1.9 μm superficially porous packing material with radially oriented pores and tailored pore size for ultra-fast separation of small molecules and biomolecules

Abstract

In this work, 1.9μm reversed-phase packing materials with superficially porous structure were prepared to achieve the rapid and high efficient separation of peptides and proteins. The silica particles were synthesized via three steps, nonporous silica particle preparation by a modified seeded growth method, mesoporous shell formation by a one pot templated dissolution and redeposition strategy, and pore size expansion via acid-refluxing. By such a method, 1.9μm superficially porous materials with 0.18μm shell thickness and tailored pore diameter (10nm, 15nm) were obtained. After pore enlargement, the formerly dense arrays of mesoporous structure changed, the radially oriented pores dominated the superficially porous structure. The chromatographic performance of such particles was investigated after C18 derivatization. For packing materials with 1.9μm diameter and 10nm pore size, the column efficiency could reach 211,300 plates per m for naphthalene. To achieve the high resolution separation of peptides and proteins, particles with pore diameter of 15nm were tailored, by which the baseline separation of 5 peptides and 5 intact proteins could be respectively achieved within 1min, demonstrating the superiority in the high efficiency and high throughput analysis of biomolecules. Furthermore, BSA digests were well separated with peak capacity of 120 in 30min on a 15cm-long column. Finally, we compared our columns with a 1.7μm Kinetex C18 column under the same conditions, our particles with 10nm pore size demonstrated similar performance for separation of the large intact proteins. Moreover, the particles with 15nm pore size showed more symmetrical peaks for the separation of large proteins (BSA, OVA and IgG) and provided rapid separation of protein extracts from Escherichia coli in 5min. All these results indicated that the synthesized 1.9μm superficially porous silica packing materials would be promising in the ultra-fast and high-resolution separation of biomolecules.