Application of capillary reversed-phase high-performance liquid chromatography to high-sensitivity protein sequence analysis

Abstract

A continuous gradient elution method for capillary column (< 0.32 mm I.D.) liquid chromatography was developed. Gradient eluent from a microbore liquid chromatograph was split ahead of the injector so that an accurate percentage (2–3%) of the mobile phase delivered by the pump flowed through the capillary column. The outlet of the column was connected to a length of 0.075 mm I.D. fused-silica capillary tubing which, in turn, was connected to a 6-mm optical path length longitudinal capillary flow cell. Fused-silica capillary columns of 0.32 mm I.D. were slurry-packed efficiently with 7-μm spherical, 300 Å pore size, C 8 bonded-phase particles, and evaluated in terms of their ability to resolve mixtures of proteins, peptides or phenylithiohydantoin (PTH)-amino acid derivatives. The gradient elution profiles agreed with those obtained using microbore (<2.1 mm I.D.) and larger bore columns. The minimum detectable amounts for proteins and PTH-amino acids on 0.32 mm I.D. capillary columns were 50 pg and 25 fmol, respectively. At a flow-rate of 3.6 μl/min, proteins and peptides were recovered from the capillary columns in volumes of about 2–8 μl. The use of a multiple-wavelength, forward-optics detector for identifying tryptophan- and tyrosine-containing peptides is discussed.