Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC - - Chromatography Online
Enantiomer and Topoisomer Separation of Acidic Compounds on Anion-Exchanger Chiral Stationary Phases by HPLC and SFC


LCGC Europe
Volume 25, Issue 11, pp. 600-611

NH4OOCH was added to the methanolic modifier at two different concentrations: 0.35% and 0.20%. As shown in Table 4, the enantioselectivity was unaffected by the concentration of NH4OOCH. The higher concentration of NH4OOCH led to reduced retention times at about 10% with minor diminution in resolution degree of the enantiomers. The concentration of 0.35% NH4OOCH in MeOH seems to be a good choice for generic method development. Caution would be needed if significantly higher salt concentrations are required, because of the unknown solubility of the salt in SF-CO2.


Table 6: Comparison between QN-AX and QD-AX columns in SFC. Modifier: MeOH/FA/NH4OOCH (100/0.40/0.35 v/v/m).
It is worth noting that a binary mixture of methanol/FA with no salt is not a stable system as a result of the rapid esterification reaction. It would lead to an increasing sample retention over time for a given analysis. The presence of the salt NH4OOCH in the modifier plays the role of the "stabilizer" of the system and is essential for reproducible chromatographic results.

Elution of Strongly Retained Compounds in SFC: From the experimental data the mixture of MeOH/FA/NH4OOCH at the proportion of 100/0.40/0.35 v/v/m represents a reasonable compromise in terms of elutropic strength and can be used as a starting point for SFC method development. However, a few of the acidic compounds under investigation showed stronger retention on the columns and could not be (completely) eluted with such a methanolic modifier in the gradient as described before.

In this scenario, efficient elution could be achieved by using a high percentage of the modifier (50–60%) containing slightly higher concentrations of the acid and salt additives and, if necessary, increasing the flow rate. Some chromatographic results obtained under the enhanced eluting conditions are presented in Table 5.

It is worth mentioning that the chiral stationary phases show relatively slow mass transfer kinetics. This drastic variation of the resultant resolution degree in dependence on the flow rate is shown in Figure 5. Thus, an effective means for improving the enantiomer separation is to reduce the flow rate.

Elution Order and Complementarity: As described in Figure 1, QN and QD are diastereomers. Chromatographically, the two CSPs behave as "pseudoenantiomers" because the stereoselectivity is under the control of C8 and C9 which have the opposite configurations.

Consequently, the elution order (EO) of the enantiomers is reversed on these two columns, as described previously for the HPLC mode (Figure 2). In the current SFC investigation, such a phenomenon was monitored by injecting one of the pure enantiomers which were available in our laboratory (Table 6). Coincidently, all the four D enantiomers examined were first eluted on QN AX and secondly eluted on QD AX. The feasibility of choosing the column in terms of elution order is undoubtedly of value especially for applications in which one enantiomer is present at a very low percentage (< 1%). Elution of the trace enantiomer in front of the major enantiomer is usually beneficial.

Conclusions

Quinine- and quinidine-derived anion exchangers are versatile in enantiomer resolution of acidic compounds by LC and SFC. While their use in HPLC is well established, their application in SFC is still a working topic that needs to be developed.

The major factors influencing the chiral separation in SFC include the acidic additive, the salt concentration and the flow rate. The mixture of MeOH/FA/NH4OOCH 100/0.40/0.35 v/v/m seems to be a suitable modifier for the first trials of method development in SFC. It was also observed that the enantiomer separations by SFC are minorly affected by the temperature in the range of 20 °C and 40 °C. Lower modifier percentages usually lead to longer retention times but have no major effect on enantioselectivity and resolution degree. The effect of other polar organic modifiers (such as ethanol, 2-propanol and acetonitrile) as alternatives to methanol was not investigated because of their poor solubility to the salt additives and the resultant potential inconvenience in the balance of eluotropic strength of the mobile phase.

These chiral columns exhibit interesting selectivities in various separations beyond enantiomer resolution. The remarkable topoisomer selectivity of quinine carbamate phases was illustrated herein as an example. The applications of QN AX and QD AX columns in non-chiral applications has not been fully explored and deserves further investigation.

Pilar Franco received her PhD degree in Pharmacy from the University of Barcelona, Spain. After two years postdoctoral work in the University of Vienna, Austria, under the supervision of Professor Wolfgang Lindner, she joined Chiral Technologies Europe, Strasbourg, France. She is now Manager of Technical Operations in the same company. Her main interests include the applications of chiral selectors for the resolution of enantiomers at analytical and preparative scale.

Tong Zhang received her PhD degree in Organic Chemistry from the University of Bordeaux I, Bordeaux, France. After three years postdoctoral work at Ciba Geigy, Basel, Switzerland, under the supervision of Dr Eric Francotte, she joined Chiral Technologies Europe, Strasbourg, France. She is now the R&D Manager in the same company. Her main interests include development and applications of chiral stationary phases for resolution of stereoisomers by chromatography.

Andrea Gargano has a Master in Pharmaceutical Chemistry and Technology from the University of Pavia, Italy. Currently he is a PhD student in analytical chemistry at the University of Vienna, Austria. His expertise includes liquid chromatography, HILIC, mixed mode, chiral chromatography on silica-based stationary phases and bioanalytics on organic monoliths.

Marek Mahut received his PhD in 2010 from the Department of Analytical Chemistry, University of Vienna (Austria). His PhD work focused on separation media and methods for plasmid DNA analysis. He then occupied a position in analytical development with the Austrian pharmaceutical company Sanochemia Pharmazeutika. He recently joined the technical R&D of Novartis Pharma in Basel, Switzerland.

Michael Lämmerhofer earned his PhD in Pharmaceutical Chemistry at the University of Graz, Austria. He was coworker of Professor W. Lindner (University of Vienna, Austria) until 2011 and from 1999 to 2000 he was post-doc at the Department of Chemistry of the University of California, Berkeley, USA, with Prof Frantisek Svec. He is now Professor for Pharmaceutical (Bio)Analysis at the University of Täbingen, Germany. His research interests include the development of functionalized separation materials, monoliths, nanoparticles, metabolomics and plasmid DNA analysis.

Wolfgang Lindner has occupied the prestigious chair of Analytical Chemistry at the University of Vienna, Austria, since 1996. His pioneering and ground-breaking work in molecular recognition and on stereo-selective techniques has ensured that he is recognized as the founder of such technologies on synthesis and mechanistic understanding of anion, cation and zwitterion exchange chiral stationary phases for chromatographic separations.


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