## 3 Results
### HPLC Resolution  
The first set of the HPLC tests performed in the course of this work was dedicated to research into enantioselective conditions capable of separating the enantiomers of the atropisomeric compound **BT2-T6** on an analytical scale. Different CSPs (Chiralpak IB-3, Chiralpak IA, Chiralpak IC, and Chiralpak IG) were selected and screened by using low-polarity normal-phaseuents. Non-standard (dichloromethane and acetone) and standard alcoholic (methanol, ethanol, and 2-propanol) solvents in a mixture with n-hexane were investigated in binary and ternary mobile phase systems. The best results in terms of enantioselectivity and analysis times were obtained employing the 100 mm \(\times\) 4.6 mm Chiralpak IB-3 column and the mixture n-hexane-methanol-dichloromethane 90:5:5 (\(\
u/\
u/\
u\)) as the mobile phase. Under these conditions, very high enantioselectivity (\(\alpha\) = 3.11) and resolution (Rs = 13.03) factor values were achieved within 7 min. In the Chiralpak IB-3 CSP, the cellulose tris(3,5-dimethylphenylcarbamate) polymeric selector is immobilized onto 3 \(\upmu\)m silica particles. In the successive step of optimization of the enantioselective method, a temperature-dependent study was performed. The enantioseparation and resolution factors were calculated between 15 \({}^{\circ}\)C and 35 \({}^{\circ}\)C in 10 \({}^{\circ}\)C increments. As can be seen in Figure 2, the enantioseparation process occurred within the enthalpy-controlled domain [21] and the enantioselectivity decreased as the temperature increased. By fixing the column temperature at 15 \({}^{\circ}\)C, the enantioseparation and resolution factor values of **BT2-T6** increased to 4.07 and 19.13, respectively, (Figure 2) without excessively extending the analysis time (less than 10 min). Comparing these chromatographic parameters with those obtained by analyzing the analog **BT2-T4** in the same conditions (i.e., 2.47 and 8.95, respectively), it is evident that the introduction of a third thiophene moiety in the wings of the atropisomeric scaffold duplicated the enantioseparation and resolution factor values. After developing the enantiomer separation method on a small scale, the next step of our work was to transfer it to a larger scale and isolate both enantiomers in multimilligram amounts. By taking into account appropriate scale factors and using semipreparativecolumns containing the same packing material as the analytical ones, the scale-up process is typically easily accomplished, especially when the values of enantioseparation and resolution are as high as those observed for **BT2-T6** on the Chiralpak IB CSP. However, to develop an effective semipreparative enantioseparation, the choice of an appropriate diluent is another key step that needs to be considered. In fact, if the solubility of the racemate in the mobile phase is low, the quantity of chiral sample loaded in a single chromatographic run is limited. In the case of coated-type polysaccharide-derived CSPs, which are substantially stable only when n-hexane, alcohols, and their mixtures are used as mobile phases, the productivity of the HPLC resolution of analytes soluble in a chlorinated solvent is low. This problem can be solved using a CSP in which the polymeric selector is immobilized onto the silica matrix, as is in the case of the Chiralpak IB-3 CSP. To evaluate the nature of the diluent to be used to dissolve the racemic **BT2-T6**, their solubility in various solvents was explored. The outcomes of this study demonstrated a low solubility (<1 mg mL\({}^{-1}\)) of the chiral compound in n-hexane-alcohol mixtures and a very high solubility (>20 mg mL\({}^{-1}\)) in dichloromethane. Therefore, to accomplish the resolution of **BT2-T6** on a semipreparative scale, dichloromethane was selected as the diluent.