Ultrahigh pressure reversed-phase capillary liquid chromatography:: Isocratic and gradient elution using columns packed with 1.0-μm particles

Fused-silica capillaries with inner diameters of 33 mu m and lengths of 25-50 cm are slurry-packed with 1.0-mu m nonporous octadecylsilane-modified (C-18) 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' approximate to 1) for small, organic analytes. Average capacity factors of analytes are found to increase linearly with applied pressure. An electrically driven constant-now 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-mu m particles. A peak capacity of 300 is demonstrated for a 30-min analysis.