The development of energy transfer immunoassay methods

The use of fluorescein and rhodamine as donor and acceptor fluorescent labels in the development of energy transfer immunoassay (ETIA) methods was evaluated by developing an assay for human serum albumin. The sensitivity of the assay was found to depend on (i) the degrees of fluorophore labelling of antibody and antigen, (ii) concentrations of labelled antibody and antigen, (iii) the fluorimeter spectral bandwidth, and (iv) whether the donor (fluorescein) was conjugated to the antigen or the antibody. These results, including those relating to the stability of the labelled immune reactants on storage, lead to the conclusion that fluorescein and rhodamine are far from ideal as donor and acceptor. Nevertheless, the application of the assay to the analysis of test serum samples gave results that compared favourably with those obtained by electroimmunoassay. Other potential donor and acceptor fluorescent labels were also investigated, viz. Ca) fluorescamine and fluorescein, (b) MDPF and fluorescein, (c) dansyl chloride and rhodamine, (d) quinacrine and fluorescein, and (e) quinacrine and rhodamine. Of these, only (a) and (b) were found to be suitable donor-acceptor pairs, and they were applied to the development of a number of assays for both low and high molecular-weight analytes. Comparative studies of fluorescamine and MDPF as donor fluorescent labels together with fluorescein as the acceptor label were performed by developing immunoassay methods for the determination of human serum transferrin in four serum samples including a blood sample from the victim of a road traffic accident. Results obtained were generally in good agreement with those found by the radial immunodiffusion method. An ETIA developed ·for nortriptyline and related tricyclic antidepressants was capable of detecting nanomolar concentrations of the drugs in pure solution and in spiked sera. Other ETIA's developed include a sandwich assay for the quantitation of human immunoglobulin A, and a direct assay for the determination of human immunoglobulin G. The Fluram as well as the MDPF enhancement phenomena were also studied in detail and applied successfully to the development of fluorescence enhancement immunoassays for nortriptyline, human serum transferrin and immunoglobulin G. Finally, the automation of an energy transfer immunoassay was successfully performed by using the principles of stopped-flow injection analysis with merging zones

POLYCYCLIC POLYAMINES: SYNTHESIS AND CONFORMATIONAL ANALYSIS

The synthesis, conformational analysis, and reactivity of a homologous series of tricyclic orthoamides is discussed. The tricyclic orthoformamides, orthoacetamides, orthopropionamides, and orthobenzamides were synthesized by the uncatalyzed condensation of macrocyclic triamines with amide acetals. The conformations were studied spectrally (IR, (\u271)H NMR, (\u2713)C NMR, DNMR) and by the application of empirical force field calculations (MM2). In most (but not all) cases the minimized conformations as generated by MM2 were found to be in agreement with the experimentally determined conformations. The alkylation, acylation, and hydrolysis of these compounds is also discussed. Efforts towards the synthesis of the spherically shaped host molecule 1,5,9,13,-tetraazatricyclo{7.7.3.3(\u275,13)}-docosane are described. A classical acylation-reduction sequence was employed in this synthesis. Cyclizations were carried out under high dilution conditions. The design and construction of a new high dilution apparatus is described. High yields of monomeric cyclic intermediates were obtained. Monomeric cyclic intermediates were purified by preparative gel permeation chromatography (GPC). The modification of a Waters 200 analytical GPC unit are described as are the column packing procedures for preparative GPC columns

Analytical Pyrolysis Principles and Applications to Environmental Science

Over the past half century, analytical pyrolysis has proven itself to be an effective means for the semiquantitative characterization of complex macromolecular organic substances. It has been demonstrated that instruments such as Py-FID, Py-MS, and in particular, Py-GC/MS can provide valuable geochemical insights when applied to a wide variety of problems in environmental science. The more widespread use of analytical pyrolysis methods in the evaluation of environmental pollution is recommended, because of their relatively low cost and information-rich results. Pyrolysis is the heating of organic substances in an inert, oxygen-free atmosphere, thereby avoiding combustion. When performed on a large scale, pyrolysis is involved in industrial processes as diverse as the manufacture of coke from coal and the conversion of biomass into biofuels. In contrast, analytical pyrolysis is a laboratory procedure in which small amounts of organic materials undergo thermal treatment, the products of which are subsequently quantified and/or characterized, for example, by gas chromatography. The pyrolysis may be performed “off-line” or “on-line.” In the off-line case, pyrolysis occurs in stand-alone reactor. The pyrolysis products are then extracted or trapped manually prior to further evaluation by chromatographic or other means. In on-line methods, the pyrolysis reactor is coupled directly to the analytical system, be it the injector of a gas chromatograph or a detector such as a flame ionization device or a mass spectrometer, with the pyrolyzate swept along its course by inert carrier gas. In some cases, a trapping mechanism such as cryofocusing is employed, which can permit the use of multiple detection or analytical systems. On-line methods typically only require milligram or even submilligram quantities of sample. Samples may be analyzed with little pretreatment, thereby minimizing the use of hazardous solvents in the spirit of environmentally conscious “green chemistry.

Analysis of Secondary Amino Group Containing Drugs in Bulk Powder and Their Dosage form (Pharmaceutical Formulations)

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Growth of unsaturated, cyclic, and polycyclic aromatic hydrocarbons: Reactions under the conditions of the interstellar medium

Hydrocarbons, in particular polycyclic aromatic hydrocarbons (PAHs), have been long discussed to be carriers of interstellar infrared (IR) emission and ultraviolet (UV) absorption features. Yet, their origin in dense phases of the interstellar medium (ISM), such as molecular clouds, remains unclear. In this work, growth mechanisms based on ion-molecule reactions between cationic PAHs/hydrocarbons and methyne (CH) were investigated. The reaction type and the precursor were derived and selected from known chemical and physical properties of the ISM. These chemical reactions were characterised by calculating branching ratios (based on cross sections) and capture rate coefficients, minimum reaction paths, reaction enthalpies, thermal equilibrium constants, and microcanonic isomerisation and radiative deactivation rate coefficients. In order to cope with the variety of reaction parameters, a hierarchic workflow scheme was set up. First, the reaction potential energy surface was sampled by molecular dynamics simulations. Then, minimum energy paths of the most probable reaction channels were investigated. Finally, molecular and kinetic properties of stationary points were calculated. The quantum chemical level of theory was increased at each step from DFTB (tight-binding density-functional), to DFT, and finally to post-Hartree-Fock methods. Results on CH based hydrocarbon growth showed the transition from non-cyclic hydrocarbons to cyclic and aromatic structures and from cyclic to polycyclic aromatic hydrocarbons. Additionally, the reactive collisions between hydrocarbons and CH were found to produce sufficient energy for isomerisation and fragmentation processes even at ultra low temperatures. In all, the results indicate that methyne might be a proper precursor for the formation of large interstellar PAHs.Kohlenwasserstoffe, insbesondere polyzyklische Kohlenwasserstoffe (engl. PAHs), werden seit einigen Jahren als Mitverursacher interstellar IR-Emissions- und UV-Absorptionsbanden angesehen und diskutiert. Dabei ist die Herkunft dieser Moleküle in den dichten Phasen des interstellaren Mediums (ISM) aber noch nicht aufgeklärt. In dieser Arbeit wurden daher die Bildungsmechanismen, welche auf Ion-Molekül-Reaktionen zwischen kationischen PAHs und Kohlenwasserstoffen und dem Molekül CH beruhen, untersucht. Sowohl der Reaktionstyp als auch der Präkursor wurden anhand von bekannten physikalischen und chemischen Eigenschaften des ISM abgeleitet und ausgewählt. Die Analyse der chemischen Reaktionen basierte auf Berechnungen zur Produktzusammensetzung und Einfangsratenkoeffizienten (welche wiederum aus berechneten Reaktionsquerschnitten hervorgingen) Minimumenergiepfade (MEP), Reaktionsenthalpien, thermische Gleichgewichtskonstanten und mikrokanonische Isomerisierungs- und Strahlungsdeaktivierungs-Ratenkoeffizienten. Um der Vielzahl an Reaktionsparameter gerecht zu werden, wurden die Berechnungsmethoden entsprechend eines hierarischen Fließschemas kombiniert. Hierzu wurden zuerst durch Molekulardynamik-Simulationen die Reaktionspotentialenergieflächen abgerastert. Auf der nächsten Stufe wurden statistisch bedeutsame Reaktionskanäle bezüglich ihrer Minimumenergiepfade untersucht. Den Abschluss bildete die Berechnung molekularer und kinetischer Charakteristika stationärer Punkte auf einem MEP. Entsprechend dieses Schemas wurde die quantenchemische Genauigkeit auf jeder Stufe von approximativer DFT über DFT zu post-Hartree-Fock verändert. Die Ergebnisse des CH-basierten Kohlenwasserstoffwachstums zeigten einen Übergang von nichtzyklischen zu zyklischen and aromatischen Strukturen, sowie von zyklischen zu polyzyklischen Kohlenwasserstoffen. Außerdem zeigte sich, dass reaktive Kollisionen zwischen Kohlenwasserstoffen und CH auch bei Tiefsttemperaturen immer ausreichend Energie für Isomerisierungs- und Fragmentationsprozesse liefert. Die Ergebnisse dieser Arbeit lassen den Schluss zu, dass CH ein geeigneter Präkursor für die Bildung großer interstellarer PAH ist

Selective [3+2] and [3+3]-Cycloaddition Reactions of Nitrones

Cationic chiral dirhodium(II,III) carboxamidates, obtained from the oxidation of the corresponding dirhodium(II,II) carboxamidates by nitrosonium salts, are efficient promoters in asymmetric Lewis acid catalyzed reactions. High regiocontrol and stereocontrol have been achieved with the cationic chiral dirhodium(II,III) carboxamidate whose ligand is (R)-menthyl (S)-2-oxopyrrolidine-5-carboxylate in 1,3-dipolar cycloaddition reactions of nitrones with α,β-unsaturated aldehydes. In addition, higher rates and selectivities have been obtained in hetero-Diels-Alder and carbonyl-ene reactions with the diastereomeric catalyst having the (S)-menthyl (S)-2-oxopyrrolidine-5-carboxylate ligand. Dramatic solvent influences on reaction rates and selectivities characterize the catalysis of cationic chiral dirhodium(II,III) carboxamidates, and these influences are explained by competitive coordination of solvent to catalyst and by the influenced coordination angle of the aldehyde substrate relative to catalyst by the solvent environment. Rhodium vinylcarbenes, generated from the reactions between vinyldiazoacetates and dirhodium catalysts, are highly reactive intermediates. Through reacting rhodium vinylcarbenes with nitrones, we have discovered a [3+3]-cycloaddition pathway; and by using chiral dirhodium carboxylates as the catalysts, a highly enantioselective [3+3]-cycloaddition of nitrones with vinyldiazoacetates has been achieved. The products of this [3+3]-cycloaddition are 3,6-dihydro-1,2-oxazines, which are versatile intermediates for the synthesis of α-substituted β-amino acids and related compounds that are not easily accessible by other methods. The broad scope of cyclic and acylic nitrones that are applied demonstrates the power of this methodology. The limitation of this [3+3]-cycloaddition methodology is the requirement of using the β-TBSO-substituted vinyldiazo compounds as the rhodium vinylcarbene precursors. Although vinyldiazoacetates without the β-TBSO substituent are not reactive for the [3+3]-cycloaddition with nitrones, we have discovered an alternative reaction pathway with an unsubstituted vinyldiazoacetate. The reaction occurs with a dirhodium vinylcarbene-induced [3+2] nitrone cycloaddition, followed by subsequent cascade carbenoid aromatic cycloaddition/N-O cleavage and rearrangement. In this cascade process, both the [3+2]-cycloaddition of nitrones with a rhodium vinylcarbene and the [1,7]-oxygen migration with N-O cleavage are unprecedented in the literature. The complexity of the reaction pathway and the uniqueness of the formed heterocyclic products are of great interest to synthetic chemists