## Alternative Organic Solvents in RP-HPLC
### Ethanol  
#### 2.1.1 Chromatographic Properties  
Ethanol (EtOH) is one of the greenest organic solvents, which makes it a particularly desirable solvent for green liquid chromatography [9]. Compared to ACN and MeOH, EtOH is less toxic and has a lower vapor pressure, which leads to less evaporation, and consequently, to lower inhaled quantities. EtOH is also widely available and less expensive (particularly EtOH 96%) than ACN and MeOH, which promotes its use in resource-limited laboratories, particularly in developing countries [3]. Moreover, due to its environmentally compatible waste, EtOH has lower disposal costs compared to ACN and MeOH, which is advantageous for sensitive areas where chemical-waste disposal is still too expensive. From a chromatographic point of view, EtOH has similar properties to ACN and MeOH. For example, [PERSON] et al. [28] studied the adsorption characteristics in RP-HPLC of an EtOH/water mixture on an octadecylsilyl (ODS)-silica gel column, and compared them to corresponding results obtained from MeOH/water and ACN/water mixture-based mobile phases. In the three chromatographic systems, they found a similar mechanism of surface diffusion with regard to enthalpy-entropy compensation and linear, free-energy relation. Based on this study, quite similar contributions of adsorption mechanisms were demonstrated. From this point of view, similar separation mechanisms should be expected using these different solvent mixtures. Also, EtOH/water mixtures were evaluated by [PERSON] et al. [29], as reversed-phase mobile phases on both C8 and C18 columns. They showed that, by substituting ACN or MeOH with EtOH as a green modifier, similar peak efficiency was obtained for the chromatographic separation of a mixture containing neutral and basic compounds. As confirmed later by other investigators, satisfactory performance could be achieved with EtOH [30]. In terms of selectivity, EtOH is in the same group as MeOH, according to [PERSON] et al. 's [2] classification of organic solvents, which means that they have a similar proton acceptor, proton donor, and dipole moments. In addition, EtOH has a higher eludotropic strength, which means that a lower percentage of EtOH than MeOH is needed in the mobile phase for comparable retention times [26]. However, EtOH has two main drawbacks which can hinder its use in RP-HPLC [16]. The first drawback is that the UV cut-off of EtOH (210 nm) is higher than that of MeOH and ACN [2], which can result in elevated background noise and an important drift of baseline when gradient elution is used, leading to a reduction in sensitivity when UV detection is hyphenated. Nevertheless, the high UV cut-off of EtOH may not be a problem if compounds have strong UV chromophores, or if mass-spectrometry (MS) detection is used. The important number of applications reported in Table 1 proves that this limitation can be overcome. The second drawback comes from the viscosity of EtOH/water mixtures, which is higher than that of MeOH/water and ACN/water mixtures for equivalent eludotropic strength at room temperature. Such high viscosity leads to high backpressures, and thus, limits the use of EtOH with conventional LC systems (400 bar) [17]. The main strategy to overcome the high pressures generated by viscous mobile phases is the use of UHPLC systems, which can support high pressures above 1000 bar. However, even if UHPLC instruments are widely available on the market, these instruments are expensive, and thus, less accessible to limited-resource laboratories. Another way to overcome the high pressure generated by ethanol-based mobile phases is the use of columns packed with superficially porous particles, which are also compatible with conventional LC instruments. The development of columns packed with such particles was considered a breakthrough in column technology, since it limits backpressures created, and thus, allows the use of high mobile phase flow rates, while maintaining column efficiencies [31].