# Selected Chromatographic Methods for Determining the Biological Activity of Substances
## 3 Chromatography in the Determination of Lipophilicity of Compounds
In the extraction system, these molecules accumulate at the interface and, for this reason, their concentration in the volumetric phases in the extraction system is underestimated. To determine the acid-base properties of compounds with large molecules, which play a major role in the distribution of substances, pH gradient elution chromatography is used. Compounds that can be present in ionic form show different lipophilicity in different pH environments [62; 63]. New descriptors, CHI log_D_ and Chrom log_D_ (_D_ partition coefficient), were created to better describe the lipophilic properties of substances, taking into account their ionisation in the substance's active medium. These can be derived from the CHI value using the following relationship [60]:  
* CHI log_D_ = 0.054 ChI \(-\) 1.467--obtained on the basis of 98 compounds. * Chrom log_D_ = 0.088 ChI \(-\) 2--obtained on the basis of 40,000 compounds. * The Chrom log_D_ values are about two times higher than the log_D_ values of octanol water. Although the n-octanol-water partition coefficient characterises the lipophilic properties of substances, it is not suitable for estimating the uptake and distribution of molecules ionised at a physiological blood plasma pH (7.4). Analysing the log_kw_ values obtained in different biomimetic chromatographic systems can provide us with information on the interactions of substances with cell membranes. [PERSON] et al. compared the log_P_ values obtained in the n-octanol-water system with the log_kw_ values of substances obtained in systems with stationary IAM and C-18 phases for compounds with different acid-base properties from different pharmacological classes and showed that these values correlate differently, indicating different interactions of substances with non-polar phases and IAM phases [64; 65]. The lipophilicity of substances can also be determined in micellar biomimetic systems [51; 66; 67; 68; 69; 70]. Micellar liquid chromatography (MLC) is reversed-phase chromatography with C-18 stationary phase (most often used) or C-8 or RP-NH_2 or RP-CN stationary phase and mobile phase--aqueous surfactant solutions above the critical micelle concentration (\(cmc\)). In such a system, there are two media that mimic biological membranes--the stationary phase (RP) and the micelles in the mobile phase. The retention of substances in micellar systems depends on the interaction between stationary phases, mobile phases, and micelles (Figure 4). The retention in MLC depends on the concentration of the micelles in the mobile phases and is described by [PERSON]'s equation:  
\[\frac{1}{k}=\frac{1}{k_{m}}+\frac{K_{AM}}{k_{m}}[M] \tag{3}\]  
where \([M]\)--concentration of the micellated surfactant, which corresponds to the difference between the total concentration of the surfactant (\(C_{S}\)) and its critical micelle concentration (\(Cmc\)), \([M]\) = \(Cs\)--\(Cmc\), \(k\)--retention factor of the substance in the mobile phase with the concentration \([M]\), \(k_{m}\)--retention factor of the substance at zero concentration of the surfactant \(Ccmc\). Based on this relationship, the interaction of the substance with the micelles (\(K_{AM}\) value) and with the RP stationary phase (\(k_{m}\) value) can be determined. In micellar systems, [PERSON]'s equation provides us with information about the interaction of substances with two models of biological membranes, namely the stationary phase and the micelles. It was found that the log\(k_{m}\) values obtained in micellar systems with C-18 mobile phase differ from the log\(k_{w}\) values obtained in RP systems [71]. The reason for this is that the stationary phase is modified by surfactant molecules, and this modification changes its character. Linear correlations with a large fitting coefficient were detected between the log\(k_{m}\) values obtained for the test substances and the log\(k_{w}\) values obtained in systems with the IAM stationary phase, indicating the similarity of the interactions of these substances with the IAM phase and the BRIJ-35 monomer-modified C-18 phase.