A column benchmark was performed for the reversed-phase high performance liquid chromatography (RP-HPLC) separation of a peptide mixture. Columns were packed in the 50 × 4.6-mm configuration with various 5 um, C18 sorbents. Performance was evaluated by using different mass loads and mobile phase conditions, which consisted of either 0.1% TFA, 0.02% TFA, or 0.1% formic acid. All columns were compared using three parameters: column peak capacity, USP tailing factor, and the retention of extremely hydrophilic peptides. We also evaluated the effect of mass load on column peak capacity. Peak capacity is important for current applications of peptide separation, which include the purification of natural and synthetic peptides as well as peptide mapping. Column performance under low TFA (0.02%) and 0.1% formic acid conditions is critical for liquid chromatography-mass spectrometry applications.