Preparation and Characterization of HPLC Stationary Phases Modified with Peptide Containing Unnatural Amino Acid

In this study, a new stationary phase modified with a peptide containing 2-aminoisobutyric acid (Aib, an unnatural amino acid) was prepared, and its separation behavior in HPLC was investigated. Peptide Aib 3mer (L-Leu-L-Leu-Aib) and peptide Aib 6mer (L-Leu-L-Leu-Aib-LLeu-L-Leu-Aib) were introduced into aminopropylsilyl silica gel (APS) and packed into a stainless-steel column. The effect of organic solvent content in the mobile phase on the retention of hydrophobic compounds was investigated for the prepared columns. The results showed a typical reversed-phase mode separation behavior, in which retention decreased with increasing organic solvent content. Next, the effect of organic solvent content in the mobile phase on the retention of hydrophilic compounds was investigated. As a result, the separation behavior in the region where the organic solvent content in the mobile phase is high follows the HILIC mode, where the retention of hydrophilic compounds increases with increasing organic solvent content. All of this demonstrates the versatility of the prepared new stationary phase and its efficiency in separating both hydrophobic and hydrophilic compounds.