# Selected Chromatographic Methods for Determining the Biological Activity of Substances
## 3 Chromatography in the Determination of Lipophilicity of Compounds
The simplest biomimetic system is the chromatographic system, which involves a stationary phase; a non-polar phase (usually C-18, which imitates the non-polar part of the cell membrane); and a mobile phase (usually water or a buffer solution with a pH of 7.4), which imitates physiological fluids. The determination of kw values directly in the RP system is not possible for most substances due to the hydrophobicity of the stationary phase and the zero elution power of water in \(RP\) systems. Therefore, the log\(k_{w}\) values are determined by extrapolating the straight-line dependence of the log\(k_{w}\) value on the content of organic modifiers in the aqueous-organic mobile phase. This is achieved using the Soczewinski-Wachtmeister equation [57] (Figure 3). \[\text{log}k=\text{log}k_{w}-\text{s}\,\phi \tag{2}\]  
where log\(k\) is a retention factor of the substance obtained in a chromatographic system with a mobile phase with the concentration of the organic modifier \(\phi\); s represents the slope of the straight line; log\(k_{w}\) is the log of the value of the retention factor of the substance in water when water is acting as the mobile phase. Three parameters that describe the lipophilicity of substances can be derived from the Soczewinski-Wachtmeister equation. The first is the retention factor \(k_{w}\), which can be identified based on the partition coefficient of the substance \(P\). The second is the s value, which informs us about the non-polar part of the substance, and the last parameter (CHI (0)) is the amount of organic modifier in the mobile phase where the retention factor is equal to one [58,59]. The CHI values range from 0 (for hydrophilic) to 100 (for more lipophilic) substances. In extreme cases involving very hydrophilic or lipophilic substances, these values can be exceeded. However, it should be noted that substances with such extremely lipophilic  
Figure 3: Lipophilicity descriptors obtained from the Soczewinski–Wachtmeister equation (Equation (2)). Figure 2: Relationship between log\(P\) and log\(k_{w}\). properties have unfavourable pharmacokinetic parameters. To avoid a series of isocratic measurements, fast gradient chromatography was proposed to determine the chromatographic hydrophobicity index (CHI). In the RP system with fast gradient elution, the compounds hardly move in the column until the corresponding concentration of the mobile phase is reached. A straight line relationship was observed between the CHI values obtained during isocratic elution and the retention times of the compound during fast gradient elution [26; 60; 61]. It was found that although correlations between log_kw_ and log_P_ n-octanol-water are observed, the values of log_kw_ and log_P_ differ for some reasons:  
* Chromatography is a dynamic process, whereas the extraction process is a static equilibrium process;
* The chromatographic process of substance distribution between the stationary and the mobile phases takes place on a much larger surface than in the extraction method (as in the body);
* The C-18 phase has a more structured structure than liquid n-octanol (the structure of the stationary C-18 phase is more similar to the structure of the cell membrane than n-octanol). When comparing the log_P_ values with the log_kw_ values, it was found that significant differences occur with proton donor compounds, since the -OH group of n-octanol is a proton acceptor, whereas the C-18 ligands of the stationary phase are not. The values of the obtained lipophilicity descriptors depend on the type of organic modifier. Linear relationships were observed, with large fitting coefficients between CHI log_P_ and log_P_o/w_ (n-octanol-water) values obtained in the chromatographic system in which the organic modifier was acetonitrile. When methanol was used as the organic modifier, the relationship was parabolic [60]. For large amphiphilic molecules such as peptides and macrolides, the n-octanol-water system is not used to determine the partition coefficient \(P\) value.