HYDROPHILIC-INTERACTION CHROMATOGRAPHY OF PEPTIDES ON HYDROPHILIC AND STRONG CATION-EXCHANGE COLUMNS

Hydrophilic-interaction chromatography (HILIC) was recently introduced as a potentially useful separation mode for the purification of peptides and other polar compounds. The elution order of peptides in HILIC, which separates solutes based on hydrophilic interactions, should be opposite to that obtained in reversed-phase chromatography, which separates solutes based on hydrophobic interactions. Three series of peptides, two of which consisted of positively charged peptides (independent of pH at pH < 7) and one of which consisted of uncharged or negatively charged peptides (dependent on pH), and which varied in overall hydrophilicity/hydrophobicity, were utilized to examine the separation mechanism and efficiency of HILIC on hydrophilic and strong cation-exchange columns.