Development and evaluation of fast separation behaviors of cyclodextrin-based chiral stationary phases using sub-2 μm particles

In recent years, the emergence of Ultra High Pressure Liquid Chromatography (UHPLC) systems that can handle pressures up to 15000 psi or more has enabled the utilization of sub-2 μm column particles. UHPLC has extended the LC technique to new limits and has brought about benefits to many applications requiring shorter analysis time, reduced solvent consumption and higher sensitivity. Meanwhile, chirality is an important consideration in the pharmaceutical industry. As such, incorporation of sub-2 μm particles into enantioselective LC would afford impetus to the acceleration of drug discovery and relevant pharmacological research. However, the application of sub-2 μm columns in chiral separation is still limited, particularly in the employment of chiral stationary phase (CSP) approach, which has been established as a versatile and efficient method for chromatographic enantioseparation. Chiral columns based on sub-2 μm particles are not commercially available as yet. The current project therefore aimed at development of efficient chiral columns using sub-2 μm particles and investigation of their enantioseparation characteristics. In the current dissertation project, there are two aspects to deal with: i) preparation and evaluation of sub-2 μm to sub-1 μm particles as column support materials for efficient enantioseparation; ii) effective immobilization approaches of chiral selectors on small particles. In regards to column particles, the application of commercial sub-2 μm particles and some mesoporous silica particles developed in-house with sub-1 μm sizes were investigated. Mesoporous silica particles synthesized using templates are expected to be superior column materials on account of their notable physicochemical features. Three SBA-15 type particles with diameter around 1 μm were obtained with the aid of KCl, ethylene glycol and co-surfactants, respectively. The pressure drop vs. flow rate curve revealed that these mesoporous silica particles with organized through-pores were capable of circumventing the problem of high back-pressure encountered with reduced particle sizes to some extent. Two distinct particles (SBA-15a and SBA-15b) were chosen for consecutive enantioseparation evaluations. The SBA-15a particles are rod-like with good uniformity, large pore volume (1.37cc/g) and large pore size (7.5 nm), while the SBA-15b particles are spherical with comparable mesostructure properties to that of the commercial particles but possess higher bed permeability. Cyclodextrins (CDs) and derivatives were chosen as chiral selectors in this work due to their good chiral resolving ability as well as the diverse strategies available for immobilization. In our work, two click reaction approaches, Cu (I) catalyzed click cycloaddition of alkynyl and azido groups and a thermal free radical thiol-ene addition, were used to chemically immobilize the CD moieties onto the support material. Using commercial sub-2 μm particles, four CDs CSPs were prepared by immobilizing native α-, β- and γ-CD (CSP-1a-c) and phenylcarbomoylated β-CD CSP (CSP-1d) via cycloaddition reactions. The surface loading of CD selectors was significantly reduced with the increase of the molecule size of the selectors. The enantioseparation behavior of corresponding columns was investigated. When compared to the HPLC (4.6 mm i. d. × 150 mm) counterpart, the short (2.1 mm i. d. × 50 mm) sub-2 μm β-CD column exhibited comparable efficiency but faster analysis speed, thus validating the advantages of using smaller particles. The effects of CD analogues, buffer and immobilization strategies were studied. The cavity size was found to play crucial roles in the inclusion behaviors determined mainly by the steric match with analytes. In addition, it was found that the type of buffer and immobilization methods could affect the enantioseparation efficiency significantly. Phenylcarbamoylated β-CD, regarded as an effective selector, was also employed in this work. Some neutral and basic compounds were well-resolved by using this selector. Chiral columns based on mesoporous silica particles generated in-house with particle sizes of sub-1 μm were also evaluated. Native β-CD molecules were anchored onto SBA-15a particles to afford CSP-2b, following the same procedure as that of CSP-1b. Subsequently, enantioseparation of some flavonoids and dansyl amino acids (DAAs) was carried out on this column (CSP-2b). Compared with CSP-1b, a slight decrease in resolution was observed on the CSP-2b column, which was probably due to the poor packing and lower column permeability. Perphenylcarbamoylated β-CD CSP from SBA-15b particles (CSP-2d) were prepared through a thiol-ene addition route, which showed accentuated loading of cyclodextrin derivative (38 μmol/g), nearly twice that of CSP-1d (20 μmol/g). This might be attributable to its higher surface area (480 m2/g) as well as the smaller steric hindrance of the immobilization reaction. Enantioseparation of some neutral and basic compounds were also conducted on CSP-2d, which demonstrated good separation efficiency and column stability. Moreover, baseline enantioseparations were all completed within 10 min. Our results thus demonstrated the promising application of the small spherical mesoporous silica as a chromatographic support in high pressure liquid chromatography systems.Doctor of Philosophy (CBME-CBE