Over the past decades, high-performance liquid chromatography (HPLC) has become one of the most versatile and widely used analytical techniques, with critical applications in pharmaceutical science, environmental monitoring, and biochemical research. Among its various operational modes, reversed-phase HPLC (RP-HPLC) remains the most prevalent, offering broad flexibility in selectivity and efficiency. The wide range of stationary and mobile phases available has established RP-HPLC as a standard platform in numerous analytical workflows. As method development has advanced, the roles of various operational parameters influencing chromatographic performance have been increasingly well understood [1].

Column temperature is now recognized as a critical factor in robust RP-HPLC method development [2]. Temperature influences chromatographic behavior by affecting solute retention, selectivity, peak shape, and mass transfer kinetics [3]. Typically, increasing column temperature reduces analyte retention by lowering mobile phase viscosity and enhancing mass transfer. Guillarme et al. [4] demonstrated the substantial influence of temperature on analyte–stationary phase interactions, resolution, and peak symmetry, confirming its utility as a powerful optimization parameter.

For ionizable analytes, temperature further impacts retention by altering the apparent acid dissociation constant (pKa) of solutes, thereby shifting their ionization states during separation. The retention and selectivity of weak acids and bases are therefore inherently temperature-dependent [5], [6]. Even small temperature changes can lead to notable shifts in retention time and elution order [7]. As a result, non-linear and often sigmoidal trends are commonly observed in van ’t Hoff plots of ionizable compounds [8]. To improve retention modeling under such conditions, the concept of a temperature-dependent “chromatographic pKa” has been introduced [8], and non-linear retention models – such as the Pappa–Louisi absorption model – have shown improved fits over conventional linear van ’t Hoff expressions [9].

Several studies have also emphasized the thermodynamic underpinnings of these phenomena, relating chromatographic retention to acid–base equilibria through Gibbs free energy and enthalpy changes [6], [7], [8], [10]. These insights have expanded our understanding of the interplay between solvent composition, ionization, and retention behavior in aqueous–organic mobile phases, and provided analytical chemists with more refined models for predicting retention shifts.

Despite these advances, the combined influence of temperature on both solute ionization and retention has not been fully exploited for strategic optimization in HPLC method development. In many previous studies, the combined effect of temperature and pH has been demonstrated using analytes with markedly different chemical structures, where differences in retention are often more easily attributed to gross structural features. In contrast, the present work focuses on a series of structurally related compounds – positional isomers – where subtle differences in ionization and interaction with the stationary phase can be amplified through thermal modulation. This work was motivated by our observations during method development for hydroxylated metabolites of oxindole derivatives [11]. During method development, several analytes exhibited atypical retention behavior, with both retention times and elution order of ortho-, meta-, and para-chlorobenzyl piperazine isomers changing dramatically in response to column temperature. These findings prompted a deeper thermodynamic analysis of the interplay between temperature, protonation equilibria, and chromatographic retention.