Determination of total ammonium-nitrogen and free ammonia in a fermentation medium by sequential injection analysis

Summary Automated methods for the determination of ammonium and ammonia are reviewed, and techniques based on gas diffusion using a semi-permeable membrane were selected for the determination of both total ammonium-nitrogen and free ammonia in fermentation samples. A simple and robust instrument based on sequential injection analysis (SIA) consisting of a piston pump and two selector valves was used. Two different methods of ammonia detection, the Berthelot method and detection using pH-indicators, have been evaluated and compared. The indicator method showed higher reproducibility and the range of determination could easily be adjusted to match the concentrations of the samples. The range of determination could be adjusted from 0.05 mmol/l to 350 mmol/l, depending on choice of acceptor solution. This method has been evaluated with fermentation medium samples and tested on-line in a yeast fermentation process.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Automated method, based on micro-sequential injection, for the study of enzyme kinetics and inhibition.

A micro-reactor system with continual spectrophotometric detection has been operated in Sequential Injection lab-on-valve (SI-LOV) mode and applied to enzyme kinetics and inhibition studies, using acetylcholinesterase (AChE) and angiotensin-converting enzyme (ACE) as model systems. With the advantages of automation, real-time kinetic measurement, and thorough mixing, the SI-LOV micro-reactor system allows for the monitoring of initial reaction rates and determination of reactant concentrations in the reaction mixture, both of which are essential for the determination of kinetic constants for enzymes and inhibitors. Enzyme, substrate, and inhibitor are precisely metered by the syringe pump and delivered to a stirred micro-reactor, followed by a reference scan that establishes the baseline for the following reaction rate measurement. Michaelis constants (Km) for AChE and ACE were determined to be 0.16 mM and 0.30 mM, respectively, which are consistent with literature values. The type of inhibition (competitive, uncompetitive, or mixed), the dissociation constants for the inhibitors, and the inhibitor dose-response curves were also determined.     

Sequential Injection Photometric Determination of Molybdenum Using Organic Wetting Film Extraction

Wetting film extraction was combined with colorimetry to determine nanogram amounts of molybdenum(VI). The simple extraction procedure enhanced sensitivity and selectivity while maintaining a high sample throughput. Extraction and back extraction steps were exploited to exclude interference from the 31 metal species and 11 anions tested. In the first step, molybdenum(VI) was extracted into a toluene film as an ion paired complex. Molybdenum(VI) reacted with thiocyanate to form anionic molybdenum(V) and/or molybdenum(VI) thiocyanate complexes. The complexes were extracted into a toluene film containing tetraheptylammonium bromide as ion pairing reagent. The thiocyanate ligands were displaced by 1,5-diphenylcarbazone (DPC) to form a more intensely colored complex (λ_max= 540 nm). DPC was introduced in the back extraction solvent, methanol. The relative standard deviation was 2.5% for 50 ng ml⁻¹of molybdenum(VI) (n= 10) at a rate of 25 samples h⁻¹. The detection limit (3 × baseline noise) was 2.5 × 10⁻⁸M.     

Synthesis and binding affinities of novel SRIF-mimicking beta-D-glucosides satisfying the requirement for a pi-cloud at C1

[reaction: see text] The synthesis of four bioactive analogues of the somatostatin (SRIF-14) mimetic, beta-d-glucoside (+)-2, in which the C1 indole side chain is replaced with indole surrogates, has been achieved. These congeners, possessing the naphthyl, benzothiophene, benzyl, and benzofuran substituents, were predicted to satisfy the electrostatic requirements of the tryptophan binding pocket of SRIF. Unlike the previously described C4 picolyl and pyrazinyl congeners, these ligands bind the hSST4 receptor.     

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.     

Flow/sequential injection determination of gaseous ammonia with a glass diffusion denuder

A new combination of a flow/sequential injection method for the analysis of ammonia has been developed. Gaseous ammonia is selectively absorbed in a phosphoric acid coated glass tube and determined with Berthelot reagents by flow injection. The combination of the gas diffusion denuder sampler with flow injection makes this method very sensitive and selective. The limit of detection of 0.15 mug NH(3).     

Measurement of cellular stimulation through monitoring pH changes by bead injection fluorescence microscopy

This work presents a method for extracellular and intracellular pH measurements in live cells based on a combination of the bead injection (BI) technique and fluorescence microscopy. For extracellular pH measurement, cells are grown on fluorescent beads, packed into a small column by a sequential injection instrument, and fluorescence intensity from the beads stained by the indicator is recorded by a fluorescence microscope. The method is applied to quantifying carbachol stimulation of Chinese hamster ovary (CHO) cells transfected with the m1 muscarinic receptor and is verified by a glucose depletion experiment. The results yield an EC(50) value of 1 muM for carbachol, which is in reasonable agreement with the literature value 3 muM determined by an existing potentiometric technique for measuring acid release. The intracellular measurement utilizes CHO M1 cells growing on non-fluorescent beads. For this method the cells rather than the beads are stained by incubating them in a solution of the fluorescent pH indicator BCECF. The cells are also stimulated with carbachol and the intracellular pH dependent fluorescence from the cells is recorded. The results show dependence between intracellular pH changes and carbachol concentration and yield an EC(50) value of 4 muM.     

Ring inversion process for a series of 3,5‐dialkyl‐1‐oxa‐3,5‐diazacyclohexanes: 1H DNMR study and semiempirical calculations

The ring inversion process for a series of 3,5‐dialkyl‐1‐oxa‐3,5‐diazacyclohexanes was studied using proton dynamic nuclear magnetic resonance (¹H DNMR) spectroscopy in conjunction with semiempirical calculations. At low temperature, the ring methylene protons decoalesced into two AB spin systems in a 2:1 ratio. Lineshape simulations of the DNMR spectra provided first‐order rate constants for magnetic exchange. The energy barrier for each inversion reaction was calculated from the respective rate constants. In general, as the size of the N‐alkyl group increased, the barrier to ring inversion decreased. A similar trend was seen in semiempirical calculations that modeled the ring inversion process. Copyright © 2010 John Wiley & Sons, Ltd.     

Optical properties of the quasi-two-dimensional dichalcogenides 2H-TaSe and 2H-NbSe

We present a comprehensive analysis of the optical constants of the two-dimensional dichalcogenide materials 2 H - TaSe ₂ and 2 H - NbSe ₂ , in an attempt to address the physics of two-dimensional correlated systems. The title compounds were studied over several decades in frequency, from the far-infrared to the ultraviolet. Measurements with linearly polarized light have allowed us to obtain both the in-plane and out-of-plane components of the conductivity tensor. Although the electromagnetic response of dichalcogenides is strongly anisotropic, both the in-plane and out-of-plane components of the conductivity tensor share many common features, including the presence of a well-defined metallic component, as well as a “mid-infrared band”. We discuss the implications of these results in the context of the spectroscopic results of other classes of low-dimensional conductors such as the high-temperature superconducting cuprates. In particular, the analysis of the redistribution of the spectral weight as a function of temperature, as well as the behavior of the quasiparticles relaxation rate, points to significant distinctions between the charge dynamics of dichalcogenides and other classes of low dimensional conductors. Received 28 October 2002 / Received in final form 10 March 2003 Published online 23 May 2003 RID="a" ID="a"e-mail: degiorgi@solid.phys.ethz.ch