Quantitative evaluation of reversed-phase packing material based on calcium carbonate microspheres modified with an alternating copolymer

Considering the vulnerability of silica gel to alkaline mobile phases, a highly alkaline stable stationary phase for HPLC is required to separate basic compounds with high separation efficiency. To address this issue, we have developed a high alkaline stable packing material (CaCO₃-PMAcO) based on mesoporous calcium carbonate microspheres modified with poly(maleic acid-alt-1-octadecene). In this study, we report further investigation of the separation performance of CaCO₃-PMAcO column by systematically evaluating the effects of particle size and chromatographic conditions. Based on the theory of the van Deemter equation, the separation efficiency was related to the size of CaCO₃-PMAcO particles (2.9 – 5.7 µm). The evaluation of thermodynamics of retention by changing the column temperature from 20 °C to 45 °C implied that the retention mode was dominated by hydrophobic interaction associated with the exothermic enthalpy changes (-11.1 to -12.5 kJ/mol). The results of column selectivity tests revealed that the CaCO₃-PMAcO column had hydrophobic selectivity comparable to C₁₈ silica gel columns (α_P/B; CaCO₃-PMAcO column: 1.53, C₁₈ column: 1.69), and higher shape/steric selectivity (α_Tri/Ter; CaCO₃-PMAcO column: 1.56, C₁₈ column: 0.955). In practice, the CaCO₃-PMAcO column could be applied to the separation of not only alkylbenzenes and polycyclic aromatic hydrocarbons, but also to basic tricyclic antidepressants by using an alkaline mobile phase (pH 12).