4.1.10. Determination of Lipophilicity by Reversed-Phase Chromatography

The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis was performed on a Merck–Hitachi LaChrom Elite System (Merck, Darmstadt, Germany) with diode array detector L-2455, thermostat L-2300, pump L-2130, and autosampler L-2200 and with the Waters XTerra MS RP-18 (3.5 μm, 150 × 4.6 mm) chromatographic column (Waters, Milford, MA, USA) as the stationary phase. The mobile phase consisted of methanol, water, and 0.1% (v/v) formic acid and was degassed by the use of the built-in membrane degasser. The methanol gradient grade for HPLC was purchased from Merck (Merck, Darmstadt, Germany) and formic acid from Polish Reagents (Polish Reagents, Gliwice, Poland); double-distilled water was used. Then, 5 μL of methanolic solutions (0.1%; m/v) of selected samples were applied to the chromatographic column by the use of an autosampler Hitachi L-2200 (LaChrom Elite, Hitachi-Merck, Darmstadt, Germany). The analysis was performed with the flow rate of 0.7 mL min⁻¹ in isocratic mode using various concentrations of organic modifier (methanol) in binary polar mobile phases; percentages of methanol in water were 45–70% (%, v/v) and changed by 5% per step. Chromatograms were detected at 254 nm and the temperature of the column was 25 °C. All experiments were repeated in triplicate, and the final results were taken to be the arithmetic means. Dead time was measured by the use of uracil (Calbiochem. Merck, Darmstadt, Germany). Statistical and regression analyses were performed using Statistica (ver. 13.3 for Windows). The chromatographic lipophilicity parameters (logkW) for selected samples were obtained by the extrapolation of the retention parameter logk to pure water, according to Equation (1):

where logkW is the value of the retention factor of a substance in pure water, S is the slope of the regression curve, and ϕ is the concentration of the organic modifier [33]. The values of logk were calculated based on the raw HPLC data using the formula (2):

where tR is the retention time and t0 is the dead retention time (determined for uracil).