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Optimization of chromatographic parameters for chiral separation of biologically active compounds
Novák, Martin ; Kučera, Radim (advisor) ; Nobilis, Milan (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Bc. Martin Novák Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: Mgr. et Mgr. Rafael Doležal, Ph.D. Title of diploma thesis: Optimization of chromatographic parameters for chiral separation of biologically active compounds. The diploma thesis was focused on the development of a HPLC-UV method for the determination of K 1277 enantiomers of systematic name N-(2-((6-chloro-1,2,3,4-tetra- hydroacridin-9-yl)amino)hexyl-2-amino-3-(1H-indole-3-yl) propylamide dihydrochloride, which is one of the compounds from the tacrine-tryptophan hybrids group. These tacrine-tryptophan hybrids could be considered as promising candidates of potential drugs against Alzheimer's disease. The thesis brings an explanation of basic characteristic of chiral molecules, principles of chiral separation, pathophysiology, clinical manifestation and treatment of Alzheimer's disease and short characteristic of tacrine-tryptophan hybrids in the theoretical section. The aim of my diploma thesis was to find the optimal chromatographic conditions for separation of K 1277 enantiomers synthesized from tacrine and tryptophan fragments. The experimental part deals with the development of the chiral...
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Selectively substituted cyclodextrins for analytical and pharmaceutical applications
Benkovics, Gábor ; Jindřich, Jindřich (advisor) ; Lhoták, Pavel (referee) ; Šindelář, Vladimír (referee)
3 Selectively substituted cyclodextrins for analytical and pharmaceutical applications Abstract This thesis is focused on the selective modification of cyclodextrins, and its primary aim is the preparation and characterization of mono- and persubstituted derivatives of cyclodextrins in a regioselective and straightforward manner. The work is divided into two main parts describing synthetic strategies and applications of modified cyclodextrins with one or several substituents, respectively. The first section deals with the introduction of a single chromophoric moiety on the cyclodextrin scaffold such as cinnamyl, rhodaminyl, fluoresceinyl and eosinyl groups. The complete set of monocinnamyl-α-cyclodextrin regioisomers has been prepared by direct alkylation, and the self-assembling properties of the corresponding regioisomers were thoroughly investigated by dynamic light scattering and NMR experiments. These investigations revealed that the different isomers (mono-6-O-, mono-2-O- and mono-3-O- cinnamyl-α-cyclodextrin) form distinct supramolecular species through intermolecular association. A fast method for the unambiguous identification of the pure regioisomers has also been developed based on a series of 2D NMR measurements. Xanthene-modified β-cyclodextrins, other representatives of monosubstituted...
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Analysis of biologically active compounds using high performance separation methods
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...
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Sulfobutylether-β-cyclodextrin as a chiral selector for separation of amino acids and dipeptides in HPLC
Procházková, Hana ; Kalíková, Květa (advisor) ; Vozka, Jiří (referee)
The main aim of this work was to prepare and characterize a new chiral stationary phase (CSP). The CSP was prepared by dynamic coating of sulfobutylether- β-cyclodextrin (SBE-β-CD) on a strong anion-exchange stationary phase (SP). The selectivity and stability of the newly prepared CSP were tested on the sets of chiral and achiral analytes. The next separation system used was composed of C18 SP with the addition of SBE-β-CD as a chiral selector into the mobile phase. The set of chiral analytes contained amino acids phenylalanin, tyrosine, tryptophan, their derivatives and dipeptides glycine-DL-tryptophan and glycine-DL- phenylalanin. Four groups of mixtures of blocked and unblocked dipeptide isomers were tested for achiral separations. Dipeptides used for the mixtures had the same molecular formula but the sequence of amino acids was reversed. Measurements were carried out in reversed phase separation mode and hydrophilic interaction liquid chromatography. Mobile phases composed of methanol as an organic modifier and four different aqueous parts: (i) deionized water, (ii) aqueous solution of formic acid (pH 2.10), (iii) 20mM ammonium acetate buffer (pH 4.70) and (iv) 10mM ammonium acetate buffer (pH 8.80) in various volume ratios. Newly prepared SBE-β-CD CSP was more suitable for separations of...
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Chiral separation of diquats and determination of stability constants of their complexes with cyclodextrins by capillary electrophoresis
Bílek, Jan ; Kašička, Václav (advisor) ; Jelínek, Ivan (referee)
Capillary zone electrophoresis was used for chiral separation of eleven diquat derivatives. These N-heteroaromatic dications containing structural motif of 2,2'-bipyridine have recently been studied for their interesting electrochemical properties as well as for the axial chirality of their molecules. The combination of these properties could potencially lead to interesting applications in the future. For enantioseparation of diquats (DQ) commercially available randomly sulfated α-, β-, and γ-cyclodextrins with high degree of substitution were used. A succesfull chiral separation was achieved using all of the three sulfated cyclodextrins as chiral selectors (CS). Baseline enantioseparation was achieved for 82 %, 91 % respectively 100 % of the analyzed DQ in the presence of HS-α-CD, HS-β-CD, HS-γ-CD respectively. The highest separation efficiency and resolution were obtained in the backround electrolyte containing 22 mmol/L NaOH, 35 mmol/L H3PO4 (pH2,5) and 6 mmol/L HS-β-CD. Using three available nonracemic DQ an identification of the particular M- and P-enantiomers was done for the three corresponding DQ structures. Apparent stabillity constants of complexes of the DQ derivatives with above mentioned cyclodextrins as CS were determined by means of capillary affinity electrophoresis. The stability...
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Characterization of new chiral separation systems for HPLC
Vozka, Jiří
The dissertation thesis is focused on the physico-chemical characterization of interaction mechanisms of chiral stationary phases based on derivatized cyclofructans. Correct interpretation of retention and enantiodiscrimination processes substantially facilitates the development and optimization of new enantioselective methods using cyclofructan-based chiral stationary phases. At first, the interaction mechanisms of three commercially available cyclofructan-based stationary phases were studied in normal-phase mode of liquid chromatography. Namely, systems using chiral stationary phases based on dimethylphenyl carbamate cyclofructan 7, R-naphtylethyl carbamate cyclofructan 6 and isopropyl carbamate cyclofructan 6 were studied. Linear free energy relationship model was used as a basic tool for characterization of interactions on the stationary phases. The mentioned model revealed that the main interactions contributing to retention in cyclofructan-based systems are hydrogen bond acidity and dipolarity/polarizibility, while dispersion interactions cause decrease of retention. The impact of oligosaccharide skeleton of the cyclofructan selector on the enantioselectivity was elucidated by the comparison with seemingly analogous cyclodextrin-based chiral stationary phases. Cyclofructan-based chiral...
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Cellulose-based chiral stationary phases for reversed phase HPLC
Plecitá, Denisa ; Kalíková, Květa (advisor) ; Riesová, Martina (referee)
This diploma thesis is focused on the comparison of enantioselective potential of chiral stationary phases based on derivatized cellulose by high performance liquid chromatography (HPLC). Polysaccharide - based chiral stationary phases are suitable choice for enantioseparation of various chiral compounds. In this work Chiralpak IB column containing cellulose tris(3,5-dimethylphenylcarbamate) selector immobilized onto silica gel and Chiralpak IC column containing cellulose tris(3,5- dichlorophenylcarbamate) selector immobilized onto silica gel were used. Their ability of enantioseparation was tested on 28 structurally different chiral analytes. Reversed- phase separation mode was used for enantioseparation. Mobile phases were composed of organic modifiers acetonitrile or methanol and the aqueous part was selected according to the nature of analytes. Acidic analytes were separated in mobile phases containing aqueous solution of formic acid (pH 2.1). Analytes of bifunctional nature were separated in the presence of aqueous solution of formic acid (pH 2.1), 10 mM ammonium acetate buffer (pH 8.8) or 100 mM solution of KPF6. Mobile phases containing 10 mM ammonium acetate buffer (pH 8.8) or 100 mM solution of KPF6 were used for enantioseparation of alkaline chiral analytes. Twelve chiral analytes were...
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Newly synthesized permanently positively charged monosubstituted beta-cyclodextrin derivatives as chiral selectors in capillary electrophoresis.
Havlíková, Martina ; Bosáková, Zuzana (advisor) ; Čabala, Radomír (referee)
This diploma thesis deals with the application of two permanently positively charged monosubstituted β-cyclodextrin derivatives (PEMEDA-β-CD, PEMPDA-β-CD) as chiral selectors in capillary electrophoresis. Use of PEMPDA-β-cyclodextrin in capillary electrophoresis has not been reported in literature. Properties of PEMEDA-β- cyclodextrin are already known, but its application for separation of amino acid enantiomers has not been published yet. Cyclodextrin derivatives were tested as additives in different buffers of different pH and with eventual addition of organic modifier. As suitable background electrolyte 15 mmol·l-1 borate buffer, pH = 9.5 without organic modifier was chosen. Furthermore the influence of chiral selector on separation and eventual enantioseparation of chosen analytes was evaluated. Addition of cyclodextrin derivatives in concentration range 0.0 - 5.0 was tested. Fourteen anionogenic analytes, including native amino acids, N- blocked amino acids and profens, were detected with UV-VIS detector at optimal wavelength 214, 254 or 280 nm. Both chiral selectors were suitable for enantioseparation of N-boc-D,L-tryptophan, which was baseline separated at concentration of selector as low as 0.5 mmol·l-1 . Tested amino acids blocked with terc-butoxycarbonyl and D,L-ketoprofen were partially...
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Characterization of new chiral separation systems for HPLC
Vozka, Jiří ; Tesařová, Eva (advisor) ; Sýkora, David (referee) ; Petr, Jan (referee)
The dissertation thesis is focused on the physico-chemical characterization of interaction mechanisms of chiral stationary phases based on derivatized cyclofructans. Correct interpretation of retention and enantiodiscrimination processes substantially facilitates the development and optimization of new enantioselective methods using cyclofructan-based chiral stationary phases. At first, the interaction mechanisms of three commercially available cyclofructan-based stationary phases were studied in normal-phase mode of liquid chromatography. Namely, systems using chiral stationary phases based on dimethylphenyl carbamate cyclofructan 7, R-naphtylethyl carbamate cyclofructan 6 and isopropyl carbamate cyclofructan 6 were studied. Linear free energy relationship model was used as a basic tool for characterization of interactions on the stationary phases. The mentioned model revealed that the main interactions contributing to retention in cyclofructan-based systems are hydrogen bond acidity and dipolarity/polarizibility, while dispersion interactions cause decrease of retention. The impact of oligosaccharide skeleton of the cyclofructan selector on the enantioselectivity was elucidated by the comparison with seemingly analogous cyclodextrin-based chiral stationary phases. Cyclofructan-based chiral...
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