## 2 Methods for Determination of Lipophilicity
### Indirect Experimental Determination of Lipophilicity
#### 2.3.2 Reversed-Phase High-Performance Liquid Chromatography
CHI corresponds to the percentage of organic modifier required to achieve equal distribution of the analyte between the stationary and the mobile phase. CHI values are obtained by measuring the retention times in gradient mode (\(\mathrm{t_{g}}\)) and then inputting the \(\mathrm{t_{g}}\) into the equation [19]:  
\[\mathrm{CHI}=\mathrm{a}\ \times\ \mathrm{t_{g}}+\mathrm{b} \tag{8}\]  
where \(\mathrm{a}\) and \(\mathrm{b}\) are regression constants for the slope and intercept obtained by linear regression of \(\mathrm{t_{g}}\) of reference substances against the CHI of the reference substances. For measuring \(\log\mathrm{P}\) with RP-HPLC, the following features need to be considered:  
* Precision: triplicates should be performed, and the obtained values of the \(\log\mathrm{P}\) values should be within a range of \(\pm 0.1\log\) units [26];
* Sensitivity: HPLC enables \(\log\mathrm{P}\) to be determined over a range of about 0-6 [30];
* Specificity: the presence of impurities may difficult the peak assignment and the interpretation of the obtained values [26];
* Accuracy: the obtained \(\log\mathrm{P}\) values can be within \(\pm 1\log\) unit of the shake-flask value [26]. To control the above-mentioned features, the choice of suitable stationary and mobile phases plays a pivotal role. Various stationary phases have been applied to evaluate indirectly isotropic lipophilicity [34]. Most of the chromatographic methods for measuring lipophilicity use RP silica-based stationary phases containing long-chain hydrocarbon, like octyl (C8) and octadecyl (C18), bound onto silica (Figure 3) [19; 26; 30; 81]. Silica is the most preferred packing material for RP columns, but it has the inconvenience of possible silanophilic interactions between the analyte and the remaining free silanol sites of the stationary phase. These interactions, more prominent in positively charged compounds, can produce distortions in peak shape and a considerable increase in the compound retention, which affects their \(\log\mathrm{P}\) determination [19; 27]. In addition, these stationary phases are not stable at pH above 7.5 and can even break down [115]. OECD recommends that the stationary phases should have a minimal percentage of polar groups as they may impair the performance of the RP-HPLC column [30]. However, to overcome this problem, the best recommendation is to use highly end-capped columns for the RP-HPLC method, especially for basic compounds under high pH conditions [77; 166]. One of the strategies for end-capping is to use base-deactivated silica stationary phases where the end-capping of the silanol residues is performed by the treatment with trimethylchlorosilane [86]. Another strategy to suppress silanophilic effects is the use of polar embedded stationary phases. Here, electrostatic shielding is achieved by the incorporation of functional groups, such as amide or carbamate, at the alky chains [86]. LC-ABZ\({}^{+}\) and Discovery-RPAmide-C16 are two examples of polar embedded stationary phases which have been used for lipophilicity determination [19; 86]. However, other secondary interactions between these polar groups and certain analytes may occur. Many of these end-capped columns are still limited to pH below 8 because of their instability in an alkaline medium. Masking the silica units with hydrophobic ethylene groups prevents them from dissolution, and the structural ethylene bridges increase the column stability at a wide pH range (1-12) [19; 166]. Acquity BEH Shield RP18 and XBridge Shield RP18 column are two examples of ethyl bridged columns that can be used to measure the lipophilicity not only for neutral and acidic compounds but also for basic ones [19; 129]. Polymer-based stationary phases, composed of a rigid and porous cross-linked polymer, are another type of phases aimed at suppressing silanophilic effects. Polystyrene-divinylbenzene (PS-DVB) and octadecyl-poly(vinyl alcohol) (ODP) are two examples of these types of stationary phases that have been successfully used for log P determination.