Preparation Of 5 μm Monodispersed Fully Porous Silica Microspheres By Polymer Template Method And Its Application In Liquid Chromatography

High Performance Liquid Chromatography（HPLC）is an important method for the separation and analysis of complex samples.Chromatographic column is the core of HPLC separation technology,and the development of Chromatographic packing is the main driving force of the development of HPLC technology.The performance of Chromatographic matrix is directly related to the separation performance of Chromatographic packing,which is the basis for the preparation of efficient separation media.Silica microspheres are the most widely used Chromatographic matrix because of their higher mechanical strength,larger specific surface area and easier surface modification.Fully porous silica microspheres are still the mainstream liquid chromatography matrix due to their high porosity,large specific surface area and high loading capacity.The preparation of high performance monodisperse porous silica microspheres to achieve efficient and rapid separation of components has always been the goal of chromatographers.In view of the existing problems in the preparation process of fully porous silica gel microspheres,such as uneven sphericity,poor monodispersity,wide particle size distribution,and difficult to accurately control the pore size.this study used porous polymer microspheres as a template to realize the controllable preparation of 5μm fully porous silica microspheres.And we studied the effects of the amount of porogens,different amino functionalization reagents,the ratio of template microspheres and ethyl orthosilicate（TEOS）and the calcination temperature on the pore size of porous silica microspheres.By optimizing the experimental conditions,silica microspheres with different pore sizes were prepared to separate of proteins and small molecules.The surface modification of silica microspheres was used to characterize the Chromatographic separation performance of small molecules and proteins in reverse phase chromatography mode.The results show that this method can be used to control the pore size of all porous silica microspheres.The stationary phase of liquid chromatography prepared by the porous silica microspheres in this study can be used for the efficient separation of proteins and small molecules.The articles includes the following four parts:1.Introduction.The development of silica matrix and the preparation methods of porous silica microspheres are reviewed.The types and development history of silica matrix were introduced,and the advantages and disadvantages of different preparation methods of porous silica microspheres were discussed and analyzed,such as polymer template method,sol-gel method,spray drying method and polymerization induced colloid aggregation method.2.Preparation and characterization of 5μm fully porous silica microspheres.The 5μm porous P_GMA-EDMA polymer microspheres were prepared by seed swelling method and amino-modified with tetraethylenepentamine（TEPA）.Macroporous silica microspheres with good monodispersity and particle size of 5μm were successfully prepared by using amino functionalized polymer microspheres as template.The effects of the amount of porogens,different amino functionalization reagents,the ratio of P_GMA-EDMA template microspheres to TEOS and the calcination temperature on the pore size of porous silica microspheres were studied.Silica microspheres with different pore sizes were prepared by using different amino functionalization reagents.The morphology and structure of the prepared silica microspheres were characterized by infrared spectroscopy,thermogravimetric analysis and scanning electron microscopy.The pore size of the prepared silica microspheres was 69 nm and the specific surface area was 108.6 m²/g by mercury injection method.3.Application of macroporous silica microspheres in protein separation.The surface modification of macroporous silica microspheres were modified with different chain length alkylsilanization reagents(C₄H₉SiCl₃,C₈H₁₇SiCl₃ and C₁₈H₃₇SiCl₃).The standard proteins were separated respectively by three kinds of reverse phase bonding packings were obtained.The results showed that the prepared reverse phase Chromatographic packings had a good separation effect on the proteins,and the three columns could completely separate the seven standard proteins,and the separation effect was better than the commercial column.The results showed that Silica-C4 column was more suitable for protein separation because of its higher separation efficiency and mass recovery..4.Preparation and application of small pore size silica microspheres in small molecules separation.Small pore size silica microspheres with particle size of 5μm were successfully prepared by using TMA functionalized polymer template microspheres.The silica microspheres was modified by octadecyl trichlorosilane and packed in Chromatographic column.In a Chromatographic column,three groups of polar and nonpolar small molecules are separated in reverse phase mode.And through Chromatographic parameters,such as resolution,the number of theoretical plates and separation factor evaluated of the Chromatographic column separation performance.The results showed that the prepared reverse phase chromatographic column had a good separation effect on the three groups of small molecule solutes,which was similar to the commercial ODS column.Taking hexobenzene as solute,calculated it’s the number of theoretical plates can reach 69753 plates/m,with good separation degree and separation reproducibility.The above results indicated that the stationary phase prepared with small pore size silica microspheres as the matrix could be used for the separation of small molecules.However,compared with commercial column,the retention of solute in the sample was weaker,which might be due to the lower C18 bond density on the surface of silica microspheres.We need to further optimize the experimental conditions.In conclusion,through selecting different amino functionalization reagents,we prepared macroporous and small pore size monodispersed fully porous silica microspheres with polymer template method.And they can be used as a substrate for the preparation of stationary phase in liquid chromatography to achieve the efficient separation of proteins and small molecule solutes.