Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: Electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.

Measuring the interaction forces between protein inclusion bodies and an air bubble using an atomic force microscope

Interaction forces between protein inclusion bodies and an air bubble have been quantified using an atomic force microscope (AFM). The inclusion bodies were attached to the AFM tip by covalent bonds. Interaction forces measured in various buffer concentrations varied from 9.7 nN to 25.3 nN (+/- 4-11%) depending on pH. Hydrophobic forces provide a stronger contribution to overall interaction force than electrostatic double layer forces. It also appears that the ionic strength affects the interaction force in a complex way that cannot be directly predicted by DLVO theory. The effects of pH are significantly stronger for the inclusion body compared to the air bubble. This study provides fundamental information that will subsequently facilitate the rational design of flotation recovery system for inclusion bodies. It has also demonstrated the potential of AFM to facilitate the design of such processes from a practical viewpoint.

Model for gas-liquid flow through wet porous medium

A method involving bubbling of air through a fibrous filter immersed in water has recently been investigated (Agranovski et al. [1]). Experimental results showed that the removal efficiency for ultra-fine aerosols by such filters was greatly increased compared to dry filters. Nuclear Magnetic Resonance (NMR) imaging was used to examine the wet filter and to determine the nature of the gas flow inside the filter (Agranovski et al. [2]). It was found that tortuous preferential pathways (or flow tubes) develop within the filter through which the air flows and the distribution of air and water inside the porous medium has been investigated. The aim of this paper is to investigate the geometry of the pathways and to make estimates of the flow velocities and particle removal efficiency in such pathways. A mathematical model of the flow of air along the preferred pathways has been developed and verified experimentally. Even for the highest realistic gas velocity the flow field was essentially laminar (Re approximate to 250). We solved Laplace's equation for stream function to map trajectories of particles and gas molecules to investigate the possibility of their removal from the carrier.

Genetic algorithms for design of liquid retaining structure

In this paper, genetic algorithm (GA) is applied to the optimum design of reinforced concrete liquid retaining structures, which comprise three discrete design variables, including slab thickness, reinforcement diameter and reinforcement spacing. GA, being a search technique based on the mechanics of natural genetics, couples a Darwinian survival-of-the-fittest principle with a random yet structured information exchange amongst a population of artificial chromosomes. As a first step, a penalty-based strategy is entailed to transform the constrained design problem into an unconstrained problem, which is appropriate for GA application. A numerical example is then used to demonstrate strength and capability of the GA in this domain problem. It is shown that, only after the exploration of a minute portion of the search space, near-optimal solutions are obtained at an extremely converging speed. The method can be extended to application of even more complex optimization problems in other domains.

Conductivity, NMR and crystallographic study of N,N,N,N-tetramethylammonium dicyanamide plastic crystal phases: an archetypal ambient temperature plastic electrolyte material

N,N,N,N-Tetramethylammonium dicyanamide (Me(4)NDCA) has been examined via differential scanning calorimetry (DSC), thermogravimetric analysis, conductivity, single crystal X-ray diffraction and H-1 nuclear magnetic resonance (NMR) analyses, and was found to be highly conductive in the solid state (sigma = 10(-3) S cm(-2) at 420 K) and to also exhibit unusual plastic crystal behaviour. To investigate the correlation between such behaviour and the occurrence of molecular rotations in the crystal, H-1 NMR second moment measurements are compared with calculated values predicted from the crystal structure. While DSC analysis indicates a number of solid-solid transitions at ambient temperatures, subsequent H-1 NMR analysis of the Me4N+ cation shows that a variety of rotational motions become active at low (

Electrooxidation of glyphosate herbicide at different DSA (R) compositions: pH, concentration and supporting electrolyte effect

This paper has investigated the electrochemical oxidation of glyphosate herbicide (GH) on RuO(2) and IrO(2) dimensionally stable anode (DSA (R)) electrodes. Electrolysis was achieved under galvanostatic control as a function of pH, GH concentration, supporting electrolyte, and current density. The influence of the oxide composition on GH degradation seems to be significant in the absence of chloride; Ti/Ir(0.30)Sn(0.70)O(2) is the best electrode material to oxidize GH. GH oxidation is favored at low pH values. The use of chloride medium increases the oxidizing power and the influence of the oxide composition is meaningless. At 30 mA cm(-2) and 4 h of electrolysis, complete GH removal from the electrolyzed solution has been obtained. In chloride medium, application of 50 mA cm(-2) leads to virtually total mineralization ( release of phosphate ions = 91%) for all the evaluated oxide materials. (C) 2008 Elsevier Ltd. All rights reserved.

Platinum nanoparticles embledded in layer-by-layer films from SnO2/polyallylamine for ethanol electrooxidation

Self-assembled films from SnO2 and polyallylamine (PAH) were deposited on gold via ionic attraction by the layer-by-layer(LbL) method. The modified electrodes were immersed into a H2PtCl6 solution, a current of 100 mu A was applied, and different electrodeposition times were used. The SnO2/PAH layers served as templates to yield metallic platinum with different particle sizes. The scanning tunnel microscopy images show that the particle size increases as a function of electrodeposition time. The potentiodynamic profile of the electrodes changes as a function of the electrodeposition time in 0.5 mol L-1 H2SO4, at a sweeping rate of 50mVs(-1). Oxygen-like species are formed at less positive potentials for the Pt-SnO2/PAH film in the case of the smallest platinum particles. Electrochemical impedance spectroscopy measurements in acid medium at 0.7 V show that the charge transfer resistance normalized by the exposed platinum area is 750 times greater for platinum electrode (300 k Omega cm(2)) compared with the Pt-SnO2/PAH film with 1 min of electrodeposition (0.4 k Omega cm(2)). According to the Langmuir-Hinshelwood bifunctional mechanism, the high degree of coverage with oxygen-like species on the platinum nanoparticles is responsible for the electrocatalytic activity of the Pt-SnO2/PAH concerning ethanol electrooxidation. With these features, this Pt-SnO2/PAH film may be grown on a proton exchange membrane (PEM) in direct ethanol fuel cells (DEFC). (c) 2008 Elsevier B.V. All rights reserved.

Quantification of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high-performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. Important factors in the optimization of SBSE efficiency such as pH, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) assured recoveries ranging from 72 to 86%, except for phenytoin (62%). Separation was obtained using a reverse phase C-18 column with UV detection (210 nm). The mobile phase consisted of water: acetonitrile (78:22, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.08-40.0 mu g mL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125-40.0 mu g mL(-1) for phenytoin, The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.0, 4.0 and 20.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 8.8% and all inter-CVs were less than 10%. Limits of quantification were 0.08 mu g mL(-1) for carbamazepine, carbamazepine-10,11-epoxide and phenobarbital and 0.125 mu g mL(-1) for phenytoin. No interference of the drugs normally associated with antiepileptic drugs was observed. Based on figures of merit results, the SBSE/HPLC-UV proved adequate for antiepileptic drugs analyses from therapeutic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (c) 2008 Elsevier B.V. All rights reserved.

Hyaluronidase Behavior at the Air/Liquid and Air/Lipid Interfaces and Improved Enzymatic Activity by Its Immobilization in a Biomembrane Model

Bovine testicular hyalurphidase (BT-HAase), a tetrameric enzyme responsible for randomly hyaluronic acid, catalytic hydrolysis, was successfully immobilized on Langmuir- Blodgett films prepared with the sodium salt of dihexadacylphosphoric acid, (DHP-Zn(II)) ending with dipalmitoylphosphatidylcholine, DPPC. Data of protein, adsorption at the air-liquid interface by means of pendant drop shipe analysis and interaction of the protein with Langmuir monolayers of DPPC, using a Langmuir trough, have provided information. about the conditions to be used in the protein immobilization. The dynamic surface pressure curves obtained from pendant drop experiments for the enzyme in buffer solutions indicate that, within the range of concentration investigated in this study, the enzyme exhibits the largest induction time at 5 mu g L(-1) attributed to diffusion processes. Nevertheless, it seems that, at this concentration, the most probable conformation should be the one which occupies the smallest area at pi -> 0. The surface pressure (pi) area curves obtained for BT-HAase and mixed DPPC- BT-HAase monolayers reveal the presence of the enzyme at the air-lipid interface up to 45 mN m(-1). Tests of enzymatic activity, using hyaluronic acid, HA, as the substrate, showed an increase of activity compared to the homogeneous medium. A simplified model of protein insertion into the lipid matrix is used to explain the obtained results.

In-tube solid-phase microextraction coupled to liquid chromatography (in-tube SPME/LC) analysis of nontricyclic antidepressants in human plasma

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for simultaneous determination of mirrazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline in human plasma was developed, validated and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. The quantification limits of the in-tube SPME/LC method varied between 20 and 50 ng/mL, with a coefficient of variation lower than 10%. The response of the in-tube SPME/LC method for most of the drugs was linear over a dynamic range from 50 to 500 ng/mL, with correlation coefficients higher than 0.9985. The in-tube SPME/LC can be successfully used to analyze plasma samples from ageing patients undergoing therapy with nontricyclic antidepressants. (c) 2007 Elsevier B.V. All rights reserved.

Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection

A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC-UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw-eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 mu L.), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC-UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL(-1). The LOQ values ranged from 10 to 25 ng mL(-1). The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC-UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC-UV method was applied to analysis of plasma samples from elderly depressed patients. (C) 2010 Elsevier B.V. All rights reserved.

Rifampicin determination in plasma by stir bar-sorptive extraction and liquid chromatography

A sensitive and reproducible stir bar-sorptive extraction and high performance liquid chromatography-UV detection (SBSE/HPLC-UV) method for therapeutic drug monitoring of rifampicin in plasma samples is described and compared with a liquid:liquid extraction (LLE/HPLC-UV) method. This miniaturized method can result in faster analysis, higher sample throughput, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. Important factors in the optimization of SBSE efficiency such as pH, temperature, extraction time and desorption conditions (solvents, mode magnetic stir, mode ultrasonic stir, time and number of steps) were optimized recoveries ranging from 75 to 80%. Separation was obtained using a reverse phase C(8) column with UV detection (254 nm). The mobile phase consisted of methanol:0.25 N sodium acetate buffer, pH 5.0 (58:42, v/v). The SBSE/HPLC-UV method was linear over a working range of 0.125-50.0 mu g mL(-1). The intra-assay and inter-assay precision and accuracy were studied at three concentrations (1.25, 6.25 and 25.0 mu g mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 10% and all inter-CVs were less than 10%. Limits of quantification were 0.125 mu g mL(-1). Stability studies showed rifampicin was stable in plasma for 12 h after thawing; the samples were also stable for 24 h after preparation. Based on the figures of merit results, the SBSE/HPLC-UV proved to be adequate to the rifampicin analyses from therapeutic to toxic levels. This method was successfully applied to the analysis of real samples and was as effective as the LLE/HPLC-UV method. (C) 2009 Elsevier B.V. All rights reserved.

Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been developed, validated, and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5 mM and sodium phosphate 0.25 M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine. (C) 2009 Elsevier B.V. All rights reserved.

Automated determination of rifampicin in plasma samples by in-tube solid-phase microextraction coupled with liquid chromatography

A sensitive and automated method is described for determination of rifampicin in plasma samples for therapeutic drug monitoring by in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC). Important factors in the optimization of in-tube SPME are discussed, such as coating type, sample pH, sample draw/eject volume, number of draw/eject cycles, and draw/eject flow rate. Analyte pre-concentrated in the polyethylene glycol phase was directly transferred to the liquid chromatographic column by percolation of the mobile phase, without carryover. The method was linear over the 0.1-100 mu g/mL range, with a linear coefficient value (r(2)) of 0.998. The inter-assay precision presented coefficient of variation <= 1.7%. The effectiveness and practicability of the proposed method are proven by analysis of plasma samples from ageing patients undergoing therapy with rifampicin. (C) 2011 Elsevier B.V. All rights reserved.