On-line Flow Injection Analysis Research Articles

An automatic method of on-line FIA cation-exchange pretreating and anion-exchange separating for simultaneous determination of silica and phosphate in biological materials

A flow-injection analysis (FIA) new method of a continuous ion-exchange pretreatment and separation is given for the simultaneous determination of silicate and phosphate in biological samples. To remove interfering cations, the sample solution passes through the mini cation-exchange column connected at the inlet and outlet of one of the loading loops of a two-channel valve, and it again passes through another loading loop of the valve for sampling. After the valve is switched to the inject position, the defined sample is injected and passed through the seperation column, and silicate and phosphate ions in the sample zone are separated. At the same time, the mini cation-exchange column is switched to the regeneration position and regenerated by a regenerant. By this manner, the sample pretreatment and determination can be made continuously. The approach is extendable to other anion analyses in various biological samples without matrix matching. This article gives a description of simultaneously determining silicate and phosphate in human hair, mussel, tea leaves, sargasso, and rice flour with the flow-injection system. Regression equations of the calibration curves were Y1 = 0.7183C− − 0.0621 (r = 0.9998); Y2 = 0.4324CPO43− − 0.0963 (r = 0.9998). About 30 samples can be analyzed per hour. Determination ranges are within 0.1–10 mg/L for SiO32− and 2.0–30 mg/L for PO43−. Relative standard deviations for metasilicate and orthophosphate were less than 1.8 and 1.3%, respectively. Recovery ranges of SiO32− and PO43− in the samples are 100–107% and 97–104%, respectively. The results are in agreement with those obtained by classical procedure. Matrix effects were also investigated, and it was found that metallic cations in matrix effect positively on the simultaneous determination, and the slop of the calibration curve is increased with the increase of the kind and concentration of metallic cations. Silicate contents in mussel, tea leaves, and sargasso are reported in the present work for the first time. © 1997 John Wiley & Sons, Inc. Lab Robotics and Automation 9: 55–67, 1997.

High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions.

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl-1 in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hoklsok suicide system, improving plasmid maintenance, and the inducible lac pl o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l-1 after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fedbatch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 gl-1, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions.

Dialkylphosphoric acids as carriers in separation of lanthanides and thorium on supported liquid membranes

Permeation of seven lanthanides (Ln) and thorium through a supported liquid membrane containing di-( n- octyl)phosphoric (DOPA) or di-( n-pentyl)phosphoric (DPPA) acid as a carrier has been studied as a function of the chemical composition of the system. The results have been compared with a previous study in which di-(2-ethylhexyl)phosphoric acid was used. Metal cations were transported from feed solutions of pH 1.1–4.8 (HNO 3) into strip solutions of 0.015–0.1 mol l −1 nitric acid. The ionic strength was kept constant at 0.1 mol l −1 (HNO 3, KNO 3). The initial lanthanide concentration and carrier concentration in the liquid membrane were varied from 0.5 to 500 μmol l −1 and from 0.01 to 0.5 mol l −1 respectively. To describe the mass transfer of metal cations, permeability coefficients have been determined by inductively-coupled plasma atomic emission spectroscopy or by on-line flow-injection analysis of metal concentrations in strip or feed solution. Probably as the result of a higher solubility of the carrier in aqueous media, transport of Ln with DPPA was not observed. By using DOPA, La, Ce, Pr, and Nd permeated through the membrane while transport of heavier Ln was partly or totally suppressed. This enables these four Ln (separation factor α = 3.0 for Nd and Sm) to be separated from the others. Furthermore, at a very low acidity gradient, only La (III) is transported over the membrane ( α ≥ 3.4 for La, Ce and next Ln). The seven elements from La to Tb can be separated from Th(IV) because no evidence of its permeation through the membrane was found under the conditions of Ln transport. In contrast to previous studies on Ln transport with dialkylphosphoric acid carrier, the possibility of participation of species other than Ln(AHA) 3 in the transport has been discussed. The decrease of permeability observed at higher Ln concentrations and higher pH of the feed solution has been explained as the result of formation of species, e.g. polymeric ones, that are unable to permeate through the membrane.

An on-line flow-injection analysis system for the determination of acetate

A flow-injection system for the determination of acetate is described. The method is based on the oxidation of sarcosine by immobilized sarcosine oxidase. This enzymatic reaction is competitively inhibited by acetate and the extent of the inhibition serves to calculate the acetate concentration. The effect of pH, temperature and sarcosine concentration are investigated and discussed. As an example of application, a fermentation process of E. coli was monitored with the developed system.

On-line flow injection analysis of volatile organic compounds in seawater by membrane introduction mass spectrometry

The combination of flow injection analysis with membrane introduction mass spectrometry for analysis of volatile organic compounds (VOCs) in seawater is examined and is compared to measurements made in water. Membrane introduction mass spectrometry is performed using a benchtop ion trap mass spectrometer, and characterization of various aspects of the flow injection and ion trap combination for the analysis of volatile organic compounds (including anthropogenic halocarbons) in seawater is carried out. The analyte responses are shown to be linear over several orders of magnitude (e.g. for methylene chloride), independent of seawater pH (e.g. for chlorobenzene) and independent of matrix effects for the VOCs studied. A comparison of the performance of a microporous (Teflon) membrane with that of an amorphous silicone membrane is made, and the former is shown to provide lower detection limits which are in the parts-per-trillion range (300 ppt for chlorobenzene, 190 ppt for trans-1,2-dichloroethene). The microporous membrane provides faster response times by a factor of four to five for relatively more polar compounds, such as chlorobenzene. An analysis of a seven-component mixture demonstrates the ability of this on-line combination to allow multicomponent analysis of mixtures of some complexity.

Determination of urinary albumin using high-performance immunoaffinity chromatography and flow injection analysis.

The detection of low levels of albumin in urine can be an important aid in the early diagnosis of kidney disease. In this study, an automated system for urine analysis was developed that could simultaneously measure both albumin and creatinine, an indicator of urine output and volume. Albumin was determined by use of a column that contained immobilized anti-albumin antibodies. An on-line flow injection analysis system was used to measure creatinine as this solute eluted nonretained from the antibody column. The total analysis time of the system was 5 min per injection. Limits of detection for albumin and creatinine in a 20 microL sample (at S/N = 2) were 3 and 5 mg/L, respectively. The response for these compounds covered the range of clinical interest. Within-run precision for both analytes was +/- 1-2% at normal urine concentrations. The results of this method showed good agreement with those obtained by reference techniques. This approach can be adapted for the detection of other urine components by changing the type of antibody column used in the system.

Monitoring and control of recombinant protein production

For better control of the production of a toxic product by recombinant Escherichia coli carrying three different types of multicopy plasmids, the total and viable cell concentration, total cell count (TCC), number of colony-forming units (CFU), plasmid copy numbers, intracellular enzyme activity and the concentrations of the cell metabolites in the cultivation medium were monitored. This was performed during the growth phase and after chemical induction with IPTG and temperature induction, respectively. Gene expression was carried out in a small stirred-tank reactor and in a 60-1 tower loop reactor equipped with a draught tube. Cell concentration was determined with an in situ turbidometer, TCC by off-line counting, cell viability by culture fluorescence, CFU also by off-line counting, the concentration of metabolites with off-line and on-line liquid chromatography and with off-line and on-line flow-injection analysis (FIA) and intracellular enzyme concentrations by off-line and on-line FIA. The evaluated data allowed an improvement of the production process, and increases in cell concentration and volumetric and specific productivity.

New trends in measurement and control of biotechnological processes

Abstract

On-line monitoring and control of fermentation processes by flow-injection analysis

On line flow-injection analysis (FIA) systems were developed for the real-time measurement and control of concentrations of small molecules (i.e. MW < 500) in fermentation media. An on-line tangential flow filtration device was developed to provide a particle-free sample stream to the analyzers. Analysis of fermentation supernatant is problematic because it is a complex and variable sample matrix. Using FIA techniques, in-line sample pretreatment strategies have been implemented to eliminate specific interferences. Two examples, demonstrating the use of on-line FIA for monitoring acetate and for controlling phosphate in E. coli fermentations, are discussed.

Complete on-line determination of biopolymer molecular weight via high-performance liquid chromatography coupled to low-angle laser light scattering, ultraviolet, and differential refractive index detection

An optically modified high-performance liquid chromatography refractive index detector was developed to allow complete on-line determinations for biopolymer molecular weights. On-line concentration, refractive index, specific refractive index increment (dn/dc2)mu, and Rayleigh factor were determined under flow injection analysis (FIA) and size exclusion chromatography (SEC) conditions using low-angle laser light scattering, ultraviolet, and modified refractive index detection. This instrumental system is capable of determining absolute on-line molecular weights. The error and time requirements involved in conventional methodologies for proteins have been reduced. Sample quantities have been reduced from 150 to 200 mg, in conventional off-line methods, to less than 2 mg for on-line FIA and 0.5 mg for on-line SEC, if mass absorptivities (a) are known. Otherwise, the determination of a will be the most sample-demanding step, requiring about 3 mg of the pure protein. On-line measurements of (dn/dc2)mu are in good agreement with traditional off-line values established at Donnan equilibrium (usually within 5%). In addition, this technique provides true injected mass as determined by the UV detector, after chromatographic exposure where losses may occur, which is then used in the calculation of biopolymer molecular weight.

Rapid and sensitive determination of nucleoside H-phosphonates and inorganice H-phosphonates by high-performance liquid chromatography coupled with flow-injection analysis

A coupled high-performance liquid chromatographic (HPLC) and flow-injection analysis (FIA) system was developed for the separation and detection of H-phosphonates. The H-phosphonates are separated by HPLC in the anion-exchange mode and then introduced into an on-line FIA system. In the FIA system, H-phosphonate esters and anhydrides (POP bonds are hydrolysed and oxidized to orthophosphate, the resulting orthophosphate is converted into the heteropoly blue complex and the latter is monitored spectrophotometrically at 830 nm. The system is sensitive and easy to use. The detection limit is 10 −6 M for inorganic, sugar and nucleoside H-phosphonates. The reproducibility of the peak areas was less than 1% (relative standard deviation) for all H-phosphonates.

Communication. Reverse flow injection formation of monitorands in direct and indirect on-line flow injection analysis methods

The first page of this article is displayed as the abstract.

Combination of flow injection analysis with flame atomic-absorption spectrophotometry: determination of trace amounts of heavy metals in polluted seawater

A simple flow injection system, the FIAstar unit, was used to inject samples of seawater into a flame atomic-absorption instrument allowing the determination of cadmium, lead, copper and zinc at the parts per million level at a rate of 180–250 samples per hour. Further, on-line flow injection analysis pre-concentration methods were developed using a microcolumn of Chelex-100 resin, allowing the determination of lead at concentrations as low as 10 parts per 109(p.p.b.) and 1 p.p.b. for cadmium and zinc. The sampling rate was between 30 and 60 samples per hour and the readout was available within 60–100s after sample injection; the sampling frequency depended on the pre-concentration required.