Preparation of novel chiral stationary phases based on a chiral trianglsalen macrocycle by thiol-ene click chemistry for enantioseparation in high-performance liquid chromatography

Macrocyclic compounds, with unique cyclic structures, well-defined molecular cavities and diverse functional properties, have shown broad application prospects in the fields of molecular recognition, catalysis, separation, and supramolecular chemistry. In this study, a novel chiral trianglsalen macrocycle (CTSM) was synthesized by a one-step condensation reaction of 3,3'-dihydroxy-(1,1'-biphenyl)-4,4'-dicarboxaldehyde with (1<i>R</i>,2<i>R</i>)-1,2-diaminocyclohexane. Then, the CTSM was bonded to thiol-functionalized silica by a thiol-ene click approach to prepare two chiral stationary phases (CSPs), CSP-A and CSP-B, with different spacers for high-performance liquid chromatography (HPLC). The two prepared CSPs exhibit excellent chiral separation performance, and 22 racemates were enantioseparated on these two CSPs, including alcohols, ketones, esters, phenols, organic acids, epoxides, and amines. Among them, 20 racemates achieved baseline separation on CSP-A, while 15 racemates achieved baseline separation on CSP-B. CSP-A shows better chiral separation capability than CSP-B, and most racemates obtain higher resolution (<i>R</i>_s) values because CSP-A features a cationic imidazolium spacer that enhances enantioseparation. Compared with commercial chiral columns (Chiralpak AD-H and Chiralcel OD-H), the prepared CSP-A shows peculiar advantages, achieving the separation of some enantiomers that cannot be separated or cannot be well separated by the two commercial columns. Furthermore, the effects of analyte mass, mobile phase composition, and column temperature on enantioseparation were studied. The two fabricated columns also exhibit good reproducibility and stability. After hundreds of uses of the columns, the relative standard deviations (RSDs, <i>n</i> = 5) for separations were less than 1.16% for the retention time and 3.29% for the <i>R</i>_s value, respectively. Moreover, the RSDs (<i>n</i> = 3) of the <i>R</i>_s value and retention time in terms of column-to-column reproducibility were less than 8.79% and 5.04%, respectively. This work demonstrates the promising potential of CTSM for chiral separation in HPLC.