Ion-exchange resins as stationary phase in reactive chromatography

Dynamic behavior of bothisothermal and non-isothermal single-column chromatographic reactors with an ion-exchange resin as the stationary phase was investigated. The reactor performance was interpreted by using results obtained when studying the effect of the resin properties on the equilibrium and kinetic phenomena occurring simultaneously in the reactor. Mathematical models were derived for each phenomenon and combined to simulate the chromatographic reactor. The phenomena studied includes phase equilibria in multicomponent liquid mixture¿ion-exchange resin systems, chemicalequilibrium in the presence of a resin catalyst, diffusion of liquids in gel-type and macroporous resins, and chemical reaction kinetics. Above all, attention was paid to the swelling behavior of the resins and how it affects the kinetic phenomena. Several poly(styrene-co-divinylbenzene) resins with different cross-link densities and internal porosities were used. Esterification of acetic acid with ethanol to produce ethyl acetate and water was used as a model reaction system. Choosing an ion-exchange resin with a low cross-link density is beneficial inthe case of the present reaction system: the amount of ethyl acetate as well the ethyl acetate to water mole ratio in the effluent stream increase with decreasing cross-link density. The enhanced performance of the reactor is mainly attributed to increasing reaction rate, which in turn originates from the phase equilibrium behavior of the system. Also mass transfer considerations favor the use ofresins with low cross-link density. The diffusion coefficients of liquids in the gel-type ion-exchange resins were found to fall rapidly when the extent of swelling became low. Glass transition of the polymer was not found to significantlyretard the diffusion in sulfonated PS¿DVB ion-exchange resins. It was also shown that non-isothermal operation of a chromatographic reactor could be used to significantly enhance the reactor performance. In the case of the exothermic modelreaction system and a near-adiabatic column, a positive thermal wave (higher temperature than in the initial state) was found to travel together with the reactive front. This further increased the conversion of the reactants. Diffusion-induced volume changes of the ion-exchange resins were studied in a flow-through cell. It was shown that describing the swelling and shrinking kinetics of the particles calls for a mass transfer model that explicitly includes the limited expansibility of the polymer network. A good description of the process was obtained by combining the generalized Maxwell-Stefan approach and an activity model that was derived from the thermodynamics of polymer solutions and gels. The swelling pressure in the resin phase was evaluated by using a non-Gaussian expression forthe polymer chain length distribution. Dimensional changes of the resin particles necessitate the use of non-standard mathematical tools for dynamic simulations. A transformed coordinate system, where the mass of the polymer was used as a spatial variable, was applied when simulating the chromatographic reactor columns as well as the swelling and shrinking kinetics of the resin particles. Shrinking of the particles in a column leads to formation of dead volume on top of the resin bed. In ordinary Eulerian coordinates, this results in a moving discontinuity that in turn causes numerical difficulties in the solution of the PDE system. The motion of the discontinuity was eliminated by spanning two calculation grids in the column that overlapped at the top of the resin bed. The reactive and non-reactive phase equilibrium data were correlated with a model derived from thethermodynamics of polymer solution and gels. The thermodynamic approach used inthis work is best suited at high degrees of swelling because the polymer matrixmay be in the glassy state when the extent of swelling is low.

Streptavidin - A Versatile Binding Protein for Solid-Phase Immunoassays

Streptavidin, a tetrameric protein secreted by Streptomyces avidinii, binds tightly to a small growth factor biotin. One of the numerous applications of this high-affinity system comprises the streptavidin-coated surfaces of bioanalytical assays which serve as universal binders for straightforward immobilization of any biotinylated molecule. Proteins can be immobilized with a lower risk of denaturation using streptavidin-biotin technology in contrast to direct passive adsorption. The purpose of this study was to characterize the properties and effects of streptavidin-coated binding surfaces on the performance of solid-phase immunoassays and to investigate the contributions of surface modifications. Various characterization tools and methods established in the study enabled the convenient monitoring and binding capacity determination of streptavidin-coated surfaces. The schematic modeling of the monolayer surface and the quantification of adsorbed streptavidin disclosed the possibilities and the limits of passive adsorption. The defined yield of 250 ng/cm² represented approximately 65 % coverage compared with a modelled complete monolayer, which is consistent with theoretical surface models. Modifications such as polymerization and chemical activation of streptavidin resulted in a close to 10-fold increase in the biotin-binding densities of the surface compared with the regular streptavidin coating. In addition, the stability of the surface against leaching was improved by chemical modification. The increased binding densities and capacities enabled wider high-end dynamic ranges in the solid-phase immunoassays, especially when using the fragments of the capture antibodies instead of intact antibodies for the binding of the antigen. The binding capacity of the streptavidin surface was not, by definition, predictive of the low-end performance of the immunoassays nor the assay sensitivity. Other features such as non-specific binding, variation and leaching turned out to be more relevant. The immunoassays that use a direct surface readout measurement of time-resolved fluorescence from a washed surface are dependent on the density of the labeled antibodies in a defined area on the surface. The binding surface was condensed into a spot by coating streptavidin in liquid droplets into special microtiter wells holding a small circular indentation at the bottom. The condensed binding area enabled a denser packing of the labeled antibodies on the surface. This resulted in a 5 - 6-fold increase in the signal-to-background ratios and an equivalent improvement in the detection limits of the solid-phase immunoassays. This work proved that the properties of the streptavidin-coated surfaces can be modified and that the defined properties of the streptavidin-based immunocapture surfaces contribute to the performance of heterogeneous immunoassays.

Fabrication technology and applications of fiber Bragg gratings

In this thesis theoretical and technological aspects of fiber Bragg gratings (FBG) are considered. The fabrication of uniform and chirped fiber Bragg gratings using phase mask technique has been exploited throughout this study. Different requires of FBG inscription were considered and implemented experimentally to find economical and effective procedure. The hydrogen loading was used as a method for enhancement the photosensitivity of the fiber. The minimum loading time for uniform and chirped fiber Bragg gratings was determined as 3 days and 7 days at T = 50°C and hydrogen pressure 140 bar, respectively. The post-inscription annealing was considered to avoid excess losses induced by the hydrogen. The wavelength evolution during annealing was measured. The strain and temperature sensor application of FBG was considered. The wavelength shifts caused by tension and temperature were studied for both uniform and chirp fiber Bragg gratings.

Rapid analysis of multiclass antibiotic residues and some of their metabolites in hospital, urban wastewater and river water by ultra-highperformance liquid chromatography coupled to quadrupole-linear ion trap tandem mass spectrometry

The present work describes the development of a fast and robust analytical method for the determination of 53 antibiotic residues, covering various chemical groups and some of their metabolites, in environmental matrices that are considered important sources of antibiotic pollution, namely hospital and urban wastewaters, as well as in river waters. The method is based on automated off-line solid phase extraction (SPE) followed by ultra-high-performance liquid chromatography coupled to quadrupole linear ion trap tandem mass spectrometry (UHPLC–QqLIT). For unequivocal identification and confirmation, and in order to fulfill EU guidelines, two selected reaction monitoring (SRM) transitions per compound are monitored (the most intense one is used for quantification and the second one for confirmation). Quantification of target antibiotics is performed by the internal standard approach, using one isotopically labeled compound for each chemical group, in order to correct matrix effects. The main advantages of the method are automation and speed-up of sample preparation, by the reduction of extraction volumes for all matrices, the fast separation of a wide spectrum of antibiotics by using ultra-high-performance liquid chromatography, its sensitivity (limits of detection in the low ng/L range) and selectivity (due to the use of tandem mass spectrometry) The inclusion of β-lactam antibiotics (penicillins and cephalosporins), which are compounds difficult to analyze in multi-residue methods due to their instability in water matrices, and some antibiotics metabolites are other important benefits of the method developed. As part of the validation procedure, the method developed was applied to the analysis of antibiotics residues in hospital, urban influent and effluent wastewaters as well as in river water samples

Studies of Liquid-liquid Separation in Fibre Coalescer

This thesis focuses on fibre coalescers whose efficiency is based on the surface properties/characteristics. They have the ability to preferentially wet or interact with one or more of the fluids to be separated. Thus, the interfacial phenomena governing the separation efficiency of the coalescers is investigated depending on physical factors such as flowrates, phase ratios and coalescer packing density. Design of process equipment to produce and separate of the emulsions was carried out.The experimentation was carried out to test the separation efficiency of the coalescing medias, namely fibreglass, polyester I and polyester II. The performances of the coalescing medias were assessed via droplet size information. In conclusion, the objectives (design of process equipment and experimentation) were achieved. Fibre glass was the best coalescing media, next was polyester I and then finally polyester II. Droplets sizes increased with decreased flowrates and increased packing density of the coalescer. Phase ratio had effect on the droplet sizes of the feed but had no effect on the coalescence of droplets of the feed.

Reversed phase HPLC determination of tamoxifen in dog plasma and its pharmaco-kinetics after a single oral dose administration

The analytical method developed to evaluate tamoxifen in dog plasma samples was precise, accurate, robust and linear in the range of 5-200 ng/mL. The limits of detection and quantification were 0.981 ng/mL and 2.97 ng/mL, respectively. Besides, the intra-day precision and accuracy variations were 8.78 and 10.16%, respectively. Tamoxifen concentrations were analyzed by combined reversed phase liquid chromatography and UV detection (lambda=280 nm). The study was conducted using an open randomized 2-period crossover balanced design with a 1-week washout period between the doses. This simple, rapid and selective method is suitable for pharmacokinetic, bioavailability and bioequivalence studies.

Determination of etoricoxib in human plasma using automated on-line solid-phase extraction coupled with LC-APCI/MS/MS

A liquid chromatography-tandem mass spectrometry method with atmospheric pressure chemical ionization (LC-APCI/MS/MS) was validated for the determination of etoricoxib in human plasma using antipyrin as internal standard, followed by on-line solid-phase extraction. The method was performed on a Luna C18 column and the mobile phase consisted of acetonitrile:water (95:5, v/v)/ammonium acetate (pH 4.0; 10 mM), run at a flow rate of 0.6 mL/min. The method was linear in the range of 1-5000 ng/mL (r²>0.99). The lower limit of quantitation was 1 ng/mL. The recoveries were within 93.72-96.18%. Moreover, method validation demonstrated acceptable results for the precision, accuracy and stability studies.

Recovery of organic acids with liquid-liquid extraction contactors

Liquid-liquid extraction is a mass transfer process for recovering the desired components from the liquid streams by contacting it to non-soluble liquid solvent. Literature part of this thesis deals with theory of the liquid-liquid extraction and the main steps of the extraction process design. The experimental part of this thesis investigates the extraction of organic acids from aqueous solution. The aim was to find the optimal solvent for recovering the organic acids from aqueous solutions. The other objective was to test the selected solvent in pilot scale with packed column and compare the effectiveness of the structured and the random packing, the effect of dispersed phase selection and the effect of packing material wettability properties. Experiments showed that selected solvent works well with dilute organic acid solutions. The random packing proved to be more efficient than the structured packing due to higher hold-up of the dispersed phase. Dispersing the phase that is present in larger volume proved to more efficient. With the random packing the material that was wetted by the dispersed phase was more efficient due to higher hold-up of the dispersed phase. According the literature, the behavior is usually opposite.

Highly selective transport of mercury(II) ion through a bulk liquid membrane

In this work carrier-facilitated transport of mercury(II) against its concentration gradient from aqueous 0.04 M hydrochloric acid solution across a liquid membrane containing isopropyl 2-[(isopropoxycarbothiolyl)disulfanyl]ethane thioate (IIDE) as the mobile carrier in chloroform has been investigated. Sodium thiocyanate solution (1.6 M) was the most efficient receiving phase agent among several aqueous reagents tested. Various parameters such as investigated. Under optimum conditions the transport of Hg(II) across the liquid membrane is more than 97% after 2.5 h. The carrier, IIDE, selectively and efficiently could able to transport Hg (II) ions in the presence of other associated metal ions in binary systems.

Quantitative determination of acetaminophen, phenylephrine and carbinoxamine in tablets by high-performance liquid chromatography

An alternative methodology for analysis of acetaminophen (Ace), phenylephrine (Phe) and carbinoxamine (Car) in tablets by ion-pair reversed phase high performance liquid chromatography was validated. The pharmaceutical preparations were analyzed by using a C18 column (5 μm, 300 mm, 3.9 mm) and mobile phase consisting of 60% methanol and 40% potassium monobasic phosphate aqueous solution (62.46 mmol L-1) added with 1 mL phosphoric acid, 0.50 mL triethylamine and 0.25 g sodium lauryl sulfate. Isocratic analysis was performed under direct UV detection at 220 nm for Phe and Car and at 300 nm for Ace within 5 min.

Determination of phenobarbital in human plasma by a specific liquid chromatography method: application to a bioequivalence study

A liquid chromatography method was developed and validated for the determination of phenobarbital in human plasma using phenytoin as internal standard. The drugs were extracted from plasma by liquid-liquid extraction and separated isocratically on a C12 analytical column, maintained at 35 ºC, with water:acetonitrile:methanol (58.8:15.2:26, v/v/v) as mobile phase, run at a flow rate of 1.2 mL/min with detection at 205 nm. The method was linear in the range of 0.1-4 μg/mL (r²=0.9999) and demonstrated acceptable results for the precision, accuracy and stability studies. The method was successfully applied for the bioequivalence study of two tablet formulations (test and reference) of phenobarbital 100 mg after single oral dose administration to healthy human volunteers.

Development and validation of a high performance liquid chromatographic method for determination of cyclosporine-A from biodegradable intraocular implants

An HPLC method was developed and validated aiming to quantify the cyclosporine-A incorporated into intraocular implants, released from them; and in direct contact with the degradation products of PLGA. The separation was carried out in isocratic mode using acetonitrile/water (70:30) as mobile phase, a C18 column at 80 ºC and UV detection at 210 nm. The method provided selectivity based on resolution among peaks. It was linear over the range of 2.5-40.0 µg/mL. The quantitation and detection limits were 0.8 and 1.2 µg/mL, respectively. The recovery was 101.8% and intra-day and inter-day precision was close to 2%.

Chemical stability of enalapril maleate drug substance and tablets by a stability-indicating liquid chromatographic method

The chemical stability of enalapril drug substance and tablets was studied by a stability-indicating liquid chromatographic method. Stress testing was performed on drug substance under various conditions. Accelerated stability testing was carried out for different formulations of enalapril tablets. Chromatographic separation was achieved on a RP-18 column, using a mobile phase of methanol phosphate buffer at 1.0 mL min"1 and UV detection. Degradation of the drug substance was greater under hydrolytic conditions. After 180 days of accelerated stability testing most enalapril tablets showed more than 10% of degradation. Enalapril drug substance and tablets showed instability under stress and accelerated testing respectively, with possible implications on the therapeutic activity.

High performance thin layer chromatographic determination of nifedipine in human serum after liquid-liquid extraction

A method using HPTLC for quantitation of nifedipine in serum was developed and validated. It includes a liquid-liquid extraction, and carbamazepine as internal standard. Chloroform: ethyl acetate: cyclohexane (19:2:2, v/v/v) was the mobile phase. The method showed good relationship (r = 0.996) (2.00 to 25.00 ng/band, corresponding to 0.02 and 0.25 ng/µL in serum). The % RSD of intra-assay and inter-assay, were between 0.57 and 3.56 and 1.16 to 3.60, respectively. LOD and LOQ were 0.72 and 0.86 ng/band, respectively. The recovery values were between 93 and 102%. Rf for nifedipine and carbamazepine were 0.31 and 0.10, respectively.

Quantification of tryptophan in plasma by high performance liquid chromatography

A simple, rapid and selective method using high-performance liquid chromatography with ultraviolet detection (267 nm) was applied for the determination of tryptophan in plasma. Separation was carried out on a C18 column (150 x 4.6 mm internal diameter) in 6 min. The mobile phase consisted of 5 mM the sodium acetate and acetonitrile (92:8, v/v). The method was shown to be precise and accurate, and good recovery of analyte was achieved, characterizing the method as efficient and reliable for use in laboratory analysis.