Extended multidimensional design space studies: Comparing volatile and non-volatile buffer systems in UPLC

Abstract

Selecting the appropriate pH modifiers for separating ionizable compounds in HPLC often presents complex challenges that are frequently overlooked by industry practitioners. In Reversed-Phase Chromatography (RPC), defining pH is particularly problematic due to the presence of organic solvents, which influence the dissociation behavior of buffering agents, ionizable analytes, and residual silanol groups on the silica base material. Additional factors such as temperature changes and varying buffering capacities further complicate the interpretation of pH-effects on the separation.

A key practical question arises: can a volatile acetate buffer effectively replace an equimolar non-volatile phosphate buffer within the same pH range? To explore this, we utilized an Analytical Quality by Design (AQbD) modeling approach with DryLab to construct and compare three-dimensional (t_G-T-pH) separation models for terazosin and selected impurities across a pH range of 6.0–8.0. These Design Space (DS) models provided a comprehensive understanding of the dynamic changes occurring within each separation system. Notably, our findings revealed not only equivalent separation performance, as indicated by overlapping Method Operable Design Regions (MODRs), but also critical insights into buffer-specific differences in the selectivity of HPLC-separations.