Adamantyl-Group Containing Mixed-Mode Acrylamide-Based Continuous Beds for Capillary Electrochromatography: Synthesis, Characterization, Optimization and Investigation of the Chromatographic Efficiency

The present work reports the synthesis, characterization, and optimization of the chromatographic efficiency of a highly crosslinked amphiphilic macroporous N-adamantyl-group containing mixed-mode acrylamide-based continuous bed synthesized for capillary electrochromatography (CEC) employing solubilization of the hydrophobic monomer by complexation with statistically methylated -CD. The work includes the investigation of the chromatographic efficiency of the synthesized monoliths dependent on the retention mode and analyte functionality. In the first part of the thesis a new synthesis procedure for amphiphilic macroporous N-adamantyl-group containing mixed-mode acrylamide-based continuous beds for capillary electrochromatography (CEC) is investigated employing solubilization of the hydrophobic monomer via 1:1 complex formation with Me--CD. For this purpose, N-(1-adamantyl)acrylamide was synthesized and characterized as a hydrophobic monomer forming a water soluble-inclusion complex with statistically Me-β-CD. Mixed-mode monolithic stationary phases were synthesized by in situ free radical copolymerization of cyclodextrin-solubilized N-adamantyl acrylamide, methacrylamide (MA), piperazinediacrylamide (PDA), and vinylsulfonic acid (VSA) in aqueous medium. Due to the amphiphilic nature of the synthesized capillaries, separations in the reversed-phase, in the normal-phase, and in a mixed-retention mode (depending on the composition of the mobile phase) are achieved for polar and non-polar neutral analytes. The stoichiometry, the complex formation constant, and the spatial arrangement of the formed inclusion complex are determined by CD modified capillary electrochromatography (CD-CEC), CD modified micellar EKC (CD-MEKC), and the application of 1H NMR and 2D-1H NOESY spectroscopy. The influence of the total monomer concentration (%T) on the chromatographic properties of the synthesized monoliths is also investigated. In the second part of the thesis the morphology and the pore size distribution of a series of amphiphilic macroporous N-adamantyl-group containing mixed-mode acrylamide-based continuous beds synthesized under variation of the concentration of the lyotropic salt ammonium sulfate (AS) in the polymerization mixture are investigated by scanning electron microscopy (SEM) and inverse size exclusion chromatography (ISEC). The impact of the concentration of the lyotropic salt AS in the polymerization mixture on the formed morphology and pore size distribution is determined. SEM photographs demonstrate the homogeneity and uniformity of the formed monolith over the length of the capillary and the covalent attachment of the formed polymer to the confining wall. Additionally, SEM photographs demonstrate a clear increase in the domain size (average size of globules + average flow through pore diameter) with increasing concentration of AS in the polymerization mixture. The pore size distribution determined with ISEC using the retention data of a series of polystyrene standards reveals the presence of two different types of pores for the synthesized capillaries: (i) pores located inside the microglobules (internal pores) and (ii) pores which are located between the microglobules (external pores). The presence of a trimodal pore size distribution is confirmed by ISEC for those monoliths which are synthesized with lower concentration of AS in the polymerization mixture, and a bimodal pore size distribution for those synthesized with higher content of AS in the polymerization mixture. In the third part of the thesis, efficiency data are gained for a series of amphiphilic macroporous N-adamantyl-group containing mixed-mode acrylamide-based continuous beds synthesized under variation of different synthesis parameters. The studied synthesis parameters are (i) concentration of ammonium sulfate, (ii) concentration of the initiator ammonium persulfate, and (iii) concentration of the negatively charged monomer vinylsulfonic acid (VSA) in the polymerization mixture. The optimization of different synthesis parameters with regard to the chromatographic efficiency under isocratic conditions for alkylphenones in the reversed-phase mode is studied employing CEC. The major conclusion was that with varied concentration of AS or varied concentration of ammonium persulfate in the polymerization mixture, a strong impact on the chromatographic efficiency is observed, while there is only a minor influence when varying the molar fraction of VSA. In addition, the absence of a significant influence by heating (Joule heating) and via extra-column band broadening effects on the determined efficiency is confirmed. In the fourth part of the thesis we studied with different classes of neutral analytes (with varied hydrophobicity) the impact of the type of retention mode (influenced by the type of analyte and the mobile phase composition) and the impact of the analyte functionality on the chromatographic efficiency and peak symmetry with a monolith synthesized with optimized parameters. With this monolithic capillary, high separation efficiencies (up to ca. 220,000 m-1) were obtained for separations of different analyte classes (alkylphenones, nitrotoluenes, and phenolic compounds with k = 0.2-0.55) in the reversed-phase mode, in the normal-phase mode, and in mixed retention-mode. For neutral alkylanilines (k < 0.25) separated in the reversed-phase elution mode, high plate numbers exceeding 300,000 m-1 were routinely obtained. For phenolic analytes, it is shown that analyte functionality and mobile phase composition have a strong influence on peak symmetry and chromatographic efficiency.