## 3 Aqueous Mobile Phases  
Using 100% aqueous mobile phases is a good way to make conventional RP-HPLC greener, as water is one of the most benign solvents. Indeed, water is readily available, inexpensive, non-toxic, non-flammable, and environmentally friendly with no disposal concerns. In addition, water does not absorb in UV down to 190 nm, so that it is possible to detect even weak chromophores [72]. Also, using water as an eluent allows the combination of LC with non-conventional detectors, (e.g., flame ionization detector (FID)) as water has no significant FID response. This is highly desirable for UV-transparent compound analysis. Moreover, the compatibility of heated water with MSdetectors has been shown to increase the ionization efficiency and signal-to-noise ratio (S/N) for some compounds [17]. Some applications report the use of totally aqueous mobile phases in RP-HPLC at relatively low temperatures for the analysis of pharmaceuticals [73; 74]. Nevertheless, such applications are limited to the separation of highly polar analytes since, in RP-HPLC, water is the weakest solvent due to its high polarity (dielectric constant, \(\varepsilon\) = 80 at 20 \({}^{\circ}\)C) [72]. Indeed, the elution strength of water at ambient temperature is usually not sufficient to elute relatively non-polar compounds in RP-HPLC. Another problem coming from the use of totally aqueous mobile phases is the phenomenon of phase collapse or phase dewetting, which occurs with alkyl bonded phases (such as C8 or C18) in RP-HPLC [75]. In fact, phase dewetting leads to chromatographic problems such as retention loss, peak tailing, non-reproducible retention times, and gradient regeneration delays. Nevertheless, this issue of phase collapse can be avoided by using columns especially designed to be compatible with highly aqueous environments (i.e., including a polar end-capping or polar embedded groups) [75]. For example, a RP-HPLC-DAD method was developed and validated using only aqueous solvents, by [PERSON] et al. [73] for paracetamol quantitation in cell culture fluid from an in vitro Blood Brain Barrier model using a C18 column with embedded polar groups (XTerra RP18 column) at 35 \({}^{\circ}\)C. The mobile phase was a 20 mM formate buffer in water at pH 4. Also, [PERSON] et al. [74] have developed and validated a simple, rapid, and environmentally friendly method for the separation of four compounds (4-aminophenol, caffeine, paracetamol, and propyphenazone) on a polyethylene glycol (PEG) stationary phase at 30 \({}^{\circ}\)C, with a low-toxicity mobile phase consisting of water and 0.04% (_v/v_) trimethylamine, whose pH was adjusted to 4.5 by means of glacial acetic acid. Then, the proposed green method was successfully applied to the analysis of active substances and one degradation product (4-aminophenol) in commercial preparations. Due to its low elution strength at room temperature, water cannot elute many organic analytes, thus, high temperatures are usually needed to increase its elution strength. Indeed, at elevated temperatures, the dielectric constant of water decreases, increasing its elution strength [34]. Chromatographic separation performed with water at high temperatures is known as superheated water chromatography (SHWC), or subcritical water chromatography (SWC) when the temperatures used are lower than the critical temperature of water (374 \({}^{\circ}\)C) [76]. At high temperature, pure water tends to have a similar polarity as eleuents typically used in RP-HPLC [72]. For example, at 150 \({}^{\circ}\)C, the dielectric constant of water is reduced, and its elution strength becomes comparable to that of a mixture of MeOH/water 50:50 (_v/v_) at ambient temperature [34]. Some comprehensive reviews about SHWC have been published [72; 76; 77]. As does HTLC, SHWC requires special instrumentation, such as a mobile phase preheater, a column heater, and a post-column effluent cooling system [34].