Design of Novel Mixed-Mode Columns for Analysis of Amino Acids Derivatized with 4-Fluoro-7-nitro-2,1,3-benzoxadiazole and the Evaluation of Retention Properties

Abstract

Three-dimensional (3D) HPLC methods are useful tools for the selective analysis of biomolecules in complicated real-world matrices, however, the columns applicable for the second dimension are still limited. In the present study, two types of mixed-mode columns (Singularity MX-102 and 103) were newly designed/developed focusing on the separation of N-protected chiral amino acids. The retention profiles of the two mixed-mode columns were evaluated using 10 proteinogenic amino acids with various chemical properties, namely, alanine, asparagine (Asn), glutamine, glycine, lysine (Lys), phenylalanine, proline, serine (Ser), threonine and valine (Val), and compared to that of a mixed-mode column (Singularity MX-001) previously adopted for the 3D-HPLC analysis of chiral amino acids. The amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence (ex. 470 nm, em. 530 nm). For the mobile phases, mixed solutions of acetonitrile/methanol containing formic acid were used. As a result, NBD-Val eluted the fastest for all the tested mixed-mode columns. However, the NBD-amino acids most strongly retained on the columns were different (Asn for MX-001, Ser for MX-102 and Lys for MX-103). Also, the elution orders of the 10 NBD-amino acids on the three mixed-mode columns were not the same as that on the reversed-phase C18 column, and characteristic elution orders and retention properties were observed for the individual mixed-mode columns. These results indicated that mixed-mode columns reported in the present study have different retention mechanisms, and the development of highly selective 3D-HPLC systems using these columns is expected.