A DryLab® AQbD approach to facilitate pH-interpretation in RPC

Berlin: – A recent collaboration involving scientists from the MOLNÁR-INSTITUTE for Applied Chromatography has achieved groundbreaking results in developing clear modelling strategies to address the longstanding challenge of selecting appropriate pH modifiers for robust separation of ionizable compounds in high-performance liquid chromatography (HPLC). The team, comprising experts from both industry and academia, focused on one of the most common practical issues in reversed-phase chromatography (RPC): the substitution of an equimolar non-volatile phosphate buffer with a volatile acetate buffer within the same nominal pH range.

The study Extended multidimensional design space studies: Comparing volatile and non-volatile buffer systems in UPLC was recently accepted for publication in the peer-reviewed Journal of Chromatography A. The research was supervised and actively supported by Dr. Imre Molnár, President of the Molnár-Institute, in collaboration with Professor Krisztián Horváth from the University of Pannonia and renowned industry expert Dr. Róbert Kormány of Budapest-based Egis Pharmaceuticals.

Modeling 3-D Design Spaces

To address the question around buffer replacement possibilities, the team employed the latest Design Space Comparison (DSC) capabilities of the Analytical Quality by Design (AQbD) software DryLab to construct and compare three-dimensional separation models based on gradient time, temperature for terazosin and selected impurities across a pH range of 6.0 – 8.0.

The acquisition of Design Spaces (DSs) provided a comprehensive understanding of the dynamic changes within each separation system and revealed not only equivalent separation performance – as indicated by overlapping Method Operable Design Regions (MODRs) – but also critical insights into buffer-specific selectivity differences in HPLC separations.

The general “pH-problem” in RPC

In routine practice, pH is typically measured in fully aqueous solutions using a hydrogen-ion selective glass electrode filled with a standard reference solution (e.g., 3 M KCl). Under these conditions, a well-calibrated pH meter provides objective and reliable results – assuming the sample is dilute, aqueous, and close to neutral pH. However, determining the acidity of a solution becomes significantly more challenging when organic solvents are introduced. Several physicochemical factors contribute to this difficulty.

First, the junction potential of the standard glass electrode becomes substantial, resulting from charge separation between the aqueous filling solution and the partially organic sample. Second, the conventional 0 – 14 pH scale expands, as organic solvents such as methanol and acetonitrile possess different autoprotolysis constants compared to water. Third, the dissociation behavior of acids and bases may shift considerably in the presence of organic solvents, deviating from their established aqueous pK_a values.

In connection with this, selecting appropriate pH modifiers for separating ionizable compounds in HPLC often presents complex challenges – challenges that are frequently overlooked by industry practitioners. This is especially true for gradient elution, where defining and measuring pH becomes particularly problematic due to the changing composition of organic solvents, which alters the dissociation behavior of buffering agents, analytes, and residual silanol groups on the silica backbone of stationary phase. Additional influences, such as temperature effects and varying buffer capacities, further complicate the interpretation of pH-related impacts on separation behavior.

As a result, tangible pH effects in RPC cannot be reliably assessed without extensive and systematic modeling support.

Comparing Buffer-Specific Separation Changes in HPLC

In response to this challenge, the presented study employed systematic modeling to assess the potential interchangeability of volatile acetate and non-volatile phosphate buffers. Researchers selected two stationary phases differing in residual silanol activity (HSS C18 and HSS C18 SB) and a simple modeling sample (terazosin), containing a few basic impurities and one neutral impurity. Following earlier research strategies, DryLab was used to construct three-dimensional (tG–T–pH) Design Spaces (DSs), generating unique chromatographic fingerprints that illustrated how separation behavior varied between different buffer systems at identical nominal pH values.

The resulting modeled resolution spaces (MODRs) demonstrated that under certain conditions, acetate and phosphate buffers could indeed be interchanged without compromising separation quality. However, even within this simplified experimental setup, distinct differences in selectivity – including an elution order reversal – were observed in other regions, making the two buffer systems non-alternatives depending on the specific pH and method conditions.

These findings underscore the multidimensional nature of chromatographic separations and highlight the necessity of advanced modeling tools to obtain reliable and predictive assessments of pH effects.

Research Summary and Outlook

To further expand the scope of the research, the study explored potential “salting-out” effects by varying the buffering cation species – specifically NH₄⁺, K⁺, Na⁺, and Li⁺ – to investigate whether such substitutions could further influence separation outcomes. Experiments were performed under a selected set of conditions using the residual silanol-rich HSS C18 SB stationary phase, applying identical buffer anions and concentrations. The resulting chromatograms revealed notable changes in both selectivity and peak shapes. Interestingly, the observed results did not fully align with the anticipated trend of increasing overall retention toward the most kosmotropic ammonium ion (Hofmeister-series). Furthermore, the basic peaks showed unusual curved retention profiles with phosphate buffers, and less consistent, more variable behavior with acetate buffers.

Overall, this research highlights the critical importance of constructing custom-built DS-models, which provide a quantitative understanding of all possible separation outcomes within the studied system. Such models offer essential support for clarifying actual chromatographic behavior, helping practitioners avoid becoming entangled in overly complex synergistic and antagonistic pH effects, and thereby streamlining practical method development.

‌About The MOLNÁR-INSTITUTE

Founded in 1981, The MOLNÁR-INSTITUTE develops DryLab®4, a software for UHPLC modelling for a world-wide market. Its powerful modules gradient editor, peak tracking, automation, robustness and Design Space Comparison allow for the most sophisticated method development as required across modern pharma industries. Analytical scientists use DryLab®4 to understand chromatographic interactions, to reduce analysis time, to increase robustness, and to conform to Analytical Quality by Design (AQbD) principles, according to the recently published ICH Q14 regulatory framework.

The MOLNÁR-INSTITUTE is a registered partner of the US-FDA, CDC and many other regulatory bodies. DryLab®4 pioneered AQbD long before regulatory agencies across the world encouraged such submissions. Widely implemented by thought leaders, the software contributes substantially to the paradigm shift towards a science and risk driven perspective on HPLC Quality Control and Assurance.

Further information at: http://www.molnar-institute.com/

Resources

Click on Journal of Chromatography: Extended multidimensional design space studies: Comparing volatile and non-volatile buffer systems in UPLC to access full study.