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Synthesis of fully porous silica microspheres with high specific surface area for fast HPLC separation of intact proteins and digests of ovalbumin

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Abstract

Fully porous silica microspheres (FPSM) with high specific surface area and hierarchical pore as matrix for HPLC were prepared. First, the porous silica nanospheres with controllable particle size and pore diameter were successfully synthesized using a dual-templating approach, the pore size of nanospheres can be increased to 18.4 nm by changing the molar ratios of octyltrimethylammonium bromide (TOMAB) and cetyltrimethyl ammonium bromide (CTAB), which is suitable for separation and analysis of biomolecules without pore enlargement. Then, the micron FPSM with hierarchical pore were synthesized by polymerization-induced colloid aggregation (PICA) using the porous nanospheres as a silicon source, which has a large mesoporous structure (35.2 nm) and high specific surface area (560 m2 g−1). Subsequently, the FPSM modified with octadecyltrichlorosilane were studied as stationary phase for separation of cytochrome C, lysozyme, ribonuclease A, and ovalbumin, bovine serum albumin, and the baseline separation of five proteins was achieved within 1 min. The prepared column was also applied to the fast separation of digests of ovalbumin, and more chromatographic peaks were obtained compared to a commercial column under the same gradient elution conditions. In addition, the static-binding capacity of the functionalized FPSM for bovine serum albumin (BSA) was measured to be 276 mg g−1, which was nearly twice the static adsorption given in literature. Therefore, these FPSM with high specific surface area and hierarchical pore structure are expected to have great potential for the separation of complex biological samples using HPLC.

A synthetic strategy was provided towards FPSM with hierarchical pores and high specific surface area using porous nanospheres as silicon source. The outstanding performance of the FPSM is that it has a high specific surface area while maintaining a large mesoporous size, which overcomes the disadvantage of sacrificing the specific surface area when increasing the pore size of porous silica microspheres prepared by using the traditional PICA method.

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Funding

This work is supported by the National Natural Science Foundation of China (21874106, 21601213), Shaanxi Provincial Science and Technology Coordinating Innovation Projects (2013SZS18-K01), Natural Science Foundation of Shaanxi Province (2018JZ2001), the Foundation of Key Laboratory in Shaanxi Province (2010JS103, 11JS097, 15JS115, 16JS116), and Scientific Research Foundation of the Zhoukou Normal University (ZKNUC2017044).

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Authors and Affiliations

  1. Henan Key Laboratory of Rare Earth Functional Materials, The Key Laboratory of Rare Earth Functional Materials and Applications, Zhoukou Normal University, Zhoukou, 466001, People’s Republic of China

    Hongjun Xia, Lin Wang, Chunyang Li, Boshi Tian, Qingfeng Li & Hui Zhao

  2. Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Institute of Modern Separation Science, Key Lab of Modern Separation Science in Shaanxi Province, Northwest University, Xi’an, 710069, China

    Hongjun Xia & Quan Bai

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  1. Hongjun Xia
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  2. Lin Wang
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Correspondence to Hui Zhao or Quan Bai.

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Xia, H., Wang, L., Li, C. et al. Synthesis of fully porous silica microspheres with high specific surface area for fast HPLC separation of intact proteins and digests of ovalbumin. Microchim Acta 187, 382 (2020). https://doi.org/10.1007/s00604-020-04327-2

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