Different multiple-injection methodologies and the partial filling technique (PFT) have been utilized for separation of pharmaceuticals by capillary elec-trophoresis. In multiple-injection capillary zone electrophoresis (MICZE), the samples and all standards, used for construction of the calibration curve, are analyzed within a single run. Four different modes of MICZE have been described by means of equations, which were experimentally verified. The developed equations facilitate the transfer from conventional single-injection CZE to one or more of these MICZE-modes, depending on the selectivity between the analyte and the injection marker. The applicability of two of these modes was then demonstrated by quantification of buserelin and salbutamol, re-spectively in commercially available pharmaceutical products. The content of buserelin in an injection solution was determined to 0.94 mg/ml, which only deviated slightly from the declared concentration (1 mg/ml). An alter-native mode of MICZE, offering a higher number of sequential sample injec-tions, was then utilized for single-run determination of salbutamol in 15 tab-lets, with a labelled content of 8 mg. The average content of the tablets was determined to 7.8 mg, with an intra-tablet variation of 3 % or less. Moreover, UV- and mass-spectrometric detection of enantiomeric amines, resolved by non-aqueous capillary electrophoresis (NACE), was demon-strated. Separation of enantiomeric amines was achieved using the chiral selector (-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid, (-)-DIKGA. Introduction of the non-volatile (-)-DIKGA into the mass-spectrometer was avoided by using the PFT, where the capillary is only partially filled with electrolyte containing the chiral selector.

<div>The first chapter reports a study of aryl nitrene intermediates. Although extensively studied over the past 30 years, phenyl nitrenes have a propensity to undergo rearrangement reactions and form polymeric tars. This is in stark contrast to the phenyl carbenes which are known to undergo several important reactions to produce a library of useful organic compounds. One such reaction is the insertion of phenyl carbenes into a double bond to produce a cyclopropane moiety. If aryl nitrenes can be exploited to conjure a similar reactivity, they would be an excellent synthetic route to produce aziridine rings which are a crucial component of many natural products. This review chapter is a collection of all the efforts that have been made in this regard.</div><div><br></div><div>In the next chapter, the electronic effect of the azide functional group on an aromatic system has been investigated by using Hammett-Taft parameters obtained from the effect of azide-substitution on the gas-phase acidity of phenol. Gas-phase acidities of 3- and 4-azidophenol have been measured by using mass spectrometry and the kinetic method and found to be 340.8 ± 2.2 and 340.3 ± 2.0 kcal/mol respectively. The relative electronic effects of the azide substituent on an aromatic system have been measured by using Hammett-Taft parameters. The σF and σR values are determined to be 0.38 and 0.02 respectively, consistent with predictions based on electronic structure calculations. The values of σF and σR demonstrate that azide acts an inductively withdrawing group but has negligible resonance contribution on the phenol. In contrast, acidity values calculated for substituted benzoic acids gives values of σF = 0.69 and σR = -0.39, indicating that the azide is a strong  donor, comparable to that of a hydroxyl group. The difference is explained as being the result of “chimeric” electronic behavior of the azide, similar to that observed previously for the n-oxide moiety, which can be more or less resonance donating depending on the electronic effects of other groups in the system.</div><div><br></div><div>Phenyl nitrenes undergo bimolecular chemistry under very specific circumstances. For example, having an oxide substituent at the para position of the phenyl ring enables the formation of an indophenol product from a photocatalyzed reaction of the nitrene. Although, this reaction has been reported before, the mechanism involved in this reaction has not been fully understood. A two-electron mechanism involving electrophilic aromatic substitution reaction has been proposed in the literature, however we found evidence that did not support this theory. Instead, we find this reaction analogous to the popular Gibbs’ reaction whose single electron transfer mechanism has been extensively studied. The following chapter encompasses a study of the mechanism of the photolysis reaction to look for evidence of a single electron transfer similar to the Gibbs’ reaction.</div><div><br></div><div>As mentioned earlier, phenyl nitrenes have a proclivity to undergo rearrangement reactions instead of exhibiting bimolecular reactivity that can lead to useful products. One of the strategies to overcome this challenge is to spatially separate the two electrons of an open-shell singlet nitrene so as to minimize electron-electron repulsion. This separation can be achieved by delocalizing the individual electrons over multiple aromatic rings and heteroatoms which can act as radical stabilizers. In this chapter, a short review of literature that sets precedence for developing a unique heteroatom containing aromatic backbone to achieve the necessary stabilization is presented. Our efforts in synthesizing the model azide precursor compound have also been discussed.</div>

<p>High resolution tandem mass spectrometry (MSⁿ) coupled with various separation techniques, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), is widely used to analyze mixtures of unknown organic compounds. In a mass spectrometric analysis, analytes of interest are at first transferred into the gas phase, ionized (protonated or deprotonated) and introduced into the instrument. Tandem mass spectrometric experiments may then be used to gain insights into structure and reactivity of the analyte ions in the gas phase. The tandem mass spectral data are often compared to those reported in external databases. However, the tandem mass spectra obtained for protonated analytes may be markedly different from those in external databases because protonation site manifested during a mass spectrometric experiment can be affected by the ionization technique, ionization solvents and condition of the ion source. This thesis focuses on investigating the effects of instrumental conditions and analyte concentrations on the protonation sites of 4-aminobenzoic acid. Reactivities of radical species were also investigated. A modified bracketing method was developed and proton affinities of a series of mono- and biradicals of pyridine were measured. In another study, a <i>para</i>-benzyne analog was generated in both solution and the gas phase and its reactivities towards various neutral reagents in the gas phase were compared to those in solution.</p> <p> Chapter 2 discusses the fundamental aspects of the instruments used in this research. In chapter 3, the effects of residual moisture in linear quadrupole ion trap on the protonation sites of 4-aminobenzoic acid are considered. Chapter 4 focuses on the use of gas-phase ion-molecule reactions with trimethoxymethylsilane (TMMS) for the identification of the protonation sites of 4-aminobenzoic acid. Further, the effects of analyte concentration on the protonation sites of 4-aminobenzoic acid are considered. Chapter 5 introduces a modified bracketing method for the experimental determination of proton affinities of a series of pyridine-based mono- and biradicals. In chapter 6, successful generation of <i>para</i>-benzynes in solution is discussed. The reactivity of a <i>para</i>-benzyne analog, 1,4-didehydrophenazine, is compared to its reactivity in the gas phase.</p>

<div>Mass spectrometry (MS) is a powerful and versatile analytical tool, especially for identification and analysis of complex mixtures. Coupling to high-performance liquid chromatography (HPLC) or gas chromatography (GC) provides additional dimension for mixture analysis. MS manipulates ionized analytes and separates them based on their mass-to-charge (<i>m/z</i>) ratios. MS is capable of providing molecular weight (MW) information by generating pseudo-molecular ions of the analytes. Detailed elemental compositions can be also obtained if high resolution MS is used. MS can also provide extensive structural information of the analyte ions. One of the most commonly used technique is tandem mass spectrometry (MSⁿ). Ions of interest are isolated and subject to sequential reactions (reactions with other molecules or dissociation reactions) to generate product ions that can provide structural information. MS is also a powerful tool for generating and studying highly reactive reaction intermediates, such as organic polyradicals.</div><div><br></div><div>The research described in this dissertation mainly focuses on the generation and gas-phase reactivity studies of different organic biradicals. Their reactions with various organic reagents are studied, and the reactivity-controlling factors are discussed. For example, the reactivity of several substituted pyridine-based biradical cations with 2,6-topology are discussed (all with singlet ground states), and their special reactivity from their excited triplet states are illustrated. Besides, several quinoline-based biradicals and cyano-substituted pyridine-based <i>para</i>-benzyne cations are also discussed. Some of the radicals (or ions) described in this dissertation are generated for the first time, i.e. the quinoline-based oxenium cations. Their structural characterization and gas-phase reactivity toward some organic molecules are discussed in the dissertation. Further, an electrophilic anion, <i>closo</i>-[B₁₂X₁₁]^- (X = Cl, Br) and its application in the activation of C-H and C-C bonds in hydrocarbon molecules are described in the dissertation.</div>

The function of many biological processes depends on the structure and composition of the biomolecules involved. Both spectroscopy and mass spectrometry provide complimentary information regarding the three-dimensional conformation and the composition, respectively, as well as many other things. Here, double resonance conformer specific spectroscopy coupled with the latest ab inito computational methods is used to make structural assignments at the atomic resolution as well obtain information regarding propensities of intramolecular interactions. Additionally, rapid cooling in conjunction with IR excitation to modulate and measure the relative populations of conformers present in the expansion. Two different designer peptide systems are studied, including an achiral acylated 𝛼-aminoisobutryic acid dipeptide (Ac-AIB2-R) with various C-terminal protecting groups (R=NHBn, NHBnF, 𝛼-methylbenzylamine) and an acylated 𝛾4-phenylalanine (Ac-𝛾4Phe-NHMe) with the a methyl amine C-terminal protecting group. Mass spectrometry is used to determine the kinetics of gas-phase covalent tagging reactions used to enhance the sequence coverage. The covalent modification reactions utilize click chemistry between NHS or HOBt substituted sulfobenzoic acid tags with nucleophiles present on the residues of the amino acids composing the backbone. Effective temperatures are approximated using the Tolmachev model, which relates the statistical average internal energy of the molecule to a temperature.

<div> <div> <div> <p>Understanding protein-protein interactions (PPIs) in a cell is essential for learning protein functions, pathways, and mechanisms of diseases. This dissertation introduces the computational method to predict PPIs. In the first chapter, the history of identifying protein interactions and some experimental methods are introduced. Because interacting proteins share similar functions, protein function similarity can be used as a feature to predict PPIs. NaviGO server is developed for biologists and bioinformaticians to visualize the gene ontology relationship and quantify their similarity scores. Furthermore, the computational features used to predict PPIs are summarized. This will help researchers from the computational field to understand the rationale of extracting biological features and also benefit the researcher with a biology background to understand the computational work. After understanding various computational features, the computational prediction method to identify large-scale PPIs was developed and applied to Arabidopsis, maize, and soybean in a whole-genomic scale. Novel predicted PPIs were provided and were grouped based on prediction confidence level, which can be used as a testable hypothesis to guide biologists’ experiments. Since affinity chromatography combined with mass spectrometry technique introduces high false PPIs, the computational method was combined with mass spectrometry data to aid the identification of high confident PPIs in large-scale. Lastly, some remaining challenges of the computational PPI prediction methods and future works are discussed. </p> </div> </div> </div>

No / Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures.

The diploma thesis deals with determination of organic compounds in the tar. It is focussed on polycyclic aromatic hydrocarbons, BTEX and n-alkanes. The tar samples were collected by Faculty of Mechanical Engineering BUT Brno during two days and under diferent conditions of biomass gasification. The samples were filtrated and refined using column chromatography on silicagel sorbent. Gas chromatography with mass detection was chosen to determine polycyclic aromatic hydrocarbons and gas chromatography with flame ionisation detection was chosen to determine BTEX and n-alkanes.

This master’s thesis is focused on optimizing of the analytical method for the determination of selected drugs - estrone, estradiole, ethynylestradiol, estriole, diethylstilestrole, norethindrone, mestranole and progestrone in wastewater using liquid chromatography with mass spectrometry (LC/MS). Solid phase extraction (SPE) using Oasis HLB cartridges was chosen extraction method Different mobile phase, temperature, flow rates and mobil phase gradient were during the process of optimization. Samples of the wastewater were collectedat the inflow and outlow of the wastewater treatment plant in Břeclav and in University of Veterinary and Pharmaceutical Sciences Brno. Drugs were isolated from wastewater and preconcentrated using solid phase extraction (SPE) and determined by high performance liquid chromatography with mass spectrometric detection (HPLC/MS).

The sensitivity, speed, and reproducibility of modern mass spectrometers enable in-depth new functional looks into the cellular proteome. Thousands of proteins can be detected in a single sample. In Co-Fractionation Mass Spectrometry (CF-MS) method, the input sample is fractionated by any biochemical method of choice. The reduced complexity of each fractionated sample leads to better proteome coverage. The separation profiles provide functional information on the proteins. This application has been used to predict organelle localization based on co-purification with marker proteins. More recently, CF-MS is being used to measure the apparent masses and determine the localization of soluble or membrane-associated protein complexes. This Ph.D. dissertation focuses on the extension of the boundary of CF-MS application to learn how protein complex evolution and protein complex composition have been accomplished. In the first part of this dissertation, the data will be presented on the degree to which variation in protein oligomerization across plant species is present, how proteomics in phylogenetic analysis (phyloproteomics/evolutionary proteomics) helps understand the evolutionary changes, and how oligomerization drives neofunctionalization during plant evolution. The latter part will describe that CF-MS coupled with multiple orthogonal chromatographic separations increases the resolving power of the profiling technique, enabling the composition of protein complexes to be predicted in the subaleurone layers of rice endosperm. Lots of novel protein complexes involved in RNA binding protein, translation, and the tissue-species metabolism will be discussed.