Peptides and proteins were separated by reversed-phase chromatography on a 30 x 4.6 mm I.D. column packed with non-porous crosslinked polystyrene particles having a mean particle diameter of 3 micron and a rugulose surface. The polymeric support did swell slightly in organic solvents, but the estimated 5-8% change in particle diameter did not adversely affect the efficiency of the column which was used repeatedly with gradient elution from water to organic solvent under conditions typically employed in reversed-phase chromatography. In these experiments, the pH of the eluent was varied in a wide range in order to compare the effect of acidic and alkaline eluents on the separation of protein and complex peptide mixtures. The column showed no deterioration even after extensive exposure to alkaline mobile phases. The retention behavior of sixteen proteins having widely different pI values was studied as a function of the eluent pH. The chromatographic system exhibited large selectivity differences upon changing the pH of the eluent from 2 to 11. Analytical information about peptide and protein mixtures could therefore be enhanced by using eluents at the pH extremes. At the pH extremes of 2 and 11 peak sharpness and protein mass recovery were found to be superior to those obtained with neutral eluents. Usually the column temperature was held at 80 degrees C and typical analysis times ranged from 30 s to 10 min as illustrated by chromatograms of protein mixtures and by peptide maps. With regular use under such conditions the column showed no deterioration after three months.