## 2 Methods for Determination of Lipophilicity
### Indirect Experimental Determination of Lipophilicity
#### 2.3.7 Micellar Liquid Chromatography (MLC)  
Micellar liquid chromatography (MLC) is a variant of RP-LC in which a surfactant at a concentration above the critical micellar concentration is added to the mobile phase, leading to the formation of micelles within the aqueous mobile phase (Figure 4c) [194; 213]. MLC can be coupled to TLC, HPLC, and over-pressured-layer chromatography using micellar mobile phases [213].  
In MLC, the micellar aggregates act as partition sites in the mobile phase, while the surfactant monomers present in the mobile phase modify the stationary phase either by both hydrophobic and/or silanophilic adsorption [203]. Due to the amphiphilic character of surfactants, the micellar mobile phase mimics the phospholipidic bilayer structure of biological membranes, while the modified stationary phase mimics not only the ordered array of the hydrophobic chains of phospholipids, mainly through the C18 carbon chains but also the polar heads of phospholipids, mainly through the adsorbed surfactant monomers [214]. During the elution, the solute can establish interactions with the micellar core, the micellar surface, the polar head of the surfactant bound to the stationary phase, and the alkyl chains bound to the stationary [194]. Therefore, solute retention involves the establishment of two different equilibriums: one between the bulk aqueous phase and the surfactant-coated stationary phase and another between the aqueous phase and the micelles [203]. The outcome index of MLC (log kMLC) is a metric of anisotropic lipophilicity as it depends on hydrophobic, electronic, and steric interactions [215]. Due to its similarity with biological membranes and extracellular fluids, MLC is also known as biopartitioning micellar chromatography [203].  
Since the first reports of MLCs in the early 1980s, modifications of MLC have been reported [194]. These modifications are aimed to solve the decreased column efficiency in MLC due to slow mass transfer. The slow mass transfer is attributed to the poor wettability of a purely aqueous mobile phase and modification of the stationary phases by the adsorption of surfactant monomers [216]. To improve column efficiency, the main approaches are the addition of organic modifiers, the increase of column temperature, and decreased flow rate.  
Exploiting the ability to mimic the phospholipid barriers, log k\({}_{\text{MLC}}\) values have been used to predict several pharmacokinetic parameters, such as human oral absorption (HOA) [196; 197; 198; 199], blood-brain barrier (BBB) permeability [196], plasma protein binding (PPB) [200; 201], the volume of distribution [200; 201], and therapeutic parameters [200; 201]. When compared to other chromatographic techniques, MLC appears to be in the mid-ground between IAM and RP-HPLC. As an example, the lipophilicity of a set of fused azaisocytosine analogues was evaluated using RP-HPLC, IAM, and MLC [193]. For all compounds under study, the log k\({}_{\text{MLC}}\) values were higher than log k\({}_{\text{W-IAM}}\) but lower than log k\({}_{\text{W}}\). In another example, the lipophilicity of a set of 88 compounds was evaluated using IAM and MLC [203]. Here, the obtained log k\({}_{\text{MLC}}\) values covered a narrower range than the obtained log k\({}_{\text{W-IAM}}\) values (log k\({}_{\text{W-IAM}}\) range: from \(-\)1.15 to 3.98, log k\({}_{\text{MLC}}\) range: from 1.84 to 3.98). These differences are attributed to the strong electrostatic interactions between positively charged drugs and the IAM phase and to the improved distribution of lipophilic compounds in the micellar MLC mobile phase. The chromatographic retention factors were used to predict cell permeability, HOA, and PPB. MLC retention factors demonstrated a better predictor capacity than IAM for cell permeability, while similar results were found for the remaining parameters. The advantage of the MLC is that it uses a conventional and easily available RP column, which is usually available in any analytical laboratory, while IAM requires a specific and expensive column.