## 2 Methods for Determination of Lipophilicity
### Indirect Experimental Determination of Lipophilicity
#### 2.3.2 Reversed-Phase High-Performance Liquid Chromatography
Silanophilic effects are suppressed because they do not have any polar site, and they can be used at a wide range of pH values (pH 1-13) [19; 95; 205]. However, these columns often have a longer retention time when compared to the silica-based ones [115] and the retention mechanism may be controlled by different structural properties of the polymer [19]. Regarding the mobile phase, the most extensively used are binary mixtures of water or buffer with an organic modifier [27]. HPLC-grade modifier and distilled water should be used to prepare the eluting solvent, which is degassed prior to use [30]. The use of binary mixtures is a requirement as the use of only water in RP silica-based columns is not feasible [61]. With high percentages of water, more than 98%, C18 and C8 columns exhibit poorly reproducible retention due to ligand collapse and the decreased contact between the mobile phase and the stationary phase [206]. For the lipophilicity assessment, methanol is considered the most suitable organic modifier. Being a protic solvent, methanol can interact with free silanol groups suppressing this way the silanophilic effects [166]. In addition, methanol forms a monolayer that mimics the _n_-octanol/water interface of the shake-flask method [27]. For lipophilicity determination, the volume fraction of methanol should be above 25-30% [30]. At a high-volume fraction of methanol, the elutent mixture is so unlike water; the phase aimed to be mimicked by the mobile phase, that the correlation between retention times and lipophilicity will be poor [77]. Acetonitrile is another organic modifier used for lipophilicity determination. When acetonitrile is used as an organic modifier instead of methanol, the correlation between retention times and organic modifier percentage in the mobile phase is generally better described by a quadratic model instead of a linear model [71; 166]. In addition, the correlation between log P and log k\({}_{\rm W}\) was better when methanol was used instead of acetonitrile [71]. Additives to the organic modifier-water mixture can be used, particularly when dealing with dissociable compounds [19]. Hydrophobic amines, such as triethylamine, _n_-decylamine, and \(N\),_N_-dimethyloctylamine, are the traditional examples of additives added to the mobile phase. These masking agents can reduce silanophilic interactions and are very useful when basic compounds are analyzed [19]. Ionic liquids, such as 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF\({}_{4}\)]), are greener alternatives to the traditional additives [19; 72]. These additives are usually composed of relatively large asymmetric organic cations and inorganic/organic anions, meaning that both anion and cation can participate in the retention mechanism [19]. Therefore, the inclusion of a masking agent can selectively affect the compound retention times, and it should only be used on justifiable occasions [77]. In a similar way, ion suppressors can be added to the mobile phase to suppress the dissociation of ionizable compounds. Ion suppression is achieved by using buffer solutions based on phosphate, phosphate-buffered saline (PBS), or morphilinepropanesulfonic acid (MOPS) [19]. _n_-Octanol itself can be used as an additive to provide a better correlation with lipophilicity. The idea behind it is to render octanol-like characteristics to the column by saturating the column with _n_-octanol and then use of _n_-octanol saturated water as eluent. However, the inclusion of \(n\)-octanol as an additive should be tested. Some reports show better correlations between log P and log k\({}_{\rm W}\) if better if \(n\)-octanol is supplementarily added [72; 77] while others show the opposite trend [174]. Regarding the detection, most of the reported LC methods use UV-Vis or diode-array detectors (DAD), while mass spectrometry (MS) detection is only scarcely used (Table 1).