Micro sequential injection: fermentation monitoring of ammonia, glycerol, glucose, and free iron using the novel lab-on-valve system

Using an integrated lab-on-valve manifold in a microfluidic sequential injection format (microSI), automated sample processing has been developed for off-line and on-line monitoring of small-scale fermentations. Spectrophotometric assays of ammonia, glucose, glycerol, and free iron were downscaled to use micro-quantities of commercial reagents. By monitoring the reaction rate, the response curves in a stopped-flow mode generate linear calibration curves for ammonia [r2 = 1.000 (0.9% SE)], glycerol [r2 = 0.999 (1.1% SE)], glucose [r2 = 0.999 (1.1% SE)], and free iron [r2 = 0.999 (1.5% SE)]. Since sample dilution and reagent quantities are easily adjusted within the programmable SI format, the lab-on-valve system can accommodate samples over a wide concentration range (ammonia: 3-1200 ppm; glycerol: 20-120 ppm; glucose: 35-1000 ppm; and free iron: 80-400 ppm). This work demonstrates the key advantages of miniaturization through the reduction of sample and reagent use, minimizing waste and providing a compact yet reliable instrument. The lab-on-valve manifold uses a universal hardware configuration for all analyses, only requiring changes in software protocol and choice of reagents. All of these features are of particular importance to small-scale experimental fermentation where multiple analyte analyses are needed in real-time using small sample volumes. It is hoped that this first real-life application of the lab-on-valve manifold will serve not only as a model system to downscale assays in a practical fashion, but will also inspire and promote the use of the integrated microSI manifold approach for a wider range of biotechnological applications.     

A sequential injection method for the determination of Tween-80 in natural water samples using a fluorescence enhancement of the dye Eosin-B.

A simple sequential injection (SI) method is reported for the determination of the non-ionic surfactant polyoxyethylene sorbitan monoleate (Tween-80). The proposed method is based on a fluorescence enhancement of the probe 4',5'-dibromo-2',7'-dinitro fluorescein, disodium salt (Eosin B) in the presence of a surfactant. The procedure is optimized using the univariate method of optimization. The optimum operating conditions are as follows: reagent concentration, 40 ppm; pH, 4.5; aspirated volumes of sample and reagent, 120 microL and 100 microL, respectively. With the optimum conditions described, linear calibration curves were obtained from 10-200 ppm. The detection limit was 1.7 ppm and the maximum relative standard deviation of the method was 2.7% (n = 5). The fluorescence excitation and emission were fixed at lambda(ex) = 545 mn and lambda(em) = 585 nm. The method was successfully applied to the determination of (TW-80) in natural water samples.     

Simultaneous determination of diethylene glycol and propylene glycol in pharmaceutical products by HPLC after precolumn derivatization with p-toluenesulfonyl isocyanate

A simple and reliable HPLC method was developed for the simultaneous quantitative analysis of diethylene glycol (DEG) and propylene glycol (PG) in pharmaceutical products by precolumn derivatization. The derivatization reagent p-toluenesulfonyl isocyanate (TSIC, 10 microL, 20% in ACN v/v) was added to 100 microL of the sample, and then 10 muL of water was added. The resulting derivatives were separated using a C(18)analytical column and a mobile phase composed of 0.01 M KH(2)PO(4)buffer (adjusted to pH 2.5 with phosphoric acid) and ACN (47:53 v/v) at 1 mL/min and 25 degrees C. For detection, UV light at 227 nm was used. The derivatization conditions including reaction time, temperature, and concentration of TSIC were optimized. The calibration curves were linear from 0.062 to 18.6 microg/mL (r(2)= 0.9999) and from 0.071 to 21.3 microg/mL (r(2) = 0.9999) for DEG and PG, respectively. The RSD values of intra- and interday assays were all below 4% for DEG and PG. The proposed method was then successfully applied to analyze two Armillarisin A injection samples and two spiked syrup samples.     

Spectrophotometric determination of procaine hydrochloride in pharmaceutical products using 1,2-naphthoquinone-4-sulfonic acid as the chromogenic reagent

Spectrophotometric determination of procaine hydrochloride is described. The procaine hydrochloride reacts with 1,2-naphthoquinone-4-sulfonic acid in pH 3.60 buffer solution to form a salmon pink compound, and its maximum absorption wavelength is at 484 nm, epsilon(484)=5.22 x 10(3).The absorbance for procaine hydrochloride from 0.30 to 100 microg ml(-1) obeys Beer's law. The linear regression equation of the calibration graph is C=19.23A-0.03, with a linear regression correlative coefficient is 0.9996, the detection limit is 0.28 microg ml(-1); recovery is from 98.0 to 105.2%. Effects of pH, surfactant, organic solvent, foreign ions, and standing time on the determination of procaine hydrochloride have been examined. This method is rapid and simple, and can be used for the determination of procaine hydrochloride in injection solution of procaine hydrochloride. The results obtained by this method agreed with those by the official method (dead-stop titration).     

A sequential injection method for the fluorimetric determination of aluminum in drinking water using 8-hydroxy-7-(4-sulfo-1-naphthylazo)-5-quinoline sulfonic acid

A robust and simple sequential injection (SI) method for the assay of aluminum ions in drinking water is described. The method is based on the complex formation between aluminum and 8-hydroxy-7-(4-sulfo-1-naphthylazo)-5-quinoline sulfonic acid (HSNQ). The fluorescence of the complex is monitored at an emission wavelength of 492 nm with excitation at 357 nm. The HSNQ concentration, aspirated reagent and sample volumes were optimized simultaneously using 3(3) full factorial design. The optimum operating conditions are aspirated sample and reagent volumes of 90 and 70 microL, respectively, and HSNQ concentration of 20 microM. With these conditions linear calibration curves were obtained from 100 to 800 ppb. The detection limit was 4 ppb. The maximum relative standard deviation of the method was 1.43% (n=5). The method was successfully applied for the determination of aluminum in drinking water samples.     

Stopped-flow injection liquid-liquid extraction spectrophotometric determination of palladium in airborne particulate matter and automobile catalysts

A stopped-flow injection liquid-liquid extraction (SF-EX-FIA) spectrophotometric method is reported for the determination of palladium(II), using the 2,2'-dipyridyl-2-pyridylhydrazone (DPPH) as a color forming reagent. The colored complex Pd(II)-DPPH was extracted in CHCl(3) and the absorbance was monitored at 560 nm. An injection valve was used as a commutator in order to combine the stopped-flow technique with liquid-liquid extraction FI system. The calibration graph was linear up to 12 mg l(-1) (s(r)=0.27%; r=0.9999) with a detection limit of c(L)=0.007 mg l(-1). The sampling rate was 20 injections per hour. The proposed method has been successfully applied to the determination of palladium in airborne particulate matter (APM) and in automobile exhaust gas converter catalysts.     

Sequential injection technique for automated titration: spectrophotometric assay of vitamin C in pharmaceutical products using cerium(IV) in sulfuric acid

For the first time sequential injection analysis (SIA) technique has been employed for titrimetry. A new SI titrimetric spectrophotometric method for the assay of vitamin C in drug formulations was explored. The method is based on the oxidation reaction of vitamin C with cerium(IV) in sulfuric acid media using a spectrophotometer as a detector with the wavelength monitored at 410 nm. A 2(3) factorial design chemometric approach was employed to study the interaction effect of the chemical and system variables, mainly cerium(IV), sulfuric acid concentrations and the flow rate. The results of the chemometric optimization revealed that the optimum operating conditions for the SI titrimetric analysis of vitamin C were 7.0 x 10(-3) M cerium(IV), 0.455 M sulfuric acid and 28.9 microL s-1 flow rate. A linear calibration plot for the determination of vitamin C was obtained in the concentration range between 30 to 200 ppm. The method was applied to the determination of vitamin C in pharmaceutical preparations and no excipient was found to pose any interference, thus rendering the method suitable for the determination of the drug in pharmaceutical preparations. The SIA method is found to be accurate when the results were statistically compared with the results obtained by the BP standard method. The SIA method is superior when compared to the conventional titration method, the BP standard method and previous methods with respect to precision and automation in solution handling.     

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).     

毛细管气相色谱法测定速效伤风胶囊中对乙酰氨基酚、咖啡因、马来酸氯苯那敏的含量

 用５％Ｐｈ－Ｍｅ－Ｓｉｌｉｃｏｎｅ毛细管色谱柱，ＦＩＤ，二阶程序升温，以正十六烷作为内标物测定速效伤风胶囊中对乙酰氨基酚、咖啡因、马来酸氯苯那敏的含量。浓度线性范围：对乙酰氨基酚４～２０ｇ／Ｌ，咖啡因０．０８４～０．４２ｇ／Ｌ，马来酸氯苯那敏０．１５～０．７５ｇ／Ｌ。平均回收率（ｎ＝５）：对乙酰氨基酚９９．６２％（ＲＳＤ＝０．４４％）咖啡因９６．４６％（ＲＳＤ＝１．３２％），马来酸氯苯那敏９８．５５％（ＲＳＤ＝０．６５％）。     

Determination of sulfate in river water by flow injection analysis

Sulfate ion in river water is determined by flow injection analysis at a rate of 30 samples per hour; the sulfate contents are typically less than 30 ppm. The reagent solution contains dimethylsulfonazo-III, barium chloride, potassium nitrate and chloroacetate buffer in 70% ($\text{v}\text{v}$) ethanol, and is saturated with barium sulfate. The aqueous carrier stream is also saturated with barium sulfate. The sample is filtered and treated with Amberlite IR120-B cation-exchanger before injection into the carrier stream, and the decoloration of the barium—dimethylsulfonazo-III complex by sulfate is measured at 662 nm. The calibration graph is linear over the range 0–30 μg ml^-1 for sulfate in water.     

Flow-injection spectrophotometric determination of glucose in blood plasma with bindschedler's green leuco base as color reagent

The hydrogen peroxide produces in the oxidation of glucos in an immobilized glucose oxidase reactor is determined by using Bindschedler's green (leuco base) as color reagent with iron(II) as catalyst; the increase in the absorbance at 725 nm is measured. For 100-μl samples, calibration was almost linear in the range 0–2.5 mg l^?1 glucose; the relative standard deviation for 1 mg l^?1 glucose was 0.6% (n=10) and the detection limit (S/N= 2) was 0.02 mg l^?1. The injection rate was 20 h^?1. Glucose was determined satisfactorily in control sera and in real blood sera.     

Monitoring of ethanol during fermentation using a microbial biosensor with enhanced selectivity

The present study is concerning the construction of ferricyanide-mediated Gluconobacter oxydans cell ethanol biosensor. The size exclusion effect of a cellulose acetate membrane was used for elimination of glucose interferences during ethanol assays in real samples. A typical response time of the biosensor was 13 s with a high sensitivity of 3.5 microA mM(-1). The microbial biosensor exhibits a very low detection limit of 0.85 microM and a wide linear range from 2 to 270 microM. The operational stability was excellent. During 8.5 h of repetitive ethanol assays, no decrease in the sensor sensitivity was observed. The biosensor was successfully used in the off-line monitoring of ethanol fermentation with a good agreement with HPLC measurements (R(2)=0.998).     

Simultaneous analysis of aspartame and its hydrolysis products of Coca-Cola Zero by on-line postcolumn derivation fluorescence detection and ultraviolet detection coupled two-dimensional high-performance liquid chromatography

An innovative two-dimensional high-performance liquid chromatography system was developed for the simultaneous analysis of aspartame and its hydrolysis products of Coca-Cola Zero. A C8 reversed-phase chromatographic column with ultraviolet detection was used as the first dimension for the determination of aspartame, and a ligand-exchange chromatographic column with on-line postcolumn derivation fluorescence detection was employed as the second dimension for the analysis of amino acid enantiomers. The fluorimetric derivative reagent of amino acid enantiomers was o-phthaldialdehyde. The hydrolysis of aspartame in Coca-Cola Zero was induced by electric-heating or microwave heating. Aspartame was quantified by the matrix matched external standard calibration curve with a linear concentration range of 0-50 μg mL(-1) (r(2)=0.9984). The limit of detection (LOD) and the limit of quantification (LOQ) were 1.3 μg mL(-1) and 4.3 μg mL(-1), respectively. The amino acid enantiomers was analyzed by the matrix matched internal standard calibration method (D-leucine as the internal standard) with a linear concentration range of 0-10 μg mL(-1) (r(2)=0.9988-0.9997). The LODs and LOQs for L- and D-aspartic acid and L- and D-phenylalanine were 0.16-0.17 μg mL(-1) and 0.52-0.55 μg mL(-1), respectively, that was 12-13 times more sensitive than ultraviolet detection. The overall analysis accuracy for aspartame and amino acid enantiomers was 90.2-99.2% and 90.4-96.2%, respectively. The overall analysis precision for aspartame and amino acid enantiomers was 0.1-1.7% and 0.5-6.7%, respectively. Generally, the extent of aspartame hydrolysis increases with the increase of electro-thermal temperature, microwave power, and the duration of hydrolysis time. D-aspartic acid and D-phenylalanine can be observed with the electro-thermal racemization at the hydrolysis temperature 120°C for 1 day and only D-aspartic acid can be observed at the hydrolysis temperature 90°C for 2 and 3 days. For the microwave induced hydrolysis, only L-aspartic acid was detected at the power 560 W for 1 min and 320 W for 3 min.     

水杨基荧光酮胶束增敏流动注射分光光度法测定微量锗

本文将锗一水杨基荧光酮一溴化十六烷基三甲基铵胶束五色体系应用于流动注射分析，建立了测定微量锗的快速方法。方法采用单通道流路及合并带流路进行试验。对有关的管路参数及试剂浓度等影响因素进行了探讨。优化的流动注射分析体系对锗的检测灵敏度高、重现性好。方法用于混合成试样及有机锗生物制品中锗的测定，结果满意。     

Fluorescence polarization is a useful technology for reagent reduction in assay miniaturization

The use of fluorescence polarization (FP) has increased significantly in the development of sensitive and robust assays for high throughput screening of chemical compound libraries during the past few years. In this study, we show that FP is a useful assay miniaturization technology for reagent reduction during high throughput screening. We developed and optimized several FP assays for binding to estrogen receptor alpha and two protein kinases with an assay volume of 100 microl. Without any re-optimization, a consistent signal window was maintained in 384- or 1536-well format when the assay volume varied from 2.5-100 microl at constant concentrations of all assay components. In contrast, the signal window decreased with decreasing assay volume at constant reagent concentration in the protein kinase C scintillation proximity assay (SPA) and prompt fluorescence assay. In addition, the effect of evaporation on the signal window was minimal for the FP assays. Our study suggests that FP is superior to SPA and prompt fluorescence in terms of reagent reduction in the miniaturized assay format.     

Determination of beta-lactams and their biosynthetic intermediates in fermentation media by pre-column derivatisation followed by fluorescence detection

This paper describes a novel and sensitive pre-column derivatisation method for the detection and quantitation of beta-lactams and their biosynthetic precursors at trace levels in fermentation media. Filtered broths from fermentations of strains of Penicillium chrysogenum and Cephalosporium acremonium, after deproteination and centrifugation, were incubated with 9-fluorenylmethylchloroformate for 5 min at 20 degrees C in 0.2 M borate buffer at pH 7.7. Following two-fold pentane extraction of the reagent hydrolysis product, the aqueous layer was injected directly onto a C18 reversed-phase column, and products were detected spectrofluorimetrically with excitation and emission wavelengths of 260 and 313 nm, respectively. Detection limits of 0.01 and 0.05 micrograms ml-1 were achieved for both 6-aminopenicillanic acid (6-APA) and isopenicillin N in borate buffer and filtered fermentation broths, respectively, using a 10-microliter injection volume. A linear calibration for 6-APA in fermentation broth was obtained for a very wide concentration range (0.05-100 micrograms ml-1). Detection limits for solutions of cephalosporin C, deacetylcephalosporin C and deacetoxycephalosporin C in broth were all 0.25 micrograms ml-1. The detection limit for the beta-lactam precursor delta-(L-aminoadipyl)-L-alpha-cysteinyl-D-valine (ACV) dimer in borate buffer was 0.5 microgram ml-1. The cephalosporins and ACV dimer gave linear plots in the ranges 3-25 and 1-100 micrograms ml-1, respectively. Repeated analysis of 6-APA at a concentration of 10 micrograms ml-1 in filtered broth gave a mean peak area of 2.5.10(6) with a standard deviation of 2.6.10(5) using a 10-microliter injection volume. Ampicillin spiked into deproteinated blood serum gave a linear calibration in the concentration range 2-100 micrograms ml-1.     

Spectrophotometric determination of aluminum in river water with bromopyrogallol red and n-tetradecyltrimethylammonium bromide by flow injection analysis

In the proposed flow injection system, the reagent solution contains bromopyrogallol red, n-tetradecyltrimethylammonium bromide and hexamine in 60% ethanolic solution, and the carrier solution contains acetate buffer, 1,10-phenanthroline and hydroxylammonium chloride. Sample solutions (160 μl) acidified by sulfuric acid are injected and the peak absorbance at 623 nm is recorded. The detection limit is about 0.001 ppm and calibration plots are linear for the ranges 0–0.3 and 0–0.1 ppm aluminum.     

Electron spin resonance for quantitative assay of chlorpromazine in drug formulations by oxidation with cerium(IV) in sulfuric acid media

For the first time electron spin resonance (ESR) spectroscopic technique has been used for a highly selective determination of chlorpromazine. The method is based on the oxidation reaction of chlorpromazine with cerium(IV) in sulfuric acid media. In this method 3.8x10(-3) mol dm(-3) cerium(IV) was used in 0.035 mol dm(-3) sulfuric acid with the ESR spectra recorded at room temperature. A calibration equation of the following form was obtained over the linear concentration range of 10-100 ppm with a correlation coefficient (r) of 0.999: A=1.355+0.0382C. The results obtained by the ESR method were found to be comparable with those obtained by the British Pharmacopoeia (BP) method. The method suffers no interferences excipients rendering the method suitable for determination of this drug in pharmaceutical preparations.     

Determination of sulphonated azo dyes in food by ion-pair liquid chromatography with photodiode array and electrospray mass spectrometry detection

An ion-pair liquid chromatography method with on-line photodiode-array and electrospray mass spectrometry detection was developed to determine 10 commonly used sulphonated azo dyes (Tartrazine, Amaranth, New Coccine, Sunset Yellow FCF, Allura Red AC, Ponceau R, Ponceau 3R, Orange I, Orange II and Metanil Yellow) in food. A reversed phase C(18) column with gradient elution was utilized to separate these compounds. Triethylamine was added in the mobile phase as an ion-pair reagent for chromatographic separation. Photodiode-array detection was employed for quantitative determination and electrospray mass spectrometry was used for identification. Good linearity (0.05-10 ppm, r(2)=0.999) and detection limit (<0.01 ppm) were determined with 5 mul injection. In addition, precision and accuracy associated with this newly developed method will be presented. A liquid extraction method was also developed to extract these dyes from different foodstuffs. The application of this method was demonstrated by analyzing sulphonated azo dyes in soft drinks, fruit jam, and salted vegetables.     

An ethanol sensor based on cataluminescence on ZnO nanoparticles

A novel gas sensor for the determination of ethanol was proposed in the present work, which was based on the generated cataluminescence emission from catalytic oxidation of ethanol on the surface of ZnO nanoparticles. The cataluminescence characteristics and the effect of different parameters on the signal intensity, such as morphology of synthesized ZnO, temperature and flow rate, were discussed in detail. Under the optimized experimental conditions, the calibration curve of cataluminescence intensity versus ethanol vapor concentration was linear in the range 1.0-100 ppm, and with a detection limit of 0.7 ppm (S/N = 3). Compared with the traditional electrical conductivity-based ZnO gas sensor for the determination of ethanol, the proposed ethanol sensor showed the advantages of high sensitivity, high selectivity and low working temperature.