Comparative study of zwitterionic Cinchonane-based ion-exchangers for liquid chromatographic enantioseparations of free fluorinated tryptophanes: mechanistic and thermodynamic insights under green mobile phase conditions

In recent years, growing awareness of environmental sustainability has driven the search for eco-friendly alternatives in analytical chemistry. In liquid chromatography, this includes replacing traditional organic solvents like acetonitrile (MeCN) with greener options that comply with environmental standards while maintaining chromatographic performance. In this study, we describe in a comparative manner the performance of four Cinchona alkaloid-based ion-exchangers for the enantioresolution of fluorinated tryptophan analogs, applying dimethyl carbonate (DMC) as a green alternative to MeCN. Quinine- and quinidine-based zwitterionic chiral stationary phases (CSPs), namely ZWIX(+), ZWIX(-), ZWIX(+A), and ZWIX(-A) were utilized with binary mobile phase systems consisting of methanol (MeOH), MeCN, and DMC. To the best of our knowledge, this is the first report of the Cinchona alkaloid-based ion-exchangers applied with DMC-based mobile phase systems. In polar ionic mode using MeOH/MeCN and MeOH/DMC mobile phases similar retention characteristics were obtained on all the Cinchona-based columns. Under the same eluent conditions, higher retention factors were observed in the presence of DMC, and in both systems, increasing the proportion of the less polar component (MeCN or DMC) in the mobile phase resulted in a significant increase in retention for all investigated tryptophan analogs, due to stronger interactions with the stationary phase. The stoichiometric displacement model described the dependence of retention on the counterion concentration with good accuracy in both mobile phase systems, and the more negative slopes observed in the case of DMC indicate the formation of stronger ionic interactions. Unlike in the MeCN-based system, the DMC-based eluents showed a general decrease in both enantioselectivity and resolution for most analytes as the DMC content increased on all four columns. Given the substantial difference in polarity between MeCN and DMC, notable variations in enantioselectivity were anticipated. Surprisingly, only modest deviations were detected when comparing mobile phases containing MeCN to those with DMC. Despite the similar chromatographic properties observed for the two solvents, the thermodynamic studies revealed significant differences between the two mobile phase systems. For MeCN-based eluents, retention times slightly decreased with temperature on most CSPs, in contrast, with DMC-based eluents, retention times increased for all analytes on all CSPs as temperature rose. Interesetingly, enantioselectivity changed differently on the studied CSPs; enthalpically driven separations were observed in the MeCN-containing systems on all zwitterionic CSPS, whereas several entropically driven separations were observed in the DMC-containing systems on the ZWIX(-) CSP. The elution order of the enantiomers remained unchanged, regardless of replacing MeCN with the greener solvent DMC or varying the eluent composition and temperature.