Chromatographic Enantioseparations in Achiral Environments: Myth or Truth?

Abstract

Direct chromatographic enantioseparations are among the most important practical tasks of chromatography. The accepted rules and concepts of stereochemistry anticipated one type of chromatographic systems applicable only to such enantioseparations consisting either of chiral stationary phase, or chiral selector present in mobile phase. In such a model of chromatographic system, both racemic and non-racemic mixtures could be enantioseparated. Over the years, however, reports on successful chiral enantioseparations in non-chiral chromatographic systems have cumulated as well. To adapt the initial model to novel experimental evidence, an assumption had to be added stating that these were only non-racemic mixtures (and not racemic ones), which could be enantioseparated in achiral chromatographic systems, by granting them an inevitable chirality from the enantiomeric excess of a more abundant enantiomer. There still exists an overlooked portion of experimental evidence for successful enantioseparations of racemic mixtures in the non-chiral chromatographic systems, which cannot be explained by the accepted model. Facing this incompatibility between the model and practical results, we reflect on how to reconcile these two by questioning the possibility of pinpointing a true racemate with the help of our imperfect analytical tools.

A Prevalent Concept of Chromatographic Enantioseparations and Its Later Modification

According to the accepted rules and concepts of stereochemistry, resolution of enantiomers can be effective only in the presence of another chiral species and there would be no discrimination in optically inactive surroundings (except in the crystalline state as achieved by Pasteur). Gil-Av opined that enantioseparations can be executed, e.g., in chromatographic systems which employ either a chiral stationary phase or a mobile phase with a chiral selector (1, 2).

It was understood that in such systems both the non-racemic mixtures and the racemates can be separated into individual enantiomers. In view of experimental evidence stepwise cumulating and showing that separation of enantiomers is also possible in completely achiral environments (i.e., those composed of an achiral stationary phase and a mobile phase lacking a chiral selector), the original rigid assumption was with time modified, as given in the classical reference by Charles and Gil-Av (3). Thus, it was eventually agreed that separation of enantiomers in achiral chromatographic systems could also be possible, yet with non-racemic mixtures alone and provided a convincing model (4), which explains that such cases could be provided. This was based on certain previous experimental reports (3, 5, 6).

Scientific Reports in Support of the Presently Accepted Model

In 2016, a review article was published by Martens and Bhushan (7), in which the authors presented the results of their survey of scientific literature dealing with the subject matter of the enantioseparations of non-racemic mixtures in achiral chromatographic systems and they summarized the available explanations contained therein. Earlier surveys on the similar subject matter and by the same authors appeared in papers (8, 9).

Accumulation of Uncomfortable Experiments

However, a certain number of reports have cumulated in the chromatographic literature over the past more than 3 decades, which originate from at least three different and unconnected laboratories, and furnish experimental evidence of the enantioseparations of the racemates in the non-chiral chromatographic systems (10–16). Moreover, these reports do not provide any theoretical justification of the obtained results and for this reason they could not be included in the review paper (7). As a matter of fact, information contained in papers (10–16) is regarded by the analytical community of chromatographers engaged in the enantioseparations as controversial and perhaps unreliable, and it certainly happens to be overlooked. Were it not a predominant opinion that the achiral chromatographic systems are unable to enantioseparate the racemates then (who knows?), perhaps some more researchers would be brave or unwise enough to report on similar phenomena observed in their own laboratories. Anyway, we have a feeling that the time has come to once again approach this overlooked information and consider it with all seriousness, as a result of thorough investigations, certainly getting confirmed in more than sufficient number of laboratory repetitions by the respective research groups.

Should We Sacrifice Racemates to Reconcile Uncomfortable Experiments With the Accepted Model?

The accepted models that justify the enantioseparation of the non-racemic mixtures with the use of an achiral chromatographic system basically ascribe this desirable performance to the enantiomeric excess of one type of enantiomers [either (R) or (S)], which introduces indispensable asymmetry to this system. Thus, in order to find theoretical justification for the reported cases of the enantioresolution of the racemates in achiral chromatographic systems (10–16), we have to scrutinize the very concept of a racemate, or more precisely, to question a possibility to experimentally pinpoint such an extreme practical case as a racemate. In our view, currently available analytical tools and methods are not sensitive enough to discriminate between a racemate and a non-racemate, i.e., to record an infinitesimal excess of one enantiomer even if specific rotation is established as equal to 0.

Moreover, the difference between optical purity and enantiomeric purity can also be a source of considerable confusion, which was demonstrated by Horeau (17). In that study, he was the first to draw public attention to possible contributions to the observed angle of rotation of the plane-polarized light from the diastereomeric association complexes” (as demonstrated upon an example of a mixture of the antipodes of α,α-ethylmethyl succinic acid). In some extreme cases, such contributions resulting in specific rotation of a non-racemic sample recorded as equal to zero could falsely suggest the presence of a racemate.

Finally, we believe that even an infinitesimal excess of one enantiomer (impossible to record with a given sample, which therefore is considered as a racemate) is in power to trigger the enantiomeric separation in an achiral chromatographic system through a molecular mechanism perhaps not clear enough at the moment, yet which should not be overlooked or a priori negated.

Conclusion

In conclusion, we express our profound conviction that the non-trivial analytical problem referring to the enantioseparation by means of chromatography in general, and to the enantioresolution of the racemates in non-chiral chromatographic systems in particular, deserves an investment of much more effort in order to clarify its possible mechanism on the molecular level.