Design And New Methods For Synthesis Of Chromatographic Separation Materials

Chromatographic separation material is the core of chromatographic technique, which is the foundation to establish and develop various separation modes. Development and preparation of separation materials with novel structures and excellent performance is one of research topics in chromatographic field. In this thesis, several novel kinds of chromatographic materials were successfully fabricated which showed excellent performance for the separation of enantiomers, basic compounds and the enrichment of glycoproteins in biological samples.Since the conventional β-cyclodextrin chiral stationary phases (β-CD CSPs) have several deficiencies in stability, synthetic routes and functional groups loading, we successfully synthesized a novel β-CD CSP by immobilization of perphenylaminocarbonyl β-CD on the 3-mercaptopropyl functionalized silica gel via thiol-ene click chemistry. This method possessed the characteristics of less reaction steps, ease of execution, facile reaction conditions and high reaction efficiency. The resulting stationary phase showed good enantioselectivities for fifteen racemates in HPLC, and the reproducibility of enantioscparation results was also satisfied.As peak tailing is always encountered in the reversed-phase chromatographic separation of basic compounds, we fabricated branched octadecyl groups modified silica-based stationary phase by sequential employment of Michael addition reaction and photoinduced thiol-yne click chemistry with 3-aminopropyl functionalized silica microspheres as initial material. In the analysis of the mixture of three basic drugs and the Corydalis yanhusuo Wang water-extracts, the obtained stationary phase showed better performance than Atlantis dC18 column in HPLC.Recently, although the hydrophilic interaction chromatography (HILIC) materials have been applied for glycopeptide enrichment, they showed poor specificity and sensitivity. We successfully synthesized poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sulfopropyl)ammonium hydroxide (PMSA) functionalized silica nanoparticles via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO2-RAFT@PMSA). The resulting SiO2-RAFT@PMSA nanoparticles showed low detection limit (10 fmol) and high recovery yield (ranged from 88.7% to 97.1%) for glycopeptide enrichment from tryptic digest of human IgG. These nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC-MS/MS runs. This result was superior to those of monolayer MSA modified SiO2@single-MSA, nonuniform multi-layer PMSA brushes coated SiO2@PMSA, and commercial ZIC@HILIC beads and Click Maltose beads.Since the hydrazine-functionalized materials currently in use were cumbersome for separation and had low content of hydrazine groups on the surface, we also fabricated hydrazine functionalized polymer brushes hybrid magnetic nanoparticles via RAFT polymerization (denoted as Fe3O4@SiO2@GMA-NHNH2). During the glycopeptide enrichment from tryptic digest of glycoproteins, the number of glycosites identified by Fe3O4@SiO2@GMA-NHNH2 nanoparticles was much higher than monolayer hydrazine groups modified Fe3O4@SiO2-single-NHNH2 nanoparticles, and even comparable to commercial Affi-Gel Hz hydrazide beads. In further analysis of hepatocellular carcinoma sample,189 glycosites were identified by the Fe3O4@SiO2@GMA-NHNH2 nanoparticles, while only 68 glycosites were identified by Fe3O4@SiO2-single-NHNH2 nanoparticles.