Chromatographic behavior of a small peptide in RP-HPLC can generally be correlated with the summation of the hydrophobic contribution from its composed amino acid residues. But this approach fails to predict the retention deviation between two sequence shuffled peptides. One set of 11-residue peptides was designed to study the conformation effect. The two sequence shuffled peptides had different conformations and showed different retention behaviors in a C18 column. The retention factors of the relatively helical peptide GELELKLKLEG (GELE) were consistently lower than those of the peptide GELKLELKLEG (GELK) at all conditions under investigation. All atom molecular dynamics (MD) simulation was performed to study the origin of the deviation in column retention. Circular dichroism spectra and MD simulation confirmed that the structure of GELE is more helical and the energy of GELE in solution is lower than that of GELK. The deformation energies calculated from MD described well the concentration effect on δCP obtained from column retention data. After analyzing the desolvation, deformation, and interaction energies after the adsorption of these two sequence shuffled peptides on C18 self assembly monolayer (SAM), it was found that the longer retention of GELK in the column was not due to its higher accessibility toward the hydrophobic stationary phase. The structurally rigid peptide, i.e., the low potential energy one, owned a lower retention time because its ability to minimize the overall adsorption energy was limited. This result suggested that the overall potential energy of peptide in solution calculated from MD simulation may be used to quantify the conformation effect.