| The target analytes in analytical chemistry are becoming more complicated with the development of metabolomics and proteomics.The complexity and diversity of samples have placed higher demands on the separation ability.Thus,developing high-selectivity,high-sensitivity,high-resolution and high-throughput analytical techniques is urgent.Both ultra-efficient analytical methods and novel separation materials have become important approaches for improving analytical efficiency.According to the van Deemter equation,the efficiency of the column is significantly affected by the chromatographic packing.In other words,using uniform chromatographic packing of small particle size dp with narrow size distribution will reduce peak broadening and,hence,increase column efficiency.UPLC,using sub-2-micron packed columns,is one of the most widely used analytical methods in modern chromatography and metabolomics.It is of great significance to develop high-throughput,high-efficient and fast analysis methods on the UPLC-MS platform for analytical chemistry.Furthermore,submicron non-porous colloidal crystal materials have unique optical properties,nano-scale pore size and regular dense structure making it the ideal material for biomolecular chromatographic separation(proteins and peptides),for which it is expected to achieve high speed and ultra-efficiency.In our research,the separation of biomolecules was applied based on a column packed with small particles(sub-2 micrometer and sub-1 micrometer).Firstly,UPLC-MS/MS with a 1.7 ?m particles packed column has been emploied as the platform to perform the separation of polar small molecules.A hydrophilic interaction chromatography(HILIC)separation method was developed and its application in metabolomics was verified Then,submicron non-porous SiO2 chromatographic columns were prepared and applied on pressurized capillary electrochromatography platform for ultra-efficient separation and analysis of peptides and proteinsThe combination of the two platforms provides separation approaches for different analytes in the samples to meet different detection demands The full thesis is divided into six chaptersThe first chapter is the introduction.Firstly,the research progress of chromatographic separation and detection technology are described,and then the separation modes of liquid chromatography and development of packing particle size are discussed in detail.Finally,the current research on biological molecular analysis using HILIC and silica colloidal crystals is introduced.In chapter 2,with the aim of separation of polar metabolites,the development and verification of separation methods for 107 polar compounds were carried out with UPLC-MS/MS technique.On the Waters Vexo TQD platform,electrospray ionization mass spectrometry(ESI-MS),both in positive and negative ionization mode,was used to establish and optimize the separation of 107 endogenous polar compounds.For the validation,accuracy,LOD,LOQ and linearity of the analytical method were evaluated.The results demonstrated a good linear relationship for the 107 analytes in their respective linear ranges.All the linear correlation coefficients were higher than 0.99.The LOQ of most metabolites was between 0.03 and 3 ?M.The RSD%values of the intra-day precision of the analytes ranged from 2.1%and 17.2%(n=6),84%of those values were less than 10%;the RSD of inter-day precision was between 3.3%and 30.5%(n=5)and 83%of those values were less than 15%;this indicates that this method could achieve the requirements of quantitative analysisIn Chapter 3,the developed HILIC-UPLC-MS/MS method was applied to quantify the level changes of metabolites in the glycolytic pathway of Huh-7 cells under the presence of the glycolytic inhibitor 3-Bromopyruvate,(3-BrPy).After the method was validated by the actual biological model,the method was applied to the TCDD cytotoxicity model to explore the level changes of polar metabolites in Huh-7 cells,and the data was compared with 1H NMR results.The developed HILIC-UPLC-MS/MS method was also applied to study the hepatotoxicity of drug-induced liver injury caused by acetaminophen(APAP)toxicological model.The results showed that a concentration of 3-BrPy above 50 ?M completely inhibited the glycolytic pathway,indicating the accuracy and reliability of the developed HILIC-UPLC-MS/MS method.In TCDD toxicity model,the results demonstrated that energy metabolism(glycolysis pathway and TCA cycle)was strongly affected in Huh-7 cells exposed to TCDD after 24 hours.In parallel,amino acids and nucleic acids levels were reduced,suggesting the consumption syndrome,which is an early sign of apoptosis.The changes in metabolite levels detected by HILIC-UPLC-MS/MS was highly consistent with the NMR results.In the APAP liver toxicity model,the glycolytic pathway,TCA cycle,fatty acid oxidative metabolism,amino acid metabolism and GSH metabolism in the liver of mice were affected.The metabolic data of HILIC-UPLC-MS/MS was compatible with the fat oxidation of CoA and acetyl-carnitine,which proved that the HILIC-MS/MS developed in this research had good practicability and reliabilityIn chapter 4,monodisperse SiO2 microspheres with a particle size range of 300 nm-900 nm were prepared by the modified Stober method and two-phase sol-gel method.The influencing factors in the preparation process,including the temperature,TEOS volume,ammonia volume and ethanol were systematically evaluated to produce microspheres with different particle sizes.The synthesis parameters were explored by an orthogonal experimental design.The poreless,monodisperse and uniform C18 SiO2 particles were characterized using scanning electron microscopy(SEM),dynamic light scattering(DLS),accelerated surface area and porosimetry,X-ray photoelectron spectroscopy(XPS)and Fourier transform infrared(FTIR)spectroscopy.The silica colloidal crystal(SCC)column was assembled by a vertical sedimentation method where the suspension concentration and solvent in the assembly were optimized.The face-centered cubic structure in the column was estimated by the blue light caused by Bragg diffraction.Since the research of SCC is still in its infancy in analytical chemistry,this study also evaluated the quality of SCC column,which may provide reference indicators for its preparationIn chapter 5,the SCC column was applied with a pressurized capillary electrochromatography platform to evaluate its stability and repeatability Then it was tested for the separation and detection of peptides and proteins,in addition to non-polar small molecules(eg.,benzene,polycyclic aromatic hydrocarbons).The EOF of the SCC column increased with the decrease of the particle size,and the EOF value of the non-porous submicron microspheres was larger than that of the fully porous microspheres with a size of?1.7?m.The porosity of the SCC column prepared in this study was between 0.26-0.30,which further indicates that the microspheres in the column are arranged in a face-centered cubic structure.The SCC columns prepared in this study showed good intra-day repeatability(RSD<1.96%)and inter-column repeatability(RSD<5.52%).The maximum efficiency of the SCC column reaches 246,000 plates/m for polycyclic aromatic hydrocarbon compounds,1,750,000 plates/m for peptides and 651,000 plates/m for monoclonal antibodies separation.These results indicated that the SCC column is a highly efficient separation medium and has great application prospects in the separation of peptides and proteinsThe sixth chapter is the summary and prospectThe research content of this thesis is the separation of biomolecules on UPLC and pCEC by sub-2 and sub-1-micron particles,which may exploit a new path for rapid and efficient separation,and provide new ideas for metabolomics and proteomics. |
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