# Selected Chromatographic Methods for Determining the Biological Activity of Substances
## 3 Chromatography in the Determination of Lipophilicity of Compounds  
In order to reach the site of action, a substance must overcome a number of biological barriers--cell membranes. In order to penetrate a cell membrane, a substance must overcome its lipophilic interior. According to the principle of 'like dissolves like', the ability of a substance to penetrate biological membranes depends on its lipophilic properties. Therefore, lipophilicity is one of the most important physicochemical properties of biologically active substances after interaction with the corresponding receptor. The ability of a substance to penetrate biological membranes has a direct influence on the processes that the substances undergo in the body--absorption, distribution and, consequently, the biological activity of the substance. For a substance to have a therapeutic effect, it must be present at the site of action in the correct concentration (the so-called therapeutic window). To summarise, lipophilicity is the property of a substance that allows us to predict the ability of that substance to penetrate cell membranes; its ADME profile, i.e., the individual processes that describe the fate of the drug in the body; and its ability to reach the site of action [10; 54]. Quantitatively, lipophilicity describes the partition coefficient of a substance between n-octanol and water, \(P\). It is one of the first parameters determined to describe the biological activity of a substance in testing different substances. Nowadays, many new substances that are potential drugs are developed every day. To be a drug, a substance must of course have pharmacokinetic properties, in addition to its effect on the body. Determining the biological activity of a substance, especially in the early stages of research, can be achieved outside living organisms, using biomimetic systems. Based on the knowledge of physiological fluid composition and cell membrane structure, a model system for n-octanol-water extraction was developed for the determination of \(P\) values. It was found that within the limits of log\(P\) 1-3, the penetration rate through biological membrane substances is directly proportional to the log\(P\) value. The partition coefficient \(P\) was first used by [PERSON] et al. to determine the lipophilicity of substances [55]. It can be determined by the extraction method (shake flash)--until recently, this was the method recommended by IUPAC. Although this method is easy to perform, it has a number of limitations: the substances used must be very pure and large amounts of the substance and non-polar organic solvents are required. Therefore, alternative methods for the determination of \(P\) values are being sought out--one of these methods is chromatography in systems that mimic the environment of the drug. [PERSON] observed a linear relationship between the partition coefficients of substances obtained in different aqueous extraction systems that differ in the organic solvents [56]. The retention factor of a substance (\(k\)) is the ratio between the amount of the substance in the stationary phase and the mobile phase and therefore log\(k\) values can be equated with log\(P\) values (Figure 2). If the stationary phase mimics cell membranes and the mobile phase mimics physiological fluids, the retention factor in such a chromatographic system can be equated to the \(P_{\alpha/\alpha}\) partition coefficient. For many substances, a linear relationship between log\(k_{w}\) in a biomimetic system and log \(P_{\alpha/w}\) has been observed, which is a special case of [PERSON]'s equation:  
\[\text{log}k_{w}=a\text{log}P_{\alpha/w}+b \tag{1}\]  
where log\(k_{w}\)--logarithm of the retention factor, \(a\), \(b\)--constants describing the rectilinear relationship (1).