Hold-up volume as a reference-state convention in Whelk-O1 chiral chromatography: From modifier excess adsorption to benzoin retention thermodynamics.

The hold-up volume Vref in liquid chromatography is usually treated as a dead-volume correction, but it also defines the reference-state convention under which adsorption and retention are quantified. This distinction becomes essential when retention factors are used for thermodynamic interpretation. To relate a dilute-solute retention factor, k, to an adsorption equilibrium constant, the retention process should be described within a total-uptake isotherm framework, in which Vref defines both the phase ratio and the dividing-plane convention. Therefore, the commonly used definition of the hold-up volume cannot automatically be assumed to provide an appropriate reference for the thermodynamic analysis of dilute-solute retention. This issue was examined on a Whelk-O1 chiral stationary phase by combining minor-disturbance measurements of modifier adsorption with benzoin retention and van't Hoff analysis. Excess adsorption isotherms of isopropanol (iPrOH) and acetone (ACE) were measured in iPrOH/n-hexane, iPrOH/water, ACE/n-hexane, and ACE/water eluents. Thermodynamic void volumes, Vth​, were determined from the reversed-phase composition paths using the pure-adsorbate endpoint condition and then applied to the corresponding normal-phase data, giving 1.793 mL for iPrOH and 1.765 mL for ACE at 303 K. These modifier-derived Vth​ values are useful for constructing modifier excess adsorption isotherms, but they were not suitable as reference volumes for weakly retained benzoin in the high-modifier region. Retention factors, enantioselectivity, apparent van't Hoff enthalpies, and enthalpic selectivity became strongly dependent on Vref​ when benzoin eluted close to the selected hold-up volume, whereas Vref≤1.5 mL gave convergent trends. Using Vref=1.406 mL as a practical working convention, ACE suppressed benzoin retention, enantioselectivity, and enthalpic discrimination more strongly than iPrOH, suggesting more effective competition for the Whelk-O1 amide-NH anchoring interaction. These results show that hold-up-volume selection can generate artificial mechanistic trends in chiral retention thermodynamics unless the reference-volume convention is consistent with the total-uptake framework used to interpret dilute-solute adsorption.