Ultrahigh-pressure reversed-phase capillary liquid chromatography: isocratic and gradient elution using columns...

Fused-silica capillaries with inner diameters of 33 microns and lengths of 25-50 cm are slurry-packed with 1.0-micron 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' approximately 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-micron particles. A peak capacity of 300 is demonstrated for a 30-min analysis.