Anal. Chem. 1999, 71, 700-708
Ultrahigh-Pressure Reversed-Phase Capillary Liquid Chromatography: Isocratic and Gradient Elution Using Columns Packed with 1.0-µm Particles John E. MacNair, Kamlesh D. Patel, and James W. Jorgenson*
Department of Chemistry, University of North Carolina at Chapel Hill, Venable Hall, CB 3290, Chapel Hill, North Carolina 27599-3290
Fused-silica capillaries with inner diameters of 33 µm and lengths of 25-50 cm are slurry-packed with 1.0-µm nonporous octadecylsilane-modified (C18) silica spheres. These columns are used to perform ultrahigh-pressure reversed-phase liquid chromatographic analyses in both isocratic and gradient elution modes. Mobile-phase pressures as high as 5000 bar (72 000 psi) are applied to column inlets to generate more than 200 000 theoretical plates in 6 min (k′ ≈ 1) for small, organic analytes. Average capacity factors of analytes are found to increase linearly with applied pressure. An electrically driven constant-flow syringe pump capable of generating mobilephase pressures as high as 9000 bar (130 000 psi) is described. This pump is used in conjunction with an exponential dilution method for the gradient separation of peptides from a tryptic digest on a 27-cm-long capillary packed with 1.0-µm particles. A peak capacity of 300 is demonstrated for a 30-min analysis. The use of small particles in HPLC has become a popular way to reduce analysis time. Most conventional pumping systems have upper pressure limits of ∼400 bar (6000 psi) confining columns packed with 1.5 µm particles to ∼3.3 cm in length. These columns enable completed analyses in a matter of minutes but provide less than 10 000 theoretical plates. Longer columns (i.e., ∼50-cm capillaries) packed with 1.5-µm reversed-phase particles generate more than 200 000 theoretical plates for small organic analytes that elute in less than 20 min (capacity factor
