Determination of the phase ratio of a dehydroabietic-acid-bonded silica-gel chromatographic stationary phase and its effect on separation thermodynamics.

Rosin-based chromatographic columns are widely used for separation purposes, but, to date, their phase ratios (Φ) have been imprecisely measured. This affects the understanding of their separation mechanism and the calculation of related thermodynamic parameters. In this study, a stationary phase was synthesized by bonding dehydroabietic acid (DA) to silica gel (Si-DO) and applied for reversed-phase liquid chromatography. The distribution coefficient (Kdm) of methyl dehydroabietate (MD), which has the same structure as the bonded phase of Si-DO, was used as a surrogate for the determination of the equilibrium coefficient (K) of Si-DO, and the Kdm values of MD in different mobile phases were measured and compared with the K values of Si-DO. It was found that the phase ratio of Si-DO varied with mobile phase composition and temperature, as shown by the Φ values: 0.039-0.122 for the methanol/water system and 0.051-0.116 for the acetonitrile/water system; in addition, the a indices were 0.552-0.757 and 0.564-0.674, respectively. The Kdm of MD was closer to the K of Si-DO than those of other surrogate models, including the octanol-water and octane-mobile phase partition coefficients. In addition, the thermodynamic parameters (ΔG°, ΔH°, and ΔS°) of n-alkylbenzenes on Si-DO were negative, indicating a spontaneous and enthalpy-driven separation process. Overall, the phase ratio of rosin-based columns is crucial for accurate thermodynamic analysis and interpretation of the separation mechanism. Finally, the MD surrogate model allows the estimation of phase ratio of Si-DO and other similar columns, providing a novel method for measuring the phase ratio of rosin-based columns and providing a validated concept and methodology for determining the phase ratios of HPLC columns.