Reversed phase liquid chromatography is a common analytical technique employed in a variety of fields as a core component in many developmental and validation methods. Traditionally, this technique uses acetonitrile/water or methanol/water mixtures as the mobile phase to elute analytes of interest via differential partitioning to and from an embedded stationary phase. These organic modifiers, however, are generally toxic and flammable. This toxicity and volatility necessitates additional care in use and disposal compared to greener solvents. In particular, the more commonly employed acetonitrile is moderately toxic and produces significantly more toxic substances upon decomposition (nominally carbon monoxide and hydrogen cyanide), which further complicates its disposal. In addition, while methanol is less toxic, this modifier is more volatile and requires significantly greater concentrations for similar eluotrophic strength. Ethanol, on the other hand, provides a greener alternative due to its low toxicity to both humans and the environment. Furthermore, the eluotrophic strength of ethanol is comparable to the acetonitrile modifier, thus requiring smaller organic concentrations than methanol. Ethanol's boiling point is also significantly greater than methanol's boiling point, thereby reducing the flammability risk versus methanol. Finally, moderate increases in temperature are found to significantly reduce the several times greater viscosity of ethanol/water mixtures versus traditional mobile phases. The application of higher temperatures also provides a secondary benefit by increasing the elution strength of the mobile phase, thereby reducing run times and, more importantly, 'organic waste'. In this work, high temperature ethanol/water mixtures were explored in-depth as a green alternative to traditional hydro-organic mobile phases. Solvent strength comparisons revealed that high temperature ethanol/water mixtures have a broad range of eluting power that exceeds 60% ambient acetonitrile/water mobile phases without requiring greater than 50% ethanol content. In addition, retention was demonstratively less sensitive to changes in temperature than to changes in organic content. From an examination of the effect of these changes on the retention mechanism by linear solvation energy relationships, it was determined that temperature altered retention more by affecting the relative difference in the hydrogen-bond acidity while ethanol content largely changed retention by modifying mobile phase cavity formation effects. From a van't Hoff analysis of the thermodynamics of transfer for a methylene substituent, the structures of the mobile phase was revealed to differ considerably. In particular, it was determined that the greater elution strength of the ethanol modifier is a result of a reduction in the favorable change in entropy by transfer of the analyte out of the mobile phase. This observation implied that a mobile phase cavity around a non-polar analyte is more stable when employing ethanol as the modifier. Finally, high temperature ethanol/water mobile phases were examined in the chromatographic approximation of pure water retention (log k'w) and subsequent estimation of the octanol/water partition coefficient (log P) via the Collander equation. Linear solvation energy relationships were employed to compare the log k'w extrapolated systems based on high temperature ethanol/water, ambient acetonitrile/water, and ambient methanol/water mobile phases. Based on the comparisons of the three organic modifiers, high temperature ethanol/water mobile phases were observed to provide the best estimation of log P. This conclusion is based on a high Collander correlation of 0.978 and a near unity cos(θ) value of 0.998 between the LSER coefficient vectors of ethanol/water log k'w and octanol/water log P systems.
