In the study of proteomics, the high efficiency separation of peptides is very important for the identification of proteins and their interactions. Therefore, the relationship between peak capacity and column length for the separation of peptides on two reversed-phase columns with different lengths by reversed-phase high performance liquid chromatography was investigated and the effect of gradient times on peak capacity and peak width was examined. The experimental results showed that column length has significant effect on peak capacity. Furthermore, longer gradient times increase not only the peak number, but also the peak width, which is very favorable for the detection of more complicated mixture of peptides when they are separated and identified by capillary liquid chromatography combined with tandem mass spectrometry.