Factors affecting retention and resolution of peptides in high-performance liquid chromatography

Abstract Retention of small peptides (20 residues or less) on reversed-phase columns can be predicted by summing the contribution to retention of each amino acid and end group. The high correlation ( r = 0.98) for 100 peptides of predicted versus actual retention times indicates that conformation and sequence have minor effects on retention. Resolution depends not only on the column, but also on the mobile phase composition and flow-rate, the gradient rate and the size and composition of the peptides themselves. These factors were studied using 9 small peptides. Column efficiency (height equivalent to a theoretical plate) in isocratic separation improved linearly with decreasing flow-rate and decreasing log molecular weight of peptide. Resolution in gradient separations was primarily a function of gradient rate; flow-rate had little effect on resolution, but did affect peak height. A compromise must be made between analysis time and resolution: separation with a given mobile phase can be improved by using slower gradients, but only at the expense of exponentially increasing analysis time.