Rapid, high-resolution HPLC separation of peptides using small particles at elevated temperatures.

Practical advantages are described for operating small-particle columns at elevated temperatures to increase the peak capacity, resolution or speed for separating peptides. Columns of small, highly-purified porous silica microspheres with a dense covalently bonded, bulky alkylsilane stationary phase permit continuous operation at temperatures of at least 90 degrees C. Operation at elevated temperatures decreases mobile phase viscosity and enhances solute diffusion, resulting in increased column plate number and separation resolution. Higher temperatures also decrease column back pressure, permitting longer columns of small particles for separating complex mixtures requiring large plate numbers. Use of a sterically protecting di-isobutyl-n-octadecylsilane stationary phase ensures stable and reproducible columns for operation at high temperatures, with the aggressive low pH mobile phase preferred for separating peptides. The monomeric nature of this phase ensures rapid mass transfer and high column efficiency.