Preparation of a chiral porous organic cage-bonded silica high performance liquid chromatography stationary phase and its application to the resolution of chiral compounds

Porous organic cages （POCs） are a new class of porous materials formed by the assembly of discrete three-dimensional cage-like molecules through intermolecular forces. They possess good solubility and well-defined intrinsic molecular cavities， making them an extremely attractive medium for chromatographic separation. In this study， a chiral porous organic cage （CC3-R， C₇₂H₈₄N₁₂） was synthesized using 1，3，5-triformylbenzene and （1<i>R</i>，2<i>R</i>）-diaminocyclohexane as raw materials， and it was then reduced by NaBH₄ to obtain RCC3-R （C₇₂H₁₀₈N₁₂）. After post-modification of RCC3-R with 5-bromo-1-pentene to introduce carbon-carbon double-bond functionalized linker arms， it was successfully bonded to the surface of thiolated silica via thiol-ene click reaction to prepare a chiral stationary phase （CSP） for high performance liquid chromatography （HPLC）. The successful synthesis of CC3-R， RCC3-R and CSP was confirmed by various characterization methods such as nuclear magnetic resonance （NMR）， Fourier-transform infrared spectroscopy （FT-IR）， high resolution mass spectrometry （HRMS）， thermogravimetric analysis （TGA）， and elemental analysis （EA）. The enantioseparation performance of the CSP was evaluated by separating various types of racemic compounds， including alcohols， esters， ketones， aldehydes， amines， and organic acids in both normal-phase （NP） and reversed-phase （RP） elution modes. The results demonstrate that the CSP achieved enantioseparation of 18 and 16 racemates in NP and RP modes， respectively. Among them， 12 racemates achieved baseline separation in NP elution mode， while 7 racemates achieved baseline separation in RP elution mode. Moreover， the CSP-packed column exhibited good complementarity in chiral separation with two widely used commercial columns， Chiralcel OD-H and Chiralpak AD-H， enabling the separation of some racemic compounds that cannot be separated on these two commercial columns. In NP elution mode， 7 of the 18 tested racemates could not be enantioseparated on Chiralpak AD-H column， and 4 of the 18 tested racemates could not be enantioseparated on Chiralcel OD-H column. In RP elution mode， 8 of the 16 tested racemates could not be enantioseparated on Chiralpak AD-H column， and 5 of the 16 tested racemates could not be enantioseparated on Chiralcel OD-H column. The effects of column temperature and injection mass on chiral separation performance of the RCC3-R column were investigated. In addition， the RCC3-R column exhibited excellent repeatability and stability. After hundreds of injections， no significant changes were observed in the retention times and resolution values of the analytes compared to the initial use of the column， with relative standard deviations （RSDs， <i>n</i>=5） of less than 0.50% and 1.30%， respectively. Furthermore， the RSDs （<i>n</i>=3） of retention times and resolution values of the racemates separated on RCC3-R columns prepared in different batches were less than 1.89% and 4.10%， respectively. This study demonstrates that the chiral POC RCC3-R is a potentially valuable chiral separation material for HPLC. It also holds significant importance for the research on novel CSPs for HPLC based on chiral POCs.