Ultrahigh-Pressure Reversed-Phase Capillary Liquid Chromatography:  Isocratic and Gradient Elution Using Columns Packed with 1.0-μm Particles

• Published in: Analytical chemistry (Washington) 1999-02, Vol.71 (3), p.700-708
• Main Authors: MacNair, John E, Patel, Kamlesh D, Jorgenson, James W
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Exact sciences and technology; Other chromatographic methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9807013

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 mobile-phase 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.