IntroductIon Reversed-phase (RP) silica columns (e.g., C18 and C8) are widely used for separating small molecules. However, these columns are unsuitable for retaining and separating highly polar compounds. Some modifed RP columns, such as the Acclaim® PolarAdvantage (PA) column, a type of polar-embedded column, retain some polar compounds. However, chromatographers often need to use a buffer, which usually impairs MS detection and results in insuffcient retention of some highly polar compounds. Hydrophilic interaction chromatography (HILIC), termed by Alpert in 1990, is a technique capable of retaining these highly polar compounds with additional benefts, including complementary selectivity compared to RP columns, enhanced sensitivity for MS detection, and simplifed sample preparation.However, due to the low-hydrophobicity surfaces associated with traditional HILIC columns (i.e., silica-, cyano-, amino-, and diol- phases), small molecules cannot be separated via hydrophobic interaction. Mixed-mode hydrophilic interaction-cation- exchange chromatography (HILIC-CEX) promotes hydrophilic interactions overlaid on ionic interactions with a cation-exchange matrix and this high-performance technique has the potential for peptide separations.25