# Stationary Phases for Green Liquid Chromatography
## 2 Purely Aqueous Conditions: Features and Requirements
High temperatures enable elution of non-polar compounds, moderate thermal conditions enable elution of weakly polar compounds, and low temperatures are sufficient for non-polar substances. Of course, this is limited by the thermal stability of the stationary phase. A higher temperature of the water changes the polarity and reduces the viscosity, but also reduces the analyte adsorption--which is exothermal--on the stationary phase. Depending on the conditions used and the authors, chromatography with water at elevated and ambient temperatures can be referred to by different terms. The modes with the description of the conditions of pure water as a mobile phase and features of stationary phases are shown in Figure 2.  
More than 30 years ago, a study was conducted on separation in pure water [37]; sadly, it did not gain popularity. Satisfactory results were obtained when the surface of the stationary phase was modified with shorter hydrophobic alkyl chains not exceeding eight carbon atoms. Later studies confirmed that free silanols at the surface of silica particles play the main part in retention when such highly polar eluents as pure water are used; however, their excessive activity adversely affects the shape of the peaks and, therefore, the separation results. The use of even shorter carbon chains (up to C4) allowed us to obtain stable stationary phases with high coverage density. The problems associated with the attachment of longer alkyl chains are then avoided, since these phases have lower coverage densities and, at low contents of organic modifier in the mobile phase, tend to form chain bonds and decrease the stationary phase solvation [38; 39]. The adverse effect of silanols on retention led to the search for new solutions, one of which is the use of polar-endcapped and polar-embedded stationary phases [40; 41; 42; 43]. More details related to the structure, capabilities, and requirements of such phases will be described in the next section of this work. An increase in water temperature causes changes in its eluctropic strength [44]. Unfortunately, the thermal stability of both column packing and analytes is a problem. In such a case, the next option is to change the mode of liquid chromatography. This issue can be overcome by using more polar stationary phases that allow for higher eluctropic strength of water. Applying a purely aqueous mobile phase at room temperature is the most environmentally friendly mode of liquid chromatography, with advantages such as lack of toxic waste and low energy consumption. Usually, ambient conditions in liquid chromatography are interpreted as analyses performed below 60 \({}^{\circ}\)C. The literature suggests a new subcategory of  
Figure 2: Characterization of stationary phases and conditions for using pure water as a mobile phase in different liquid chromatography modes. this type of analysis--water-only reversed-phase liquid chromatography (WRP-LC)--and per aqueous liquid chromatography (PALC) is also among these techniques [45]. High elution strength of water is observed in hydrophilic interaction liquid chromatography (HILIC). Hydrophilic interaction chromatography is mainly used for the separation of polar compounds. The water content in HILIC is usually low, and it is impossible to carry out HILIC with pure water, due to the elution strength being too high. An alternative to HILIC may be a silica hydride stationary phase applied in an aqueous normal-phase (ANP) system. ANP was named and described for the first time by [PERSON] et al. [46; 47]. The changes in the retention of polar compounds from 0 to 100% water concentration present bimodal curves. The highest retention is observed at both ends--for high organic solvent content similarly to HILIC, and for pure water or high-water content [48; 49; 50]. The surface properties of silica gel in the purely water mobile phase are somewhat surprising. The hydrophilic character of silica is attributed to free silanol groups. Silanols, depending on the pH of the solution, may ionize. [PERSON] et al.