Separation selectivity of C18 reversed-phase columns from different manufacturers has been compared to evaluate the applicability of our sequence-specific retention calculator (SSRCalc) peptide retention prediction algorithms. Three different versions of SSRCalc are currently in use: 300-A pore size sorbents (TFA as ion-pairing modifier, pH 2), 100 A (TFA, pH 2), and 100 A (pH 10), which have been applied for the separation of randomly chosen mixture of tryptic peptides. The major factor affecting separation selectivity of C18 sorbents was found to be apparent pore size, while differences in end-capping chemistry do not introduce a significant impact. The introduction of embedded polar groups to the C18 functionality increases the retention of peptides containing hydrophobic amino acid residues with polar groups: Tyr and Trp. We also demonstrate that changing the ion-pairing modifier to formic/acetic acid significantly reduces the algorithm's predictive ability, so models developed for different eluent conditions cannot be compared directly to each other.