Fast high performance liquid chromatography separations for proteomic applications using Fused-Core® silica particles

Area of Science:

• Chromatography
• Analytical Chemistry
• Materials Science

Background:

• Superficially porous particles (Fused-Core®) are utilized in chromatography.
• Optimizing particle design is crucial for enhancing separation efficiency and kinetics.

Purpose of the Study:

• To extend the separation range of Fused-Core particles by designing them with 160 Å pores.
• To evaluate the performance of these new particles for fast separations of peptides and small proteins.

Main Methods:

• Development of Fused-Core particles with 160 Å pores.
• Utilizing conventional High-Performance Liquid Chromatography (HPLC) instruments.
• Employing low pH mobile phases (e.g., trifluoroacetic acid) and elevated temperatures (up to 100 °C).
• Investigating the effect of ammonium formate additives on peptide peak shape.

Main Results:

• Particles exhibit superior kinetics, indicated by lower resistance to mass transfer.
• Fast separations of peptides and small proteins (<15,000 molecular weight) were achieved.
• High efficiency and low back pressure were maintained, enabling use with standard HPLC systems.
• Reproducible separations of peptides, including amyloid peptides, were demonstrated at elevated temperatures.
• Addition of 10-20 mM ammonium formate improved peptide peak shape, retention, and load tolerance.
• Peak capacities comparable to sub-2-microm particles were achieved with significantly lower back pressure.

Conclusions:

• The new 160 Å pore Fused-Core particles significantly enhance chromatographic performance for peptide and small protein analysis.
• These particles provide a robust and efficient alternative for complex mixture separations, offering high peak capacity with reduced back pressure.
• Stable bonded phases and optimized mobile phase conditions allow for high-temperature and low-pH separations, broadening the applicability of Fused-Core technology.