On-line-Bestimmung von Enzymen in der Bioproze?analytik unter Berücksichtigung von Probenahme und Flie?injektionstechnik

Summary The on-line determination of enzymes in biotechnical processes becomes an important factor with regard to process development and optimization. At present, most commonly enzymes are determined off-line in the laboratory after withdrawal of a separate sample. Wet chemical methods dominate in this respect, mainly because enzymes have to be measured according to the reaction schemes which are catalyzed by them. For an efficient process monitoring and control the time delay, the limited reliability and the man power needed for analysis of a large number of samples are crucial points. By using the technique of flow injection analysis (FIA) it should in general become easy to develop automatically operated enzyme determination procedures based on reaction schemes which can be used for fast and efficient process monitoring, providing the problems with the coupling of the analyzer at the bioreactor are solved. Continuous sampling in this respect plays a key role in developing on-line measuring techniques. This paper reviews the current status of on-line enzyme analysis, using flow-injection techniques. It is shown that the coupling problems can be solved by using a newly developed sampling module, which is based on membrane filtration. Some examples of on-line enzyme determinations in fermentation as well as in downstream processing illustrate the ease and reliability of the proposed concept for using FIA in connection with membrane separation.     

Determination of formaldehyde in frozen fish with formaldehyde dehydrogenase using a flow injection system with an incorporated gel-filtration chromatography column

A flow injection analysis (FIA) system for determination of formaldehyde in frozen fish products is described. The system provides a rapid and selective determination of formaldehyde in aqueous fish extracts by the combination of a deproteinization procedure and a stopped-flow enzymatic approach in a FIA system. The FIA system is furnished with a gel-filtration chromatography column for on-line removal of the proteins from the extract before the enzymatic analysis is performed. Compared with the standard methods for determination of formaldehyde in fish products the present method is much faster and less affected by interferences. The limit of detection for the proposed method is 2.5 mg/l of formaldehyde. The sampling frequency is about 10 determinations per hour.     

Spectrophotometric determination of uranium by flow injection analysis using U/TEVA.SpecTM chromatographic resin

A flow injection analysis (FIA) system is described for the determination of uranium. The system consists of a microcolumn packed with. U/TEVA.Spec^TM, chromatographic resin for on-line sample separation. The eluate is mixed with 4-(2-pyridylazo) resorcinol (PAR). The colored product is continuously monitored spectrophotometrically.     

Flow-injection analysis for hypoxanthine in meat with dissolved oxygen detector and enzyme reactor

A low cost flow-injection analysis (FIA) with a dissolved oxygen (DO) detector and a xanthine oxidase immobilized column for the analysis of hypoxanthine as an index to determine degree of aging in meat was developed for quality control in the food industry. In this system, hypoxanthine is oxidized by an enzyme reaction with xanthine oxidase immobilized on the column to produce xanthine. Then the catalytic reaction between hypoxanthine and DO with xanthine oxidase proceeds with the DO concentration decreasing in the stream of the flow system. Decrease in the DO concentration was monitored by a DO detector located downstream of the flow system. This decrease in DO concentration was proportional to the hypoxanthine concentration. For detecting the decreased DO concentration efficiently a flow-through cell with a polarographic-type DO sensor was specially designed. As a result, a linear working curve was obtained from 3.68 × 10⁻⁵ to 1.84 × 10⁻³ M hypoxanthine concentrations with this FIA system. We applied the present system with a DO detector for the determination of hypoxanthine in meat samples and compared the results with those obtained by the conventional HPLC method. The data obtained with the present FIA method were in fairly good agreement with those obtained by the conventional HPLC method for the meat samples. Correlation factor and regression line between the two methods were 0.998 and Y= 1.51X-32.64 respectively. We concluded that the present FIA system with a DO detector was suitable as a simple, easy to handle and reliable instrument for quality control in the food industry.     

Coupling of size-exclusion chromatography to a continuous assay for subtilisin using a fluorescence resonance energy transfer peptide substrate: testing of two standard inhibitors

Liquid chromatography (LC) was coupled on-line to a homogeneous continuous-flow protease assay using fluorescence resonance energy transfer (FRET) as a readout for the screening of inhibitors of an enzyme (e.g., Subtilisin Carlsberg). The inhibitors aprotinin (a protein of approximately 6500 g/mol) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 240 g/mol) were mixed with other, non-active compounds and separated on a size-exclusion chromatography column. After the separation, the analytes were eluted to the postcolumn reactor unit where the enzyme solution and subsequently the FRET peptide substrate were added; by measuring the fluorescence intensity the degree of inhibition was monitored on-line. As expected, only the two inhibitors caused a change in the FRET response. Detection limits for aprotinin were 5.8 microM in the flow injection analysis (FIA) mode and 12 microM in the on-line LC mode. System validation was performed by determining IC50 values for aprotinin for the FIA mode (19 microM) and the on-line mode (22 microM). These IC50 values were in line with the value determined in batch experiments (25 microM). With this system, chemical information (i.e., chromatographic retention time) and biological information (i.e., enzyme inhibition) can be combined to characterize mixtures.     

Flow injection analysis of potassium using an all-solid-state potassium-selective electrode as a detector

The concentration of potassium was determined by a combination of flow injection analysis (FIA) with an all-solid-state potassium sensor detection. The all-solid-state potassium-selective electrode possessing long-term potential stability was fabricated by coating an electroactive polypyrrole/poly(4-styrenesulfonate) film electrode with a plasticized poly(vinyl chloride) membrane containing valinomycin. The simple FIA system developed in this laboratory demonstrated sensitivity identical to that in the batch system and achieved considerably rapid assay (150 samples h⁻¹). Analyses of soy sauce and control serum samples by this FIA system yielded results in good agreement with those obtained by conventional measurements.     

Washing of sediment on filtering devices

Problems of filtration washing of sediment are considered; the brief overview of the most common filtering devices is made; the particularities of the course of the washing process on these devices are considered. It is stated that the optimal conditions for the effective and economic washing are provided by filter presses with vertical plates. The mechanism of the flow of the washing liquid through the sediment on these filters for two versions, chamber and membrane filter presses, is illustrated. The method of the objective control of the change in the concentration of agent washed out from the sediment during the process of washing of sediment on mentioned filters with the help of the industrial conductivity meter is described.     

Automation of radiochemical analysis by flow injection techniques: Am-Pu separation using TRU-resin™ sorbent extraction column

A rapid automated flow injection analysis (FIA) procedure was developed for efficient separation of Am and Pu from each other and from interfering matrix and radionuclide components using a TRU-resin^™column. Selective Pu elution is enabled via on-column reduction. The separation was developed using on-line radioactivity detection. After the separation had been developed, fraction collection was used to obtain the separated fractions. In this manner, a FIA instrument functions as an automated separation workstation capable of unattended operation.     

On-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface

A method for coupling an electrophoretic driven separation to a liquid flow, using conventional fused-silica capillaries and a soft polymeric interface is presented. A novel design of the electrode providing high voltage to the electrophoretic separation was also developed. The electrode consisted of a conductive polyimide/graphite imbedded coating immobilized onto the capillary electrophoresis (CE) column inlet. This integrated electrode gave the same separation performance as a commonly used platinum electrode. The on-column electrode also showed good electrochemical stability in chronoamperometric experiments. In addition, with this electrode design, the electrode position relative to the inlet end of the CE column will always be constant and well defined. The on-line flow injection analysis (FIA)-CE system was used with electrospray ionization (ESI)-time of flight (TOF)-mass spectrometry detection. The preparation of the PDMS (poly(dimethylsiloxane)) interface for FIA-CE is described in detail and used for initial tests of the on-column polymer-imbedded graphite inlet electrode. In this interface, a pressure-driven liquid flow, a make up CE electrolyte and a CE column inlet meet in a two-level cross (95 microm ID) in the PDMS structure, enabling independent flow characterization.     

On-line microwave oven digestion flame atomic absorption analysis of solid samples

A manifold has been developed for on-line microwave oven digestion and flame atomic absorption spectrometric (FAAS) determination of metallic elements in solid samples. The use of a closed flow system permits sample treatment before analysis by FAAS, the direct injection of slurries avoids a filtration step and the interconnection of two conventional rotary injection valves allows the rapid introduction of samples and standards. The determination of lead in sewage sludge was employed as a test system for the proposed on-line sample digestion manifold. The procedure has a limit of detection of 0.2 μg Pb g^?1.     

Development of an on-line coupling of liquid-liquid extraction, normal-phase liquid chromatography and high-resolution gas chromatography producing an analytical marker for the prediction of mutagenicity and carcinogenicity of bitumen and bitumen fumes.

A fast and fully automated system for the determination of polycyclic aromatic compounds (PACs) is described. The system has been developed to produce an analytical 'marker', correlating chemical characteristics (including PAC analysis) with mutagenicity and carcinogenicity. The products of interest are bitumen fumes, bitumen and other (heavy or even residual) oil products, regardless of their boiling range. Dimethyl sulphoxide (DMSO) extractables obtained from a flow-injection analysis (FIA) system are introduced on-line in a normal-phase liquid chromatographic (NPLC) system. Here, the PACs are separated from the DMSO and possible co-extracted heavy residual species. The final step incorporates on-line gas chromatographic analysis of the three-to-six-ring PAC fraction, followed by flame-ionisation detection for quantification. It was demonstrated that data obtained from samples in the distillate lubrication-oil range correlate well with data obtained from the manual DMSO-extraction method standardised by the Institute of Petroleum as IP346.     

Amberlite XAD resin solid-phase extraction coupled on-line to a flow injection approach for the rapid enrichment and determination of phenols in water and waste waters

A novel and expeditious approach for direct determination of phenols in water and waste waters based on solid-phase extraction coupled on-line to a flow injection analysis (FIA) manifold is described. The method employs on-line preconcentration of the phenols in an acidified sample (pH=2.0) onto a 3 cmx3 mm column packed with Amberlite XAD-4 resin. The phenols are subsequently eluted from the resin into a flowing system with an alkaline solution (pH=13) by actuating a switching valve; the eluted analytes were then quantified spectrophotometrically as the products of reaction with 4-aminoantipyrine (4-AAP) and potassium ferricyanide on passing through the flow-cell of a detector. The proposed method has a linear calibration range 0.01-1 mug ml(-1) of phenol, with a detection limit of 0.004 mug ml(-1) (S/N=3) and a sample throughput of 12 h(-1), investigated with a 4.4 ml sample volume. The relative standard deviation is 2.4% for 0.2 mug ml(-1) of the analyte. The sensitivity offered by the procedure was higher by a factor of 13 than that provided by a conventional flow injection analysis method. The analytical scheme of the proposed system is much simpler than its conventional manual counterpart due to the fact that it combines trace enrichment, sample clean-up, derivation and detection in one analytical set-up. The high speed, ease of use and automation, selectivity, and relative freedom from random contamination by sample handling make this method ideal for the phenols monitoring in water and waste waters.     

A compact flow injection analysis system for surface mapping of phosphate in marine waters

The design, construction and validation of a compact, portable flow injection analysis (FIA) instrument for underway analysis of phosphate in marine waters is described. This portable system employs gas pressure for reagent propulsion and computer controlled miniature solenoid valves for precise injection of multiple reagents into a flowing stream of filtered sample. A multi-reflection flow cell with a solid state LED photometer is used to detect filterable reactive phosphate (0.2 mum) as phosphomolybdenum blue. All the components are computer controlled using software developed using the Labviewtrade mark graphical programming language. The system has the capacity for sample throughput of up to 380 phosphate analyses per hour, but in the mode described here was operated at 225 analyses per hour. Under these conditions, the system exhibited a detection limit of 0.15 muM, reproducibility of 1.95 % RSD (n=9) and a linear response (r(2)=0.9992) when calibrated in the field with standards in the range 0.81-3.23 muM. The system was evaluated for the mapping of phosphate concentrations in Port Phillip Bay, south eastern Australia, and during the course of a 150 km cruise, 542 analyses were performed automatically. In general, good agreement was observed between analyses obtained using the portable FIA system and those obtained from manual sampling and laboratory analysis.     

Flow injection analysis for residual chlorine using Pb(II) ion-selective electrode detector

A simple flow injection analysis (FIA) system for residual chlorine in tap water has been developed by using a Pb(II) ion-selective electrode (ISE) detector. The method is based on a specific response of the Pb(II)-ISE to residual chlorine. The FIA system consists of a millivolt meter, a peristaltic pump, a Pb(II)-ISE detector and a recorder. A linear working curve between peak height and concentration of residual chlorine was obtained from 0.1 to 1 mg l(-1) for the developed FIA system. The relative standard deviation for repeated injections of a 0.2 mg l(-1) residual chlorine sample was 2%. The regression line and its correlation factor between the conventional o-tolidine colorimetric method and the present method were Y=0.75X+0.17 and 0.967, respectively, for this determination.     

Evaluation of the antioxidant activity by flow injection analysis method with electrochemically generated ABTS radical cation

A Trolox equivalent antioxidant capacity (TEAC) decolourisation assay was adapted to a flow injection analysis (FIA) system and a simple and rapid method for antioxidant activity evaluation was developed. To avoid the time consuming step of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) radical cation preparation by chemical oxidation of ABTS, as in the original TAEC assay, and hence, to shorten the analysis time, the ABTS radical cation was generated on-line by electrochemical oxidation of ABTS in the flow-through electrolysis cell forming a part of the FIA system. The proposed method was optimised with respect to a flow rate, injection volume and ABTS radical cation/carrier ratio. Under the optimised conditions linear calibration graphs for Trolox were obtained over the range 10-100 microM, with a limit of detection 1.6 microM. Good reproducibility (relative standard deviation 1.95%) and sample throughput (32 samples per hour) were achieved. The developed method was applied to the evaluation of the antioxidant activity of pure compounds and samples of some common beverages. In both cases a good correlation between the results obtained by the proposed method and TEAC values evaluated by the classic TAEC decolourisation assay was obtained (r(2)= 0.996 for pure compounds and r(2)= 0.957 for beverage samples).     

Nephelometry and turbidimetry with paired emitter detector diodes and their application for determination of total urinary protein

Two miniature and compact optoelectronic devices fabricated by means of integration of light emitting diodes have been developed for turbidimetric and nephelometric measurements. These devices are operating according to paired-emitter-detector-diode (PEDD) principle. The detectors have been characterized using bovine serum albumin and Exton protein assay as a model analyte and a model analytical method, respectively. The developed detectors have been adapted for measurements under conditions of flow injection analysis (FIA). Under optimized conditions the turbidimetric flow system offers the range of linear response up to 400 mg L⁻¹ with the detection limit at 20 mg L⁻¹. The linear range and detection limit found for optimized nephelometric FIA system are 15–500 mg L⁻¹ and 8 mg L⁻¹, respectively. The PEDD-based FIA systems with the detector operating according to both modes of measurements have been successfully applied for urinalysis offering total protein determination at physiological and pathological levels with high throughput (over 60 injections per hour).     

Principles and analytical applications of acousto-optic tunable filters, an overview

Advantages of acousto-optic tunable filters have been exploited to develop novel analytical instruments which are not feasible otherwise. The instrumentation development and unique features of such AOTF based instruments including the multidimensional fluorimeter, the multiwavelength thermal lens spectrometer, the near-infrared spectrometer based on erbium doped fiber amplifier (EDFA), and detectors for high performance liquid chromatography (HPLC) and flow injection analysis (FIA), will be described.     

Flow-injection systems for determination of trace manganese in various salts by catalytic photometric detection

Two flow-injection analysis (FIA) systems for the determination of trace manganese in salts are presented using highly sensitive catalytic detection based on the oxidation of 3,4-dihydroxybenzoic acid by hydrogen peroxide. Two different approaches, the use of a large sample volume injection in a usual FIA mode (system A) and on-line coupling of a cation-exchange separation column with detection in a continuous flow system (system B), have proved very effective for eliminating the blank peak problem and thus affording direct injection of a sample solution containing a large concentration of salt. The limits of determinations are 0.04 ppm and 0.01 ppm for systems A and B respectively, when a 5 g sample is used for preparing the 100 ml sample solution. The proposed FIA systems were satisfactorily applied to the determination of manganese at 0.03-1.59 ppm in solar salts (salts made by exposing brine to the sun) with good precision.     

用流动注射在线富集分离荧光分光光度计测定垃圾渗出液中的苯胺

建立了用荧光分光光度计作为流动注射分析的检测器,用自动研制的流通池测定垃圾渗出液中苯胺的新方法。方法的线性范围为０．０７～０．５ｍｇ／Ｌ,检出限为０．０５ｍｇ／Ｌ。用于垃圾渗出液的测定,结果与ＨＰＬＣ法无显著性差异,回收率在９６％～１０４％之间,该法具有选择性好、灵敏度高、重现性好的特点。