Journal of Chromatography A, 1596 (2019) 41–53 Contents lists available at ScienceDirect Journal of Chromatography A j o ur na l ho me page: www.elsevier.com/locate/chroma Kinetic mechanism of water dewetting from hydrophobic stationary phases utilized in liquid chromatography Fabrice Gritti ∗ , Darryl Brousmiche, Martin Gilar, Thomas H. Walter, Kevin Wyndham Waters Corporation, Instrument/Core Research/Fundamental, 34 Maple Street, Milford, MA 01757, USA a r t i c l e i n f o Article history: Received 12 December 2018 Received in revised form 20 February 2019 Accepted 22 February 2019 Available online 23 February 2019 Keywords: RPLC columns Aqueous mobile phases Dewetting kinetics Dewetting mechanism Intra-particle microstructure Pore connectivity a b s t r a c t An experimental protocol was designed to accurately measure the dewetting kinetics of aqueous mobile phases from reversed-phase liquid chromatography (RPLC) columns. The protocol enables the deter- mination of the losses in the wetted surface area and internal pore volume (leading to undesirable retention losses) of RPLC columns as a function of the dewetting time. It is used to evaluate the impact of the buffer/salt concentration in water (0–100 mM), nitrogen concentration dissolved in water (0–6.7 × 10 -4 M), column temperature (300–358 K), and of the internal structure (pore connectivity) of the stationary phase on the dewetting kinetics of various RPLC packing materials. From a fundamental viewpoint, the experimental facts demonstrate that dewetting kinetics are not solely driven by the pore size of the stationary phase and the contact angle with water. Temperature has a major influence on dewetting kinetics as it controls the nucleation rate of isolated water vapor bubbles over the entire mesoporous network. Additionally, the internal microstructure of the stationary phase (characterized by its internal porosity, pore size distribution, and pore connectivity) influences the rate at which the water vapor bubbles grow and coalesce in the entire particle volume. From a more practical viewpoint, the retention loss of RPLC columns due to water dewetting can be eliminated or at least minimized by (1) adjusting the surface and bonding chemistries to reduce the receding contact angle, (2) elevating the column outlet pressure, (3) operating at the lowest possible temperature, (4) minimizing the pore connectivity of the stationary phase (e.g., by increasing the degree of surface functionalization from C 8 to C 18 -bonded phases), and (5) by degassing the aqueous mobile phase from any dissolved gases. © 2019 Elsevier B.V. All rights reserved. 1. Introduction Reversed-phase liquid chromatography (RPLC) was and still is the most popular choice of elution mode to analyze com- plex sample mixtures containing apolar and moderately polar compounds [1–3]. After nearly 50 years of development of chro- matographic particle surface chemistry, today’s RPLC columns are exceptionally clean (high purity silica with few residual metals and catalysts [4]), robust [5], they are very rapidly equilibrated with any aqueous/organic eluent mixtures, and they provide the highest efficiency in comparison to normal phase, hydrophilic interaction (HILIC), chiral, and ion-exchange chromatography [6]. For all these reasons, many practitioners even prefer RPLC over HILIC when analyzing very polar compounds. To maximize the retention of such compounds in RPLC, fully aqueous mobile phases may be needed. The utilization of 100% aqueous mobile phases has ∗ Corresponding author. E-mail address: Fabrice Gritti@waters.com (F. Gritti). been associated with anomalies and irreversible retention losses in both linear [7,8] and non-linear chromatography [9]. Early erro- neous explanations and myths for these effects were based on the so-called “phase collapse”, which would take place when alkyl- bonded chains are in contact with pure water. Phase collapse would preclude the analytes from interacting with the hydrophobic sor- bent surface. The correct and now verified explanation behind this phenomenon of retention loss was first suggested by Wal- ter et al. [10] and confirmed by Monte Carlo simulations [11,12]: the retention loss is actually explained by the dewetting of the water mobile phase from the hydrophobic mesoporous network inside the porous particles [13,14]. Indeed, the equilibrium contact angle between water and a flat hydrocarbon surface is typically around 110 ◦ [15,16], so, water does not favorably wet these sur- faces. Hydrostatic pressures up to 300 bar are then needed to force water to enter 100 ˚ Asize hydrophobic mesopores [17]. Intrusion and extrusion of water into and from mesopores, respectively, are actually observed at different hydrostatic pressures because the advancing (intrusion) and receding (extrusion) contact angles are different: they are typically around 135 ◦ and 95 ◦ for intrusion and https://doi.org/10.1016/j.chroma.2019.02.051 0021-9673/© 2019 Elsevier B.V. All rights reserved.