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The research presented in this dissertation involves the synthesis, characterization, and application of novel anionic and cationic chiral molecular micelles in capillary electrophoresis (CE) for the separation of diverse chiral compounds. Chapter 1 presents brief overview of the surfactants, micelle polymer, CE and micellar electrokinetic chromatography (MEKC). Chapter 2 describes the simultaneous enantioseparation of eight single chiral center â-blockers using two novel leucine and isoleucine based polymeric surfactants. The simultaneous enantioseparation of multichiral center bearing â-blockers, nadolol and labetalol is described in chapter 3. A synergistic approach, using a combination of polysodium N-undecenoxycarbonyl-L-isoleucinate (poly-L-SUCIL) and sulfated â-CD showed dramatic enantioseparation of four stereoisomers of nadolol. On the other hand for labetalol, enantiomeric separation remains unaffected using the dual chiral selector system. Chapter 4 deals with the enantiomeric separation of the binaphthyl derivatives that was found to be influenced by pH, type and concentration of the background electrolyte as well as concentration of the polymeric surfactant. In chapter 5, characterization of five alkenoxy leucine-based surfactants with variations in chain length (C8-C11), polymerization concentration and degree of polymerization showed significant effects on the chiral resolution and efficiency of hydrophobic â-blockers. The synthesis and characterization of two positively charged amino acid derived chiral ionic liquids (ILs) and their corresponding polymers is presented in chapter 6. Chiral separation of two acidic analyte (difficult to resolve with anionic micelles) can be achieved with both monomers and polymers of ILs. In chapter 7, the synthesis and detailed characterization of three pH independent amino acids derived (L-leucinol, L-isoleucinol and L-valinol) sulfated chiral polymeric surfactants is presented. These chiral sulfated surfactants are thoroughly characterized and the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy. The work clearly demonstrates for the first time the superiority of chiral separation in MEKC coupled to mass spectrometry at low pH. Finally, in chapter 8, six amino acid derived chiral surfactants with carboxylate and sulfate head groups were compared for enantioseparation of broad range of structurally diverse racemic compounds at neutral and basic pH conditions.

In this study, a methodology for separation of chiral molecules, by using enhanced ultrafiltration system was developed. Benzoin was the model chiral molecule studied. In the scope of developing this methodology, some parameters were investigated in the preliminary ultrafiltration experiments in order to set the operation conditions for enhanced ultrafiltration experiments. Due to the slight solubility of benzoin in pure water, 15% (v/v) Polyethylene glycol (PEG 400) and 30 % (v/v) Dimethyl sulfoxide (DMSO) were selected as cosolvents. Because of the high retention capacity of RC-10000 Da membranes for benzoin, a membrane saturation strategy was developed. In polymer enhanced ultrafiltration (PEUF) experiments bovine serum albumin (BSA) was used as ligand. Effects of ligand concentration and pH on total benzoin retention and on enantiomeric excess (ee %) were investigated. Benzoin concentration was almost kept constant at ~10 ppm and ~50 ppm for 15% (v/v) PEG 400 and 30 % (v/v) DMSO cosolvents, respectively. It was observed that the increase either in pH or in BSA concentration yielded an increase in total benzoin retention. In 15% (v/v) PEG 400-water, with BSA concentration of 10000 ppm, at pH 10, total benzoin retention reached to 48.7%. For this cosolvent, at different pH values and at different BSA concentrations, all ee % values were about or less than 10%. When 50000 ppm BSA was dissolved in 30 % (v/v) DMSO-water, total benzoin retention increased to 41.3% at pH 10 and ee % reached 16.7 % at pH 11. In enzyme enhanced ultrafiltration (EEUF) experiments, specific to benzoin, apo form of Benzaldehyde Lyase (BAL, E.C. 4.1.2.38) was used as ligand. These experiments were performed with constant ~ 10 ppm benzoin concentration in only 15% (v/v) PEG 400 &ndash / water solvent. Effect of BAL concentration on total benzoin retention and ee% was investigated. It was found that / for all the studied BAL concentrations in the range of 650- 1936 ppm total benzoin retention and ee % were kept almost constant at ~75% and ~60%, respectively.

The mechanism by which chiral selectivity takes place is complicated by the surface morphology, the possible involvement of the solvent, and the characteristics of the chiral molecules at the surface. My goal is to model and understand the factors which lead to significant discrimination in the case of three closely related chiral stationary phases: N-(1-phenylethyl)-N’-[3-(triethoxysilyl)propyl]-urea (PEPU), [(3,5-dinitrobenzoyl)-amino]-N-[3-(triethoxysilyl)propyl]-2-phenylacetamide (DNB-phenyglycine), and [(3,5-dinitrobenzoyl)amino]-N-[3-(triethoxysilyl)propyl]-4-methylpentanamide (DNB-leucine). Ab initio calculations are used to develop molecular models of these chiral selectors. These models are employed in molecular dynamics (MD) simulations, which provide the theoretical framework for modelling chiral interfaces in different solvent mixtures. The MD simulations of PEPU interfaces show that, in alcohol/water mixtures, the alcohols form domains at the interface with the hydrophobic portions of the molecule tending to orient towards the surface. This disrupts the water hydrogen bonding networks at the interface and leads to the exclusion of water from the surface region relative to the bulk. The MD simulations of DNB-phenylglycine and DNB-leucine selectors in hexane/2-propanol mixtures demonstrate that the interfaces are distinct both in terms of the selector orientations at the surface and in the number of hydrogen bonds formed with 2-propanol. This occurs despite the structural similarity between these two selectors. The interfaces are also prepared experimentally by attaching the chiral selectors onto oxidized Si(111) samples and AFM tips. In particular, for DNB-phenylglycine and DNB-leucine samples, two synthetic routes have been explored. Using AFM, the morphologies of the resulting chiral interfaces are obtained. X-ray photoelectron spectroscopy and refraction-absorption infrared spectroscopy provide information regarding the relative distribution of the compounds on the surface. Using chemical force microscopy (CFM) measurements, chiral self-selectivity is examined in various solvent mixtures. For PEPU interfaces, the extent of hydrogen bonding at the surface is the dominant contributor to the measured forces. In the case of DNB-phenylglycine and DNB-leucine, CFM measurements of the chiral self-selectivity in 2-propanol demonstrate that chiral discrimination is present in both systems, but larger forces are observed for DNB-phenylglycine, consistent with the molecular dynamics study that shows much weaker solvent interactions with this species. / Thesis (Ph.D, Chemistry) -- Queen's University, 2008-08-14 11:26:37.436

The research presented in this dissertation involves the development of chiral and achiral analysis using capillary electrochromatography (CEC) and CEC coupled to mass spectrometry (CEC-MS). Chapter 1 briefly reviews CEC fundamentals and latest development on chiral CEC and CEC-MS. The CEC-UV enantioseparations for several acidic compounds are described in Chapter 2. The optimum resolutions for these acidic enantiomers are achieved in ion-suppression mode, i.e. with an acidic mobile phase. One of major drawback in coupling CEC with MS is the bubble formation at the column outlet end, resulting in irreproducible retention time and erratic baseline, or even current breakdown. By introducing internal tapered columns, the aforementioned limitations of CEC-MS are successfully overcome in Chapter 3. The CEC-MS enantioseparation of warfarin and coumachlor is carefully investigated and applied to quantify R- and S-warfarin in human plasma. For individual enantiomers, a concentration of 25 ng/mL is detectable. To further improve the robustness of CEC-MS column, a new procedure of fabricating internal tapered columns is reported in Chapter 4. These internal tapered columns demonstrate excellent ruggedness, low background noise, and good compatibility in reversed-phase and polar organic modes of CEC-MS. In chapter 5, the feasibility of using internal tapered columns packed with vancomycin chiral stationary phase (CSP) is explored for simultaneous enantioseparation of eight â-blockers using CEC-MS. After a careful optimization of the mobile phase composition, sheath liquid and spray chamber parameter, 15 out of 16 enantiomers could be simultaneously resolved with excellent efficiency and detection sensitivity. The synthesis and characterization of sulfated and sulfonated cellulose phenylcarbamate CSPs is described in Chapter 6. The use of these CSPs, especially the sulfonated one, significantly enhances the EOF profile and sample throughput but maintain high enantiomeric resolving power under various modes of CEC and CEC-MS. By combining CEC and atmospheric pressure photo-ionization (APPI) MS, Chapter 7 demonstrates the separation and detection of mono-methylated benzo[a]pyrene (MBAP) isomers with ~100 times enhancement on detection sensitivity than CEC-UV. In Appedix 2, monolithic columns are synthesized through photopolymerized sol-gel approach and utilized for CEC and CEC-APPI-MS of polyaromatic hydrocarbons, and alkyl phenyl ketones.

This dissertation thesis composes of two parts; the first part focus on the characterization of trypsin, enzyme frequently used in proteomic research for the investigation and identification of protein sequences, and its peptide digestion kinetics. The second part is aimed to the enantioseparations of biologically active compounds. First part of this project focus on tryptic digestion of synthetic peptides and the development of HPLC method for the identification of synthetic peptides and their fragments. Using the in-solution digestion and HPLC method, relative kinetic constants were determined for problematic sequences. Amino acids responsible for the decrease in trypsin catalytic activity and their location towards the cleavage site were studied. Certain slight exopeptidase activity of trypsin was noted, especially at the end of peptide chain. Furthermore, three columns with immobilized trypsin used in HPLC were compared concerning their catalytic activity. The immobilization of enzymes on solid support is used to elevate the amount of enzyme present during digestion and to assure better repeatability and reproducibility of obtained results. Activity of a new trypsin column synthesized at the University of North Carolina at Chapel Hill was compared to two commercially available trypsin columns....

Nano-liquid chromatography (nano-LC) was selected as analytical tool to develop different methods for chiral separations. Nano-LC offers several advantages over conventional LC, e.g., low sample requirement, short analysis time, easy coupling with mass spectrometer, and use of small amount of reagents, with a consequent low environmental pollution. In case of chiral separations, where expensive chiral stationary phases or chiral mobile phase additives (CMPA) have to be employed, nano-LC results very useful since it allows to perform analysis with small amount of this costly material. Initially, a derivatized β-cyclodextrin, heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin, was employed as CMPA for the chiral separation of some non steroidal anti-inflammatory drugs. The role of different achiral stationary phases in the separative process was investigated. The employed capillary columns were packed in the laboratory, following the slurry-packing procedure. Subsequently, the performance of a reversed phase C18 particulate packed column was compared with that one of a C18 monolithic column, in combination with cyclodextrins (heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin or hydroxypropyl--cyclodextrin) as CMPA. Finally, hydroxypropyl--cyclodextrin was selected as chiral selector to prepare chiral monolithic columns by one-step synthesis. For this aim, the cyclodextrin was activated as the allyl derivative. The composition of the polymeric mixture of the continuous beds was varied... [to full text] / Skysčių nano-chromatografija buvo pasirinkta kaip įrankis kurti įvairius chiralinių junginių atskyrimo metodus. Skysčių nano-chromatografija turi eilę privalumų, lyginant su tradiciniais skysčių chromatografijos metodais, pvz.: mažą bandinio poreikį, trumpą analizės trukmę, suderinamumą su masės spektrometrija ir nedideles tirpiklių, reagentų sąnaudas, todėl mažą aplinkos taršą. Chiralinių junginių analizei atlikti, kai reikalingos brangios chiralinės nejudrios fazės ar chiraliniai judrios fazes priedai, skysčių nano-chromatografija yra ypač naudinga, nes leidžia atlikti analizę su minimaliomis šių brangių medžiagų sąnaudomis. Pirmiausia, derivatizuotas β-ciklodekstrinas, heptakis (2,3,6-tri-O-metil) - β-ciklodekstrinas, buvo panaudotas kaip chiralinis nejudrios fazes priedas kai kurių nesteroidinių priešuždegiminių vaistų enantiomerams atskirti. Buvo įštirtas įvairių achiralinių nejudrių fazių vaidmuo atskyrimo procese. Šiuo tikslu naudojant suspensinį birių dalelių pakavimo metodą laboratorijoje buvo paruoštos kapiliarinės kolonėlės. Vėliau, buvo lyginama C18 biriais sorbentais pakrautų atvirkštinių fazių ir monolitinių kapiliarinių kolonėlių skiriamoji geba, chralinias judrios fazes priedais naudojant ciklodekstrinus (heptakis (2,3,6-tri-O-metil)-β-ciklodekstriną arba hidroksipropil-β-ciklodekstriną). Galiausiai, vienpakopės polimerizacijos būdu buvo gautos chiralines kapiliarines kolonėles, chiralniu selektoriumi naudojant hidroksipropil-β-ciklodekstriną. Šiuo tikslu... [toliau žr. visą tekstą]

Nano-liquid chromatography (nano-LC) was selected as analytical tool to develop different methods for chiral separations. Nano-LC offers several advantages over conventional LC, e.g., low sample requirement, short analysis time, easy coupling with mass spectrometer, and use of small amount of reagents, with a consequent low environmental pollution. In case of chiral separations, where expensive chiral stationary phases or chiral mobile phase additives (CMPA) have to be employed, nano-LC results very useful since it allows to perform analysis with small amount of this costly material. Initially, a derivatized β-cyclodextrin, heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin, was employed as CMPA for the chiral separation of some non steroidal anti-inflammatory drugs. The role of different achiral stationary phases in the separative process was investigated. The employed capillary columns were packed in the laboratory, following the slurry-packing procedure. Subsequently, the performance of a reversed phase C18 particulate packed column was compared with that one of a C18 monolithic column, in combination with cyclodextrins (heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin or hydroxypropyl--cyclodextrin) as CMPA. Finally, hydroxypropyl--cyclodextrin was selected as chiral selector to prepare chiral monolithic columns by one-step synthesis. For this aim, the cyclodextrin was activated as the allyl derivative. The composition of the polymeric mixture of the continuous beds was varied... [to full text] / Skysčių nano-chromatografija buvo pasirinkta kaip įrankis kurti įvairius chiralinių junginių atskyrimo metodus. Skysčių nano-chromatografija turi eilę privalumų, lyginant su tradiciniais skysčių chromatografijos metodais, pvz.: mažą bandinio poreikį, trumpą analizės trukmę, suderinamumą su masės spektrometrija ir nedideles tirpiklių, reagentų sąnaudas, todėl mažą aplinkos taršą. Chiralinių junginių analizei atlikti, kai reikalingos brangios chiralinės nejudrios fazės ar chiraliniai judrios fazes priedai, skysčių nano-chromatografija yra ypač naudinga, nes leidžia atlikti analizę su minimaliomis šių brangių medžiagų sąnaudomis. Pirmiausia, derivatizuotas β-ciklodekstrinas, heptakis (2,3,6-tri-O-metil) - β-ciklodekstrinas, buvo panaudotas kaip chiralinis nejudrios fazes priedas kai kurių nesteroidinių priešuždegiminių vaistų enantiomerams atskirti. Buvo įštirtas įvairių achiralinių nejudrių fazių vaidmuo atskyrimo procese. Šiuo tikslu naudojant suspensinį birių dalelių pakavimo metodą laboratorijoje buvo paruoštos kapiliarinės kolonėlės. Vėliau, buvo lyginama C18 biriais sorbentais pakrautų atvirkštinių fazių ir monolitinių kapiliarinių kolonėlių skiriamoji geba, chralinias judrios fazes priedais naudojant ciklodekstrinus (heptakis (2,3,6-tri-O-metil)-β-ciklodekstriną arba hidroksipropil-β-ciklodekstriną). Galiausiai, vienpakopės polimerizacijos būdu buvo gautos chiralines kapiliarines kolonėles, chiralniu selektoriumi naudojant hidroksipropil-β-ciklodekstriną. Šiuo tikslu... [toliau žr. visą tekstą]

Dissertation thesis is a 5-publications' collection concerning characterization and application potential of cyclodextrins, polysaccharides and macrocyclic antibiotics based chiral stationary phases. The effects of stationary phase and mobile phase are studied. This approach ensures the complex insight into separation systems studied. Systems with different nature of chiral selector were studied by HPLC. Namely, macrocyclic antibiotics and derivatized polysaccharides were used for experiments. Former ones provided better results for enantioseparation of non-coded amino acids than latter ones. Dynamic coating procedure was used for preparation of a new chiral stationary phase. Characterization of new cationic cyclodextrin based chiral stationary phase was performed. Linear free energy relationship method was used for characterization of two different separation systems, i.e. newly prepared stationary phase and commercially available stationary phase. Based on results obtained, newly prepared stationary phase showed better results for separation of different achiral groups of analysts. New stationary phase prepared by dynamic coating was compared with chromatographic system, in which the chiral selector was used as a mobile phase additive. The chiral selector used for the two different approaches was...