDH was immobilized on controlled pore glass after silanization and glutaraldehyde activation. Injections of 50 µL standard ammonium chloride solutions to the carrier (water) produced peaks proportional to 3-1400 mM ammonia with a detection limit of 3 mM. The reagent stream delivered 0.2-0.6 mM NAD (NADH), 3.0 mM a-ketoglutarate and 4.0 mM adenosine-5'-diphosphate (ADP, activator) in 0.1 M Tris-acetate or phosphate buffer. Monitoring the decreasing NADH concentration after the enzymatic reaction produced peaks which formed the basis for measuring ammonia. Detection was made with a flow-through spectrophotometer (at 340 nm) or amperometric detector with a wall-jet phenoxazine-modified graphite electrode, held at 50 mV vs SCE (SCE). PH optima were 7.7-8.5 for the optical detector and 7.5-7.9 for the electrochemical detector. The system was applied to the determination of ammonium salt in commercial crude tryptophanase. The reactor was stable only for five days when used with Tris (pH 8.0-8.5) and stored in 0.1 M phosphate buffer (pH 7.0) at 4°C between uses but stability improved when potassium phosphate buffer (pH 7.8) was the reagent carrier and the storage buffer contained 10 mM ammonium chloride.
Ammonia Commercial product Amperometry Spectrophotometry

"Fundamental Consideration About The Feature Of Matrixes Of Immobilized Enzymes For Reaction Detector"
Bunseki Kagaku 1982 Volume 31, Issue 2 Pages 77-82
Sigeru YOSHIDA, Toshio CHO, Shingo HIROSE

Abstract: Four kinds of the immobilized matrixes (Nylon 66 tube, Nylon 6 polymerized in glass capillary, whisker-walled glass capillary and controlled pore glass beads) were used for the fundamental consideration of the application to reaction detector and discussed about their characteristics. Triethyl oxonium tetrafluoroborate was used for O-alkylation of nylon tube and nylon was further reacted with 1, 6-diaminohexane as spacer. Glucose oxidase (GOD) and cholesterol oxidase (COD) were immobilized on the nylon through bismidate I. (Nylon 66, inner diameter φ: 0.2 mm, immobilized enzyme: GOD, activity: 0.53 µmol min-1 m-1), (Nylon 66, φ : 0.2 mm, COD, 0.43 µmol min-1 m-1), (Nylon 6 polymerized in glass capillary, φ: 0.7 mm, GOD, 1.68 µmol min-1 m-1). Whisker-walled glass capillary which was made by fluoroether was reacted with dimethyl dichloro silane. COD was immobilized on it through ethylenediamine and glutalaldehyde. (Whisker-walled glass capillary, φ : 0.26 mm, COD, 3.37 µmol min-1 m-1), (glass beads, GOD, 40.5 µmol min-1 g-1), (glass beads, COD, 31.9 µmol min-1 g-1). The activities of these immobilized enzyme were measured by 4-aminoantipyrinephenol method in the presence of peroxidase. As the results, nylon tube supported enzyme had lower activity compared with other glass matrixes, but it was easier to handle than glass capillary column. Whisker-walled glass capillary column was a good matrix for immobilized enzyme as well as controlled pore glass beads because of the large surface area and of the high enzyme activity. A glass capillary (40 cm) on which enzyme was immobilized showed reduced band broadening compared with 10 cm bed reactor of glass beads packed Teflon tube (φ: 1.5 mm).
Glucose Cholesterol

"Determination Of L-glutamate Using Flow Injection Analysis With Immobilized L-glutamate Oxidase Reactor"
Shengwu Gongcheng Xuebao 1994 Volume 10, Issue 4 Pages 351-355
Li Qingshan Ye Bangce Zhang Siliang Yu Juntang

Abstract: 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°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°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.
l-Glutamate Fermentation broth

"Analysis Of Aldehydes By Micro High Performance Liquid Chromatography With Post-column Derivatization On Enzyme-immobilized Glass Beads"
Chromatographia 1988 Volume 25, Issue 6 Pages 507-510
T. Takeuchi, D. Ishii and A. Nakanishi

Abstract: Aldehydes in alcoholic beverages were determined by micro-column HPLC with post-column derivatization on enzyme-immobilized glass beads and fluorimetric detection. Various ODS stationary phases, e.g., LiChrosorb RP-18, SC-01, ODS-Hypersil and LS-222, were evaluated, and the effects of mobile phase pH and composition were studied. Post-column reaction was achieved with aldehyde dehydrogenase immobilized on to Aminopropyl-CPG (200 to 400 mesh), and detection was at 470 nm (excitation at 365 nm). Calibration graphs were rectilinear up to 200 ppm for acetaldehyde and 2-furaldehyde, and the detection limit for acetaldehyde was 2.5 ppm.
Aldehydes Beverage HPLC Fluorescence

"Urea And Lactate Determined In 1-mu-L Whole-blood Samples With A Miniaturized Thermal Biosensor"
Clin. Chem. 1994 Volume 40, Issue 12 Pages 2282-2287
Bin Xie,' Ulrika Harborn, Michael Mecklenburg, and Bengt Danielsson

Abstract: A miniaturized flow injection thermal biosensor was developed for the determination of urea and L-lactate in undiluted blood in 1 µL samples. The sensor employed a small enzyme column constructed of stainless steel tubing and microbead thermistors. Urease and lactate oxidase/catalase were separately immobilized onto controlled-pore glass beads, which, in turn, were charged into the enzyme column. With a flow rate of 70 µL/min, linear analytical ranges from 0.2 to at least 50 mmol/L and 0.2 to 14 mmol/L were obtained for urea and lactate, respectively. The relative standard deviations (CVs) for measurements of analyte in buffer were 0.91% for urea and 1.84% for lactate. For urea in whole blood, the CV for 50 determinations was 4.1%. Contrived samples containing various concentrations of urea and L-lactate in whole blood were determined with this sensor and with a spectrophotometric method. Comparisons of the results gave correlation coefficients of 0.989 and 0.984 for 30 blood urea and 30 blood lactate assays in concentrations ranging from 4 to 20.9 mmol/L and from 1.7 to 12.7 mmol/L, respectively.
Urea Lactate Whole Sensor Thermistor Clinical analysis

"Continuous-flow Assay With Immobilized Enzymes For Determining Of Inorganic Phosphate In Serum"
Clin. Chem. 1995 Volume 41, Issue 1 Pages 99-102
MD Luque de Castro, R Quiles, JM Fernandez-Romero and E Fernandez

Abstract: A flow injection method for the determination of phosphate in serum based on its effect on the enzyme activity of purine-nucleoside phosphorylase (I) is presented. I was immobilized on controlled-pore glass (CPG 120-200 mesh; Electronucleonics, Fairfield, MA, USA). Serum sample (100 µL) diluted to 25 mL with 100 mM Tris hydrochloride, pH 8.5 (reagent A) was injected into the flow injection system (diagram given) which merged with 4.75 mM inosine in reagent A. A second enzyme reaction then took place with immobilized xanthine oxidase in 1 mM ammonium sulfate and 0.5 mM sodium salicylate in 100 mM Tris hydrochloride, pH 7. The main stream was then mixed with 10 mM p-hydroxyphenylacetic acid (II) and 8 U/l peroxidase, which reacted and catalyzed the H2O2 produced in the previous step. The resultant product, the dimer of II, was monitored fluorimetrically at 415 nm (excitation at 325 nm). The calibration graph was linear from 0.1-20 µM. The within- and between-run RSD were 2.1 and 3.4%, respectively. Analytes commonly present in serum did not interfere. An automated method for the determination of inorganic phosphate based on flow injection analysis and on the use of immobilized enzymes is reported. The method features a linear range between 0.1 and 20 µmol/L with a CV < 2.1% and 3.4% for the within-run and between-run studies, respectively, and a sampling throughput of 40 h-1. The sensitivity of the method makes a 1:250 dilution of the serum samples feasible, thus making undetectable the interferences from analytes commonly present in serum. The method shows an excellent correlation with conventional automated analyzers based on the same enzymatic reaction (Hitachi, r = 0.988) but with the catalyst in solution, and with the Kodak Ektachem method (r = 0.974) based on the use of dry reagents and formation of the phosphomolybdo heteropolyacid.
Phosphate Blood Serum Fluorescence Clinical analysis

"Determination Of Total Cholesterol In Serum By Flow Injection Analysis With Immobilized Enzymes"
Clin. Chim. Acta 1987 Volume 167, Issue 1 Pages 97-104
Juan M. Fern&aacute;ndez-Romero*, M. D. Luque de Castro and Miguel Valc&aacute;rcel

Abstract: The methods described previously (J. Pharm. Biomed. Anal., 1987, 5, 333) for the determination of cholesterol(I) in serum by normal or stopped-flow flow injection analysis with fluorimetric or photometric detection and with use of soluble enzymes, have been modified for use with immobilized enzymes. Cholesterol esterase and oxidase were immobilized as described by Masoon and Townshend (Anal. Chim. Acta, 1984, 166, 111) for glucose oxidase. Calibration graphs were rectilinear from 26 to 776 µM-I (both photometric methods), 5 to 265 µM-I (normal-flow fluorimetric) and 5 to 155 µM-I (stopped-flow fluorimetric). The coefficient of variation were 1.66, 0.55, 2.48 and 2.23% for the normal-flow photometric, stopped-flow photometric, normal-flow fluorimetric and stopped-flow fluorimetric methods, respectively. Recoveries of 265 µM-I were 98 to 103 and 94 to 108% for the photometric and fluorimetric methods, respectively; the corresponding values for 26.5 µM-I were 101 to 105 and 95 to 104%.
Cholesterol, total Blood Serum Clinical analysis Fluorescence Spectrophotometry

"Enzymic Determination Of Bicarbonate In Serum By Flow Injection Analysis"
Clin. Chim. Acta 1995 Volume 235, Issue 2 Pages 169-177
R. Quilesa, J. M. Fern&aacute;ndez-Romerob and M. D. Luque de Castrob,*

Abstract: An automated method for the determination of bicarbonate in human serum is presented. The method is based on the enzymatic reaction between bicarbonate and phosphoenolpyruvate (PEP) in the presence of PEP carboxylase. A diagram is shown of the hydrodynamic flow injection system. The analytical reaction was coupled with a derivatization reaction in which the NADH consumed was monitored at 460 nm (excitation at 340 nm). The enzymes PEP carboxylase and malate dehyrogenase were immobilized on controlled-pore glass. The calibration graph was linear from 25-300 mM. Within- and between-run RSD (n = 11) were 1-2.8 and 1.32-3.58%, respectively. 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.
Bicarbonate Serum Human Fluorescence Clinical analysis

"Chelating Sorbents In Inorganic Chemical Analysis"
Croat. Chem. Acta 1998 Volume 71, Issue 1 Pages 155-178
Doina Bilba, Doina Bejan, and Lavinia Tofan

Abstract: This review is concerned with the preparation, characterization and the applications of new chelating sorbents in the separation, concentration and determination of trace metals from different complex samples.

"Biosensors In Automated Analysis Systems. 1. Determination Of Ethanol In Beer And Wine By Flow-diffusion Analysis And Amperometric Detection"
Dtsch. Lebensm. Rundsch. 1996 Volume 92, Issue 1 Pages 1-4
MOHNS J. ; K&Uuml;NNECKE W.

Abstract: The flow diffusion system employed (schematic given) included a thermostatted diffusion cell with a hydrophobic gas diffusion membrane, an enzyme reactor, and a thick-layer Pt electrode in a wall-jet flow cell. The ethanol was converted enzymatically to acetaldehyde and H2O2, and the H2O2 detected electrochemically at the Pt electrode at 700 mV. The enzyme was alcohol oxidase immobilized by the glutaraldehyde method on CPG 10 (controlled pore glass). Twenty alcohol-free beers, 16 beers and 13 wines were examined. The correlation between this method and the standard methods was very good (correlation coefficient of 0.9992). Thirty samples per hour could be analyzed and the flow analysis system had a linear range up to 15% v/v.
Ethanol Beer Wine Sensor Electrode Electrode Amperometry

"Optimization Of Enzyme Ratios In A Coimmobilized Enzyme Reactor For The Analysis Of D-xylose And D-xylulose In A Flow System"
Enzyme Microb. Technol. 1994 Volume 16, Issue 3 Pages 216-222
Elena Dom&iacute;nguez, Gy&ouml;rgy Marko-Varga, B&auml;rbel Hahn-H&auml;gerdal and Lo Gorton

Abstract: A coupled enzyme system for the detection of D-xylose and D-xylulose is presented. The system is based on three consecutive enzymatic steps. The enzymes xylose isomerase (XI), mutarotase (MT), and glucose dehydrogenase (GDH) are coimmobilized on controlled pore glass and packed in a bed reactor. The relative amount of enzymes, i.e., enzyme ratio, plays a critical role in driving the overall reaction, resulting in a system with linear response characteristics and an operational range of several orders of magnitude. Three different enzyme ratios are assayed to achieve maximum conversion efficiencies for xylose and xylulose. The highest enzyme unit ratio assayed, 13.4 of GDH to XI, gave the highest apparent pseudo-first-order rate constant showing the importance of the last enzymatic reaction in the coupled system to make the overall reaction thermodynamically favorable. A pH of 7.0 was found to be an optimum compromise for the multienzyme system. Sensitivity was dependent on NAD+ concentration. The study was carried out in a flow injection system. The optimized reactor has been applied for the catalytic detection of pentoses in flow injection analysis (FIA) and liquid chromatography (LC).
d-Xylose d-Xylulose Biological LC

"Enzyme-catalysed Determination Of The Sweetner Aspartame"
GIT Fachz. Lab. 1992 Volume 36, Issue 3 Pages 199-204
Hummel, W.;Zervosen, U.

Abstract: Details are given of a flow injection analysis method for the determination of aspartame (I) in sweeteners. Sample was dissolved in water and a portion of the solution was injected into a stream of 50 mM Tris - HCl buffer of pH 8.5 which merged with a stream of the same buffer containing 2.5 mM NAD+. The mixture flowed sequentially through columns (15 mm x 3 mm) packed with pronase, chymotrypsin and phenylalanine dehydrogenase immobilized on to controlled-pore glass and the NADH formed was measured fluorimetrically. The detection limit was 0.2 mM I and 30 samples h-1 could be analyzed.
Aspartame Artificial Fluorescence

"Determination Of Trace Amounts Of Mercury In Water Samples By Online Flow Injection Analysis Ion Exchange Preconcentration Cold Atomic Absorption Spectrometry"
Guangpuxue Yu Guangpu Fenxi 1988 Volume 8, Issue 6 Pages 39-43
Zhang, S.;Xu, S.K.;Fang, Z.L.

Abstract: The Hg in water was enriched by a factor of 40 in a column packed with porous glass (60 to 100 mesh) treated with quinolin-8-ol; 60 samples h-1 could be applied, with 90 to 96% recovery of Hg. The Hg was eluted with 2 M HCl in 0.5% thiourea, the eluate was merged with a stream of 0.5% KBH4 solution in 0.05 M KOH, and, after separation of the liquid, the vapor was analyzed by AAS at 253.7 nm. The Hg content was then calculated from a calibration graph obtained by similar enrichment and analysis of standard solution of Hg adjusted to pH 5. Tolerance levels of 23 ionic species are given; the detection limit was 2 ng l-1, and the coefficient of variation (n = 11) for 0.5 µg L-1 of Hg was 1.2% .
Mercury Environmental Spectrophotometry

"Flow Injection Amperometric Determination For Sucrose And Glucose In Sugarcane Juice And Molasses"
Int. Sugar J. 1998 Volume 100, Issue 1195 Pages 320-324
Luiz De Mattos, I.;Zagatto, E.A.G.;De Oliveira Neto, G.

Abstract: A flow injection system for amperometric determination of sucrose (I) and glucose (II) employing mutarotase and glucose oxidase immobilized on controlled-pore glass was developed. A Pt microelectrode polarized at 650 mV vs Ag/AgCl was used as the working electrode. The proposed system allows sequential determination of I (0-8.0 mM, r = 0.99973, RSD = 1.5%) and II (0 - 0.60 mM, R = 0.99852, RSD = 1.2%) and was applied to the anal. of sugarcane juice and molasses. Results were compared with those provided by HPLC, and the agreement between them corroborated the applicability of the proposed procedure for large scale anal. and(or) for quality control of ethanolic fermentation Sampling frequency was 50/h.
Sucrose Glucose Juice Food Fermentation broth Amperometry

"Flow Injection Biosensor For The Detection Of Anti-cholinesterases"
J. Biochem. Mol. Biol. 1998 Volume 31, Issue 3 Pages 296-302
Chung, Myung-Sun ; Lee, Yong-Tae ; Lee, Hye-Sung

Abstract: A potentiometric flow injection biosensor for the anal. of anticholinesterases (anti-ChEs), based on inhibition of enzyme activity, was developed. The sensor system consists of a reactor with acetylcholinesterase (AChE) immobilized on controlled pore glass and a detector with an H+-selective PVC-based membrane electrode. The principle of the anal. is based on the fact that the degree of inhibition of AChE by an anti-ChE is dependent on the concentration. of the anti-ChE in contact with AChE. The sensor system was optimized by changing systematically the operating parameters of the sensor to evaluate the effect of the changes on sensor response to ACh. The optimized biosensor was applied to the determination of paraoxon, an organophosphorus pesticide. Treatment of the inhibited enzyme with pyridine-2-aldoxime fully restored the enzyme activity allowing repeated use of the sensor.
Enzyme, anticholinesterase Paraoxon Sensor Potentiometry

"Flow Injection Chemiluminescent Determination Of Glycerol-3-phosphate And Glycerophosphorylcholine Using Immobilized Enzymes"
J. Biolumin. Chemilumin. 1997 Volume 12, Issue 1 Pages 1-5
M. Yaqoob, A. Nabi, M. Masoom-Yasinzai

Abstract: To determine glycerol-3-phosphate (I, glycerol 1-phosphate), sample (30 µL) was injected into a stream (0.7 ml/min) of 0.1 M succinate buffer of pH 6 and passed through a column (3 cm x 2.5 mm i.d.) at 30°C containing glycerol-3-phosphate oxidase immobilized on controlled pore glass. The eluate merged with a stream (0.7 ml/min) of 0.1 M carbonate buffer of pH 10.5 containing 10 µM-luminol and 10 µM-Co(II), and after travelling 2.2 cm the flow passed through a glass coil (10 cm x 1 mm i.d.) in front of a photomultiplier tube where the chemiluminescence was measured. To determine glycerophosphorylcholine (II; α-glycerophosphorylcholine) offline conversion to I using phospholipase-D was carried out in 0.1 M succinate buffer of pH 6 at 30°C for 10 min. The detection limits were 0.5 µM-I and 1 µM-II and the RSD were M and 20-100 µM, respectively. Sample throughput was 40/h. Flow injection procedures with immobilized enzyme mini-columns are described for the determination of glycerol-3-phosphate, and glycerophosphorylcholine with chemiluminescent detection. The hydrogen peroxide produced on-line is coupled with a luminol (5-amino-2,3,-dihydro-1,4-phthalazinedione) peroxidation chemiluminescent system. The detection limits for glycerol-3-phosphate and glycerophosphorylcholine are 5 x 10^-7 M and 1 x 10^-6 M respectively with RSD < 2%. The sample throughput is 40/h. The immobilized enzyme columns did not show any deterioration in activity after usage for 3 months.
3-Phosphoglyceric acid glycerophosphorylcholine Chemiluminescence

"Comparison Of Different Biosensor Systems Suitable For Bioprocess Monitoring"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 257-266
U. Bilitewski*, W. Drewes, J. Neermann, J. Schrader, R. Surkow, R. D. Schmid and J. Bradley

Abstract: To achieve effective bioprocess monitoring, sensing systems are required which are suitable for an online determination of substrates, inhibitors, nutrients or products. Such devices may utilise biochemical principles, i.e. the specific interaction of biochemical receptors with their surroundings. They can be constructed either as in situ sensors or as flow-through sensors connected to the process via sampling devices. Hence, characteristic features of an in situ glucose electrode are described, e.g. analytical range, sensitivity and stability. The sensor was based on mediated electron transfer from the enzyme glucose oxidase to the graphite electrode, the mediators being tetrathiafulvalene (TTF) or dimethylferrocene (DMF). Additionally, various flow injection analysis (FIA) systems based on oxidases, which were immobilized either on controlled pore glass or in a membrane, were characterized with respect to analytical ranges and sensitivities and applied to glucose, lactate and glutamate determinations in off-line samples taken from an animal cell cultivation.
Glucose Lactate Glutamate Fermentation broth Sensor Electrode Electrode

"Use Of A Reversibly Immobilized Enzyme In The Flow Injection Amperometric Determination Of Picomole Glucose Levels"
J. Chem. Soc. Faraday Trans. 1986 Volume 82, Issue 4 Pages 1265-1270
Catherine E. Lomen, Uditha de Alwis and George S. Wilson

Abstract: Glucose oxidase is covalently attached to anti-human IgG by using p-benzoquinone as coupling agent. The resulting conjugate is introduced into a carrier stream of 0.1 M phosphate buffer (pH 6.8) and passed through a µreactor (cf. Anal. Chem., 1985, 57, 2754) containing human IgG covalently bound to controlled pore glass (Lichrospher SI-300). The subsequent immunological reaction within the reactor tube results in the immobilization of enzyme. In the event of a loss of enzyme activity, the enzyme can be removed by dissociating the antibody - antigen complex by elution with 0.1 M phosphate buffer (pH 2.0). The microreactor can then be re-loaded with fresh enzyme. The antigen remains active in binding for >1 year. Results obtained with such a reactor for determination of glucose in four samples of human serum agreed closely with those given by a Beckman Astra instrument.
Glucose Serum Human Clinical analysis Amperometry

"Determination Of Starch And Maltose Using Immobilized Amyloglucosidase And A Glucose Electrode In A Flow Injection System"
J. Chem. Technol. Biotechnol. 1989 Volume 46, Issue 4 Pages 327-333
Lo Gorton, Roger Appelqvist, Gillis Johansson, Frieder Scheller and Dieter Kirstein

Abstract: Maltose and starch were hydrolyzed by glucan 1,4-α-glucosidase immobilized on silanized CPG-10 (pore size 13 nm, particle size 74 to 120 µm) with glutaraldehyde (pH 6.7) and packed into a packed-bed reactor (11 cm x 2.4 mm). The carrier solution (1.0 mL min-1) was 0.1 M phosphate buffer (pH 5) and the glucose formed was measured by a glucose oxidase enzyme electrode in conjunction with a potentiostat vs. an Ag - AgCl reference electrode. Steady-state signals for both maltose and starch were constant for reactor residence times between 24 and 73 s which were reduced by the addition of aldose 1-epimerase to the carrier solution Calibration graphs for 40 µL samples were rectilinear from 30 µM to 12 mM starch and from 10 µM to 5 mM maltose (calculated as glucose residues). The corresponding maximum injection frequencies were 30 and 40 samples hr-1.
Glucose Maltose Starch Electrode

"Flow Injection Analysis With Immobilized Enzymes For Process Control Of Pullulan Production By Fermentation"
J. Chem. Technol. Biotechnol. 1992 Volume 53, Issue 4 Pages 397-400
Ursula Englbrecht, Hanns-Ludwig Schmidt

Abstract: Pullulan (I) and glucose (II) are determined in filtered Aureobasidium pullulans fermentation broth after hydrolysis with α-dextrin endo-1,6-α-glucosidase and glucan 1,4-α-glucosidase immobilized on controlled-pore glass. Total II (i.e., that formed by hydrolysis and that present as such in the initial sample) is determined in a FIAstar 5020 analyzer. by oxidation with glucose 1-dehydrogenase in the presence of NAD+ and spectrophotometric monitoring of the NADH formed. Free II was determined similarly without enzymatic hydrolysis, and the I content was calculated by difference. The calibration graphs for both I and II were rectilinear over the range 2 to 20 mg l-1. One analysis takes only 10 min and the system response remains constant for 30 samples. 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. The glucose produced was oxidized by glucose dehydrogenase, and the NADH formed was 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 and 20 mg/L. The determination of glucose and pullulan took ~10 min per sample.
Pullulan Glucose Fermentation broth Sample preparation Spectrophotometry

"Determination Of Neutral Fat In Milk By Amperometric Flow Injection Analysis With Immobilized Enzyme Reaction"
J. Flow Injection Anal. 1991 Volume 8, Issue 2 Pages 136-147
Seiichi Higuchi, Kiyoshi Matsumoto and Yutaka Osajima

Abstract: The method is based on hydrolyis of neutral fat by triacylglycerol lipase (I) - carboxylesterase (II) and glycerol dehydrogenase (III)-catalyzed oxidation of glycerol, after which the NADH produced is monitored amperometrically. The cited sensor is made from I and II co-immobilized on aminopropyl-substituted controlled-pore glass and from III immobilized on Amino-Cellulofine. For application, an emulsion of milk in 6% bovine serum albumin solution is diluted with buffer solution prepared from 0.125 M carbonate buffer of pH 9.5 containing 30 mM (NH4)2SO4 and 1% of Triton X-100, and a portion is injected into the analyzer. (diagram given) for the determination of neutral fat at 25°C with the above-mentioned buffer as carrier stream at 1 mL min-1. When determining triolein, the relationship between response and concentration. is rectilinear for 0.0128 to 0.0853%; the coefficient of variation was 0.87%. Results were comparable with those obtained by the Wako triglyceride G-test method.
Triolein Fatty acids Milk Amperometry

"Flow Injection Determination Of Cyanide With Use Of An Immobilized Enzyme"
J. Flow Injection Anal. 1996 Volume 13, Issue 2 Pages 138-147
Deguchi, T.;Fukaura, K.;Norimatsu, S.;Minami, S.;Tanaka, A.;Sanemasa, I.

Abstract: Rhodanese was immobilized on 200-400 mesh aminopropyl-controlled pore glass beads (particle size 585 angstrom and surface area 45.1 m2/g) by the action of glutaraldehyde in Tris buffer of pH 8.6. Sample (100 µL) was injected into a stream of 10 mM Tris buffer of pH 8.6 at 0.7 ml/min and merged with a stream of 5 mM Na2S2O3 at 0.3 ml/min before entering the immobilized enzyme reactor (4 cm x 2 mm i.d.) at 0°C. The generated thiocyanate ion was mixed with a reagent stream of 5 mM Fe(III) in a reaction coil (2 m x 0.5 mm i.d.) and detection was at 460 nm. The calibration graph was linear from 0-1 mM cyanide, with a detection limit of 15 µM. When determining 0.5 mM cyanide, the RSD was 2.3%. Only ascorbate interfered. The immobilized rhodanese was stable for at least 10 h when used continuously. The sampling frequency was 20/h. The method was applied to the analysis of environmental and clinical samples.
Cyanide Environmental Biological material Spectrophotometry

"Sensitive, Rapid And Precise Determination Of L-glutamic Acid In Cheese Using A Flow Injection System With Immobilized Enzyme Column"
J. Food Sci. 1989 Volume 54, Issue 2 Pages 423-426
Puchades, R.;Lemieux, L.;Simard, R.E.

Abstract: Glutamic acid in cheese was determined by its reaction in a packed-bed enzyme reactor containing immobilized glutamate dehydrogenase (GIDH) in a flow injection system, measuring fluorescence of NADH generated. There was a linear relationship (r = 0.999) between logpeak height and log-glumatic acid concentration (0.01 to 0.5 mM). The detection limit was 0.005 mM for an injection volume of 88 µL. The samples frequency was 30/h nad the precision was better than 1.2% for 10 successive assays. The accuracy of the enzyme reactor-flow injection system was evaluated by comparison with HPLC results and an excellent correlation was obtained (r = 0.996). No measurable decrease in activity of the GIDH column was observed after a 3-month period. Glutamate dehydrogenase (NAD(P)+) immobilized on controlled-pore glass beads packed in a glass tube (7.5 cm x 1.7 mm) was used for the flow injection determination of glutamic acid (I) at room temp.; the NADH generated was detected fluorimetrically at 450 nm (excitation at 360 nm). The log. (peak height) - log. calibration graph was rectilinear for 0.01 to 0.50 mM I (injection volume 88 µL), and the detection limit was 5 µM. Cheese was initially extracted by the method of Harwalkar and Elliott (J. Dairy Sci., 1971, 54, 8), the extract was freeze-dried, and the residue was dissolved in 0.1 M phosphate buffer (pH 8.0) with filtration. Results correlated well with those by HPLC (r = 0.996).
l-Glutamic acid Food Fluorescence Sample preparation

"A New Chemiluminescence Analysis Apparatus Involving Solid-state Peroxyoxalate And Immobilized Fluorophores"
J. Lumin. 1988 Volume 40, Issue 41 Pages 844-845
Xiangdong Ding, Ping Wang and Guoquan Liu

Abstract: A chemiluminescence detection system for flow injection determination of H2O2 is described. The system is based on peroxyoxalate and Rhodamine B immobilized on controlled-pore glass (100 mesh; pore size 750 .angstrom.), and was used in a single-flow online arrangement with a JASCO FLC-A 700 HPLC instrument. The calibration graph was rectilinear over four orders of magnitude and the detection limit was 50 nM-H2O2. The simplicity of the system decreases its cost.
Hydrogen peroxide Chemiluminescence

"Online Trace Metal Enrichment In Flow Injection Atomic Absorption Spectrometry"
Quim. Anal. 1989 Volume 8, Issue 2 Pages 159-170
Devi, S.;Habib, K.A.J.;Townshend, A.

Abstract: Aqueous samples containing Cu2+, Cd2+, Mg2+, Zn2+, Pb2+ and Hg2+ were pre-concentrated on a mini-column of 8-hydroxyquinoline-5-sulfonic acid immobilized on to controlled-pore glass and eluted with HNO3 or HCl (depending on the metal to be analyzed) directly into a nebulizer for AAS analysis. Calibration graphs were rectilinear from 0 to 100, 100 to 1000, 20 to 220, 10 to 100, 10 to 300 and 20 to 2000 ng mL-1 of Cd2+, Pb2+, Zn2+, Mg2+, Cu2+ and Hg2+, respectively; detection limits ranged from 0.5 ng mL-1 (for Cu) to 25 ng mL-1 (for Hg). . The coefficient of variation ranged from 1.1 to 2.9% (n = 4). Aqueous samples containing Cu2+, Cd2+, Mg2+, Zn2+, Pb2+ and Hg2+ were pre-concentrated on a mini-column of 8-hydroxyquinoline-5-sulfonic acid immobilized on to controlled-pore glass and eluted with HNO3 or HCl (depending on the metal to be analyzed) directly into a nebulizer for AAS analysis. Calibration graphs were rectilinear from 0 to 100, 100 to 1000, 20 to 220, 10 to 100, 10 to 300 and 20 to 2000 ng mL-1 of Cd2+, Pb2+, Zn2+, Mg2+, Cu2+ and Hg2+, respectively; detection limits ranged from 0.5 ng mL-1 (for Cu) to 25 ng mL-1 (for Hg). . The coefficient of variation ranged from 1.1 to 2.9% (n = 4).
Copper Cadmium Magnesium Zinc Lead Mercury Spectrophotometry

"Determination Of Ammonia And Urea In Water By Flow Injection Analysis"
Quim. Anal. 1989 Volume 8, Issue 4 Pages 473-483
Izquierdo, A.;Linares, P.;Luque De Castro, M.D.;Valcarcel, M.

Abstract: Ammonia can be determined by flow injection with detection by (i) fluorimetry at 450 nm (excitation at 340 nm) after treatment with phthalaldehyde and 2-mercaptoethanol, or (ii) potentiometry in basic medium with use of a Metrohm CH-9100 NH3-selective electrode. Urea is determined similarly after online conversion into NH3 by urease immobilized on controlled-pore glass. The responses to NH3 and urea in method (i) were rectilinear for 0.05 to 1 and 0.1 to 2 µg mL-1 and in method (ii) for 0.5 to 50 and 2 to 10 µg mL-1, respectively. The corresponding coefficient of variation (n = 11) at 0.5 µg mL-1 of NH3 and 0.8 µg mL-1 of urea were 1.97, 2.07, 2.07 and 3.2%, and the sampling rates were 60, 50, 50 and 50 h-1, respectively. Tolerance ratios are tabulated; the lowest are an equivalent amount of Fe(II) and a ten-fold amount of Fe(III) in method (i) and a 100-fold amount of K, Na, Ca, Mg, Zn, Cu(II), Fe(III) or Mo(VI) in method (ii). Method (i) was applied successfully to seawater.
Ammonia Urea Sea Fluorescence Electrode Potentiometry

"Additional Advantages Of The Use Of Immobilized Enzymes: Immobilization Of β-D-galactosidase For Magnesium Determination"
Quim. Anal. 1995 Volume 14, Issue 1 Pages 31-35
Quiles Zafra, R.;Fernandez Romero, J.M.;Luque De Castro, M.D.

Abstract: The method was based on Mg activation of the β-D-galactosidase-catalyzed hydrolysis of o-nitrophenyl-β-D-galactopyranoside (I). The enzyme was immobilized on to controlled-pore glass (120-200 mesh) and packed into a glass column (100 cm x 0.5 mm i.d.). The resulting enzyme reactor was incorporated into a FIA system. Sample (50 µL) and I reagent [40 µL; 100 mM Tris hydrochloride buffer of pH 7.5 containing 4 mM DTT and 0.4 mM EGTA (buffer A) with 800 mM NaCl and 2 mM I] were injected into different streams (1.2 ml/min) of buffer A. After merging, the streams passed through a single bead string reactor, then through the enzyme reactor before merging with a stream of 1N-NaOH for detection at 405 nm. Analysis was performed at 37°C. The calibration graph was linear for 5-20 µM-Mg and the within- and between-run RSD were 0.7-2.9% and 1.7-3.2%, respectively. Sample throughput was 40/h. No significant interference from cations was observed. The method was applied to human serum with recoveries of 96-113 %.
Magnesium Serum Human Spectrophotometry

"Determination Of L-glutamate In Various Commercial Soy Sauce Products Using Flow Injection Analysis With A Modified Electrode"
World J. Microbiol. Biotechnol. 1998 Volume 14, Issue 4 Pages 543-549
Udomsopagit, M. Suphantharika, W. K&uuml;nnecke, U. Bilitewski and A. Bhumiratana

Abstract: A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of L-glutamate using L-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700 mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5 mL/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine L-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatography assay using an amino acid analyzer. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of L-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatography anal.
l-Glutamate Food Electrode

"Flow Injection Column Preconcentration Coupled With Atomic Absorption Spectrometry For Aluminum Determination"
Xiamen Daxue Xuebao (Ziran Kexue Ban) 1998 Volume 37, Issue 1 Pages 80-87
Yuan Dongxing, Ian L. Shuttler

Abstract: A method has been developed for the speciation anal. of Al in tea soup and water samples using an automated flow injection system coupled with an electrothermal atomic absorption spectrometer. Two pre-concentration materials, 8-quinolinol immobilized on controlled-pore glass and Amberlite XAD-2 copolymer, were investigated. Fast exchangeable Al, hydrophobic Al, and total Al can be determined by control the pre-concentration conditions.
Aluminum Tea Water Spectrophotometry

"Lability Of Metal Ion-fulvic Acid Complexes As Probed By FIA And DGT: A Comparative Study"
Anal. Chim. Acta 2003 Volume 499, Issue 1-2 Pages 17-28
Alison J. Downard, Jared Panther, Young-Chool Kim and Kipton J. Powell

Abstract: Two kinetic-based analytical techniques with very different time constants (flow injection analysis, FIA and diffusive gradients in thin films, DGT) have been compared in a study of metal ion lability in the systems Al3+-FA and Cu2+-FA (fulvic acid, FA). Flow injection analysis used an in-line micro-column of chelating ion exchanger to capture the labile metal fraction during the short contact time, 1-3 s, with the injected sample. The moderately labile and inert metal fractions were rejected by the ion exchanger but the former gave a colorimetric reaction down-line. The labile fraction was quantified by subsequent elution of captured metal ions and down-line analysis. The chelating resins used were oxine-derivatised fractogel (for Al3+) and oxine-derivatised controlled-pore glass (for Cu2+). Diffusive gradients in thin films utilised cross-linked polyacrylamide films as the diffusive barrier and Chelex-100 to capture the diffusing metal ions that are labile on the experiment time-scale (min). Diffusion coefficients, D, for metal-FA systems at different metal:FA ratios were measured independently in a diffusion cell. They indicated that for both the labile (Cu2+-FA) and slowly labile (Al3+-FA) systems the metal ion diffuses at a similar velocity to the FA, even when the total metal ion concentration exceeds the capacity of FA to bind metal ions in non-labile forms (FA complexation capacity, CC).Complexation capacity was used as a basis for comparison of the two techniques. It was observed that for the less labile Al3+-FA system, the DGT-labile Al3+ equated to the sum of the labile+moderately labile fractions determined by FIA. For the Cu2+-FA system the CC determined by DGT was smaller than that determined by FIA (significant at 1S.D.). Further, the results indicated that D values measured for labile metal-FA complexes in a diffusion cell may not be appropriate for interpretation of diffusion processes that occur in the DGT experiment.
Aluminum(III) Copper(II) Ion exchange

"Flame Atomic Absorption Determination Of Lead Through On-line Preconcentration By Surfactant Mediated Glass Wool Retention"
Int. J. Environ. Anal. Chem. 2006 Volume 86, Issue 1-2 Pages 45-52
Christos Z. Katsaounos, Evangelos K. Paleologos, Miltiades I. Karayannis

Abstract: This work reports on the fabrication of a mini column packed with controlled pore glass and glass wool, and its application for the on-line pre-concentration of lead with the aid of an anionic surfactant (sodium dodecyl sulphate, SDS) and AAS detection. Lead reacts with SDS in a high-ionic-strength environment, and the product is retained and pre-concentrated on a high-surface-area substrate like glass wool. The intervention of controlled pore glass increases the active surface of glasswool and prevents its accumulation into a sticky mass after wetting. Washing the mini column with a methanolic solution of HNO3, causes the retained micellar face to be eluted, and consequently the lead content can be determined with AAS. A pre-concentration factor of 50 along with a signal enhancement due to the combined presence of SDS and methanol yields a detection limit of 1.5 µg/L-1. The correlation coefficient of the calibration curve is 0.999, and the linear range 5-500 µg/L-1. The method was used for the determination of lead in water and wastewater with good results.
Lead Water Waste Spectrophotometry

"Oriented Antibody Immobilization For Atrazine Determination By A Flow-through Fluoroimmunosensor"
Fresenius J. Anal. Chem. 1999 Volume 365, Issue 8 Pages 658-662
E. Turiel, P. Fern&aacute;ndez, C. P&eacute;rez Conde, A. M. Guti&eacute;rrez, C. C&aacute;mara

Abstract: An atrazine flow-through fluoroimmunosensor was developed, based on an oriented antibody covalently bound to Protein-A (Prot-A) immobilized on Controlled Pore Glass (CPG). Atrazine was detected 'in-situ' by placing the immobilized antibody in the optical path of the flow cell. Immobilization of 30 wg of polyclonal anti-atrazine antibody on 0.5 g of Prot-A-CPG provided the highest sensitivity. The effect of several solvents on the covalently immobilized antibodies regeneration was evaluated, the optimum conditions being achieved by pumping 5% acetonitrile (pH = 3) at 0.15 mL/min for 100 s. The detection limit of the immunosensor was 0.7 wg/L and the reproducibility was 2% and 4% for 5 wg/L and 40 wg/L, respectively, in the optimum working concentration range (0.7-50 wg/L). This device allowed 12 samples per hour to be analyzed and had a life-time of 200 assays. Simazine and desisopropylatrazine (DIA) were not cross-reactive, desethylatrazine (DEA) has a cross-reactivity of 8% and propazine and prometryn of 44% and 27%, respectively. The immunosensor was applied to the determination of atrazine in tap and ground water samples spiked at the 10 and 30 µg/L concentration level.
Atrazine Ground Water Fluorescence Sensor

"Development Of A Sequential Injection Analysis System For The Simultaneous Biosensing Of Glucose And Ethanol In Bioreactor Fermentation"
Food Chem. 2003 Volume 81, Issue 1 Pages 141-146
Rui A. S. Lapa, Jos&eacute; L. F. C. Lima and Ivone V. O. S. Pinto

Abstract: An on-line sequential injection analysis system using amperometric detection for the simultaneous monitoring of glucose and ethanol in fermentations media is presented. The automatic analytical procedure developed is based in a sequential injection analysis (SIA) strategy and uses catalytic reactors of oxidase enzymes immobilized on controlled-pore glass. The proposed method allows the simultaneous determination of glucose (linear range between 5 and 750 mg L-1 with a RSD better than 1.2%) and ethanol (linear range between 0.15 and 30 mg L-1 with a RSD better than 2.2%). It was applied to monitor both species in beer fermentation. In a way to adjust the levels of ethanol present in the broth to the characteristics of the proposed system a dialysis unit was used between fermenter and SIA manifold. The sampling rate optimized was 50 samples h-1.
Glucose Ethanol Fermentation broth Beer Wort Amperometry

"Use Of A Laccase-column For Flow-injection Calorimetry"
Ann. NY Acad. Sci. 1998 Volume 864, Issue 12 Pages 493-496
Ikuo Satoh, Ikuko Sakurai

Abstract: A great number of enzyme-catalyzed reactions are accompanied by heat evolutions.1 Therefore, flow-injection analysis (FIA) based on calorimetry, that is, flow-injection calorimetry,2,3 in combination with biocatalysts as specific recognition elements gives convenient and versatile analytical methods for biorelated compounds. Nakatani and Shimizu purified a thermostable laccase from Trametes sp. and reported its enzymatic properties recently.4 The enzyme functioned as the catalyst in oxidative reactions of various kinds of dihydroxylated compounds (so-called polyphenol compounds). We tried to apply its enzymatic properties to the determination of L-ascorbate in soft drinks. In this paper, we describe the fundamental performance of the calorimetric FIA system with use of the enzymes immobilized onto porous glass beads.
Ascorbate Soft drink Calorimetry