## 3 Results
### HPLC Resolution
This solvent is particularly suitable for applications on the milligram scale, not only for its high solvation power, but also for the high degree of enantioselectivity produced when utilized as an organic solvent in the mobile phase and the ease of removing it from the eluate by evaporation. The performance of the semipreparative 250 mm \(\times\) 10 mm Chiralpak IB column was studied using a solution obtained by dissolving 50 mg of racematic racemate **BT2-T6** in 7.5 mL of dichloromethane. During loading studies, the profile of the enantiomeric peaks in the chromatogram was used as a diagnostic to assess the complete separation. It was observed that just by increasing the racemic sample load from 0.07 mg to 0.67 mg (the injection volumes were 10 and 100 \(\mu\)L), each enantiomeric peak was split into two sub-peaks of similar area (Figure 3). As the injection volume continued to increase, the elution profile became more distorted and other peaks appeared in the chromatogram. When the injection volume was 500 \(\mu\)L, corresponding to 3.34 mg of _rac_-**BT2-T6** (Figure 4) besides the peak of the enantiomers (\(P\)) and (\(M\)), three additional peaks linked to the those of the enantiomers by plateaus were clearly visible. In particular, the peak relevant to the least-retained species  
Figure 2: Chromatograms illustrating the effect of column temperature on retention, enantioseparation, and resolution. Chromatographic conditions: column, Chiralpak IB-3 (100 \(\times\) 4.6 mm, 3 \(\mu\)m); mobile phase, _n_-hexane–methanol–dichloromethane 90:55 (_v_/_v_); column temperature, as indicated in figure; flow rate, 1.0 mL min\({}^{-1}\); detection, UV (black) and CD (orange) at 380 nm. was eluted in the void time and it was composed of an enantioenriched mixture of the enantiomers (_P_) and (_M_) in a 60/40 ratio. Thus, it is clear that, although the chromatographic support is able to resolve the enantiomers of **BT2-T6**, such a desired outcome will be hindered by the occurrence of the competitive phenomena. A possible explanation for the presence of distorted peaks in Figures 3 and 4 could be sample overloading on the column. However, it is useful to remember that whereas an analytical column becomes easily overloaded because of the reduced available active sites, semipreparative columns packed with polysaccharide-based CSPs have a very high sample throughput capability. Many research groups have already reported, in numerous  
Figure 4: Aberration on the HPLC chromatogram after injection of 3.34 mg of _rac_-**BT2–T6** in 500 \(\upmu\)L. of dichloromethane. Chromatographic conditions: column, Chiralpak IB (250 \(\times\) 10 mm, 5 \(\upmu\)m); mobile phase, \(n\)-hexane–methanol–dichloromethane 90:5:5 (\(\
icefrac{{p}}{{v}}\)/\(\
icefrac{{v}}{{v}}\)); column temperature, 15 \({}^{\circ}\)C; flow rate, 5.5 mL min\({}^{-1}\); detection, UV at 360 nm. Figure 3: Elution profiles of **BT2–T6** for different sample loads. Chromatographic conditions: column, Chiralpak IB (250 \(\times\) 10 mm, 5 \(\upmu\)m); mobile phase, \(n\)-hexane–methanol–dichloromethane 90:5:5 (\(\
icefrac{{p}}{{v}}\)/\(\
icefrac{{v}}{{v}}\)); column temperature, 15 \({}^{\circ}\)C; flow rate, 5.5 mL min\({}^{-1}\); detection, UV at 360 nm. publications, the resolution of milligram amounts of the racemic compound and injection volumes of 1-5 mL without observing peak overlapping or peak splitting [11; 22; 23]. Another critical aspect that should be considered to figure out the aberration of the semipreparative chromatogram is the composition of the sample diluent. In particular, when the elution power of the diluent is stronger than that of the mobile phase, undesirable broadening of the bandwidth can occur along the column. The mismatch of elution strength between the solvents used as the diluent and mobile phase falls in our case because the sample diluent is neat dichloromethane and the mobile phase is n-hexane/methanol/dichloromethane 90:5:5 (\(v/v/v\)).