Elucidating the underlying mechanism of the total breakthrough phenomenon in reversed-phase liquid chromatography

The increasing adoption of two-dimensional liquid chromatography (2D-LC) for the separation of complex samples has driven the development of various modulation strategies to overcome solvent-strength mismatch when combining orthogonal separation mechanisms, allowing to fully exploit the separation potential of 2D-LC. Conventional approaches rely on dilution or reduction of fraction volumes prior to their transfer to the second dimension column. Such approaches often require additional hardware, leading to complicated optimization procedures and potential losses in sensitivity or separation power. Recently, total breakthrough (TB) has been proposed as an alternative strategy to deal with solvent-strength mismatch in 2D-LC. TB occurs when a large volume of solvent with a high-elution strength is injected in a (second-dimension) column, resulting in the appearance of a non-retained breakthrough peak and a retained, symmetrical analyte peak. It has been theorized that TB results from interactions between analytes and unbonded silanols on the stationary phase, but no mechanistic study of this phenomenon has been undertaken so far. In this work, the influence and role of residual silanols on the occurrence of the TB phenomenon was evaluated for reversed phase liquid chromatography (RPLC) columns with different types of endcapping. Furthermore, the impact of different silanol populations was studied by varying the pH of the employed mobile phase via suitable additives. It was demonstrated that a small and comparably acidic population of residual silanols is key in the emergence of the TB phenomenon, while complete protonation of these silanols or the presence of (less acidic) silanols inhibited the emergence of TB. TB could be induced on different stationary phase chemistries, suggesting that the phenomenon is mostly governed by the type of endcapping and the applied mobile phase conditions. Understanding the underlying mechanism allows identifying optimal conditions for the emergence of this phenomenon and will enable a more effective implementation as modulation strategy in 2D-LC in the future.