Separation of tetrahydrozoline enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of chiral recognition mechanisms.

The recognition power and affinity pattern of various cyclodextrins (CD) towards the enantiomers of tetrahydrozoline (THZ) were studied using capillary electrophoresis (CE). As expected, affinity of THZ enantiomers and selectivity of recognition towards CD derivatives was strongly dependent on the cavity size and substituent type and pattern on the CD rims. Not only were the affinity strength and selectivity of recognition affected by the size of the cavity and chemistry of the CDs but also the affinity pattern. Another interesting example of opposite affinity pattern of enantiomers towards α- and ß-CD was observed here. In addition, opposite affinity pattern of THZ enantiomers was seen towards ß-CD and its acetylated derivatives, while methylation of ß-CD did not affect the affinity pattern of THZ enantiomers. In order to get more information about structural mechanisms of the multivariate dependences mentioned above, rotating frame Overhauser enhancement spectroscopy (ROESY) and computation techniques were used. Significant differences between the structure of THZ complexes with different CDs with both methods were encountered. Good correlations between experimentally determined and computed structure of complexes, as well as between computed complex stabilities and enantiomer migration order (EMO) in CE were observed.

Separation of terbutaline enantiomers in capillary electrophoresis with neutral cyclodextrin-type chiral selectors and investigation of the structure of selector-selectand complexes using nuclear magnetic resonance spectroscopy.

The major goal of this study was to determine the affinity pattern of the terbutaline (TB) enantiomers toward α-, ß-, γ-, and heptakis(2,3-di-O-acetyl)-ß-cyclodextrins and using NMR spectroscopy for the understanding of the fine mechanisms of interaction between the cyclodextrins (CD) and TB enantiomers. It was shown once again that CE in combination with NMR spectroscopy represents a sensitive tool to study the affinity patterns and structure of CD complexes with chiral guests. Opposite affinity patterns of TB enantiomers toward native α- and ß-CDs were associated with significant differences between the structure of the related complexes in solution. In particular, the complex between TB enantiomers and α-CD was of the external type, whereas an inclusion complex was formed between TB enantiomers and ß-CD. One of the possible structures of the complex between TB and heptakis(2,3-di-O-acetyl)-ß-CD (HDA-ß-CD) was quite similar to that of TB and ß-CD, although the chiral recognition pattern and enantioselectivity of TB complexation with these two CDs were very different.

Separation of brombuterol enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes using nuclear magnetic resonance spectroscopy.

The major goal of this study was to determine the affinity pattern of brombuterol (BB) enantiomers toward various cyclodextrins (CD) and to evaluate the potential of NMR spectroscopy for understanding fine mechanisms of interactions between CDs and BB enantiomers. Separation of BB enantiomers was performed in a fused-silica capillary using a phosphate buffer, pH 2.5, at the room temperature in the normal polarity mode. It was shown once again that CE in combination with NMR spectroscopy represents a very sensitive tool for studies of affinity patterns and structure of CD complexes with chiral guests. Although opposite affinity patterns of BB enantiomers were observed toward native ß- and γ-CDs, no significant differences between the structures of the complexes of these two CDs with BB were detected by NMR spectroscopy. In contrary to this, the opposite affinity pattern of BB enantiomers toward ß-CD and its two sulfated derivatives, heptakis (2,3-O-diacetyl-6-sulfo)-ß-CD (HDAS-ß-CD) and heptakis (2-O-methyl-3,6-di-O-sulfo)-ß-CD (HMDS-ß-CD) was associated with major differences in the structure of the complexes. In addition, it was shown again that HMDS-ß-CD provides separation of enantiomers without formation of inclusion-type complex with the chiral analyte.

Separation of terbutaline enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes.

The affinity pattern of terbutaline enantiomers towards various cyclodextrins was studied using capillary electrophoresis. The affinity pattern of terbutaline enantiomers was the same towards all studied cyclodextrins except heptakis(2-O-methyl-3,6-di-O-sulfo)-ß-CD. Nuclear magnetic resonance spectroscopy was used for understanding of fine structural mechanisms of interactions of ß-cyclodextrin and its two sulfated derivatives with the enantiomers of terbutaline. The structure of terbutaline complexes with all 3 cyclodextrins studied was different from each other. In confirmation with our earlier studies it was shown again that capillary electrophoresis represents very sensitive technique for studies of affinity patterns in cyclodextrin complexes with chiral guests. Other instrumental (e.g. NMR spectroscopy and X-ray diffraction analysis) and theoretical techniques, although very useful for obtaining the information regarding the stoichiometry, binding constants and structure of intermolecular complexes, as well as about the forces involved in selector-selectand binding and chiral recognition, may sometimes fail to properly sense those fine differences in the affinity patterns. Therefore, it is recommended to use capillary electrophoresis in order to examine correctness of affinity pattern determined for intermolecular complexes of cyclodextrins with guest molecules by other instrumental or computation techniques.

Separation of enilconazole enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes by using nuclear magnetic resonance spectroscopy.

In the present study, the enantiomer migration order (EMO) of enilconazole in the presence of various cyclodextrins (CDs) was investigated by capillary electrophoresis (CE). Opposite EMO of enilconazole were observed when ß-CD or the sulfated heptakis(2-O-methyl-3,6-di-O-sulfo)-ß-CD (HMDS-ß-CD) was used as the chiral selectors. Nuclear magnetic resonance (NMR) spectroscopy was used to study the mechanism of chiral recognition between enilconazole enantiomers and those two cyclodextrins. On the basis of rotating frame nuclear Overhauser (ROESY) experiments, the structure of an inclusion complex between enilconazole and ß-CD was derived, in which (+)-enilconazole seemed to form a tighter complex than the (-)-enantiomer. This correlates well with the migration order of enilconazole enantiomers observed in CE. No evidence of complexation between enilconazole and HMDS-ß-CD could be gathered due to lack of intermolecular nuclear Overhauser effect (NOE). Most likely the interaction between enilconazole and HMDS-ß-CD leads to formation of a shallow external complex that is sufficient for separation of enantiomers in CE but cannot be evidenced based on ROESY experiment. Thus, in this particular case CE documents the presence of intermolecular interactions which are at least very difficult to be evidenced by other instrumental techniques.

Effect of pore-size optimization on the performance of polysaccharide-based superficially porous chiral stationary phases for the separation of enantiomers in high-performance liquid chromatography.

Our earlier studies on the preparation of chiral stationary phases (CSP) based on superficially porous (or core-shell) silica (SPS) particles for the separation of enantiomers in HPLC have provided proof to the advantages of such sorbents. In particular, higher enantioselectivity was observed with the columns packed with superficially porous CSP compared to the columns packed with fully-porous (FP) silica-based CSPs at comparable content of chiral selector (polysaccharide derivative) in CSP. Also, less dependence of plate height on mobile phase flow rate and higher plate numbers and resolution calculated per unit time (i.e. speed of separation) were observed with SPS-based CSPs. Thirty years of CSP development have demonstrated that wide-pore silica has to be used as a support for large molecular weight chiral selectors such as the ones based on polysaccharides. In this study the effect of pore size of the core-shell silica support and of other experimental factors on column performance is demonstrated. Reduced plate heights in the range 1.4-1.5 were obtained, as well as highly effective baseline separations of enantiomers were observed with analysis times of less than 15s.

Separation of enantiomers of chiral weak acids with polysaccharide-based chiral columns and aqueous-organic mobile phases in high-performance liquid chromatography: Typical reversed-phase behavior?

When polysaccharide-based chiral columns are used in combination with aqueous-organic mobile phases for the separation of enantiomers in high-performance liquid chromatography the separation mode is commonly called "reversed-phase" in analogy to achiral separations. In several earlier and recent studies on neutral and basic chiral analytes it was shown by our and other groups that due to multiple type of interactions involved in selector-selectand binding and enantioselective recognition with polysaccharide derivatives, the above mentioned separation system may not always behave like a reversed-phase system. In the present study additional examples of non-reversed-phase behavior are described for the first time for weak acidic chiral analytes. In addition, the reversal of enantiomer elution order was observed again for the first time for several analytes based on water-content in the mobile phase.

High-performance liquid chromatographic separations of stereoisomers of chiral basic agrochemicals with polysaccharide-based chiral columns and polar organic mobile phases.

The separation of the stereoisomers of 23 chiral basic agrochemicals was studied on six different polysaccharide-based chiral columns in high-performance liquid chromatography with various polar organic mobile phases. Along with the successful separation of analyte stereoisomers, emphasis was placed on the effect of the chiral selector and mobile phase composition on the elution order of stereoisomers. The interesting phenomenon of reversal of enantiomer/stereoisomer elution order function of the polysaccharide backbone (cellulose or amylose), type of derivative (carbamate or benzoate), nature, and position of the substituent(s) in the phenylcarbamate moiety (methyl or chloro) and the nature of the mobile phase was observed. For several of the analytes containing two chiral centers all four stereoisomers were resolved with at least one chiral selector/mobile phase combination.

Effect of basic and acidic additives on the separation of some basic drug enantiomers on polysaccharide-based chiral columns with acetonitrile as mobile phase.

The separation of enantiomers of 16 basic drugs was studied using polysaccharide-based chiral selectors and acetonitrile as mobile phase with emphasis on the role of basic and acidic additives on the separation and elution order of enantiomers. Out of the studied chiral selectors, amylose phenylcarbamate-based ones more often showed a chiral recognition ability compared to cellulose phenylcarbamate derivatives. An interesting effect was observed with formic acid as additive on enantiomer resolution and enantiomer elution order for some basic drugs. Thus, for instance, the enantioseparation of several ß-blockers (atenolol, sotalol, toliprolol) improved not only by the addition of a more conventional basic additive to the mobile phase, but also by the addition of an acidic additive. Moreover, an opposite elution order of enantiomers was observed depending on the nature of the additive (basic or acidic) in the mobile phase.

Dynamic computer simulation of electrophoretic enantiomer migration order and separation in presence of a neutral cyclodextrin.

One-dimensional dynamic computer simulation was employed to investigate the separation and migration order change of ketoconazole enantiomers at low pH in presence of increasing amounts of (2-hydroxypropyl)-ß-cyclodextrin (OHP-ß-CD). The 1:1 interaction of ketoconazole with the neutral cyclodextrin was simulated under real experimental conditions and by varying input parameters for complex mobilities and complexation constants. Simulation results obtained with experimentally determined apparent ionic mobilities, complex mobilities, and complexation constants were found to compare well with the calculated separation selectivity and experimental data. Simulation data revealed that the migration order of the ketoconazole enantiomers at low (OHP-ß-CD) concentrations (i.e. below migration order inversion) is essentially determined by the difference in complexation constants and at high (OHP-ß-CD) concentrations (i.e. above migration order inversion) by the difference in complex mobilities. Furthermore, simulations with complex mobilities set to zero provided data that mimic migration order and separation with the chiral selector being immobilized. For the studied CEC configuration, no migration order inversion is predicted and separations are shown to be quicker and electrophoretic transport reduced in comparison to migration in free solution. The presented data illustrate that dynamic computer simulation is a valuable tool to study electrokinetic migration and separations of enantiomers in presence of a complexing agent.

Enantioseparation of selected chiral sulfoxides in high-performance liquid chromatography with polysaccharide-based chiral selectors in polar organic mobile phases with emphasis on enantiomer elution order.

The separation of the enantiomers of 17 chiral sulfoxides was studied on polysaccharide-based chiral columns in polar organic mobile phases. Enantiomer elution order (EEO) was the primary objective in this study. Two of the six chiral columns, especially those based on amylose tris(3,5-dimethylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate) (Lux Cellulose-4) proved to be most successful in the separation of the enantiomers of the studied sulfoxides. Interesting examples of EEO reversal were observed depending on the chiral selector or the composition of the mobile phase. For instance, the R-(+) enantiomer of lansoprazole eluted before the S-(-) enantiomer on Lux Cellulose-1 in both methanol or ethanol as the mobile phase, while the elution order was opposite in the same eluents on amylose tris(3,5-dimethylphenylcarbamate) with the S-(-) enantiomer eluting before the R-(+) enantiomer. The R-(+) enantiomer of omeprazole eluted first on Lux Amylose-2 in methanol but it was second when acetonitrile was used as the mobile phase with the same chiral selector. Several other examples of reversal in EEO were observed in this study. An interesting example of the separation of four stereoisomers of phenaminophos sulfoxide containing chiral sulfur and phosphor atoms is also reported here.

HPLC separation of enantiomers of chiral arylpropionic acid derivatives using polysaccharide-based chiral columns and normal-phase eluents with emphasis on elution order.

The separation of enantiomers of ten chiral arylpropionic acid derivatives was studied on six different polysaccharide-based chiral HPLC columns with various normal-phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on the effect of the chiral selector and mobile phase composition as well as of the separation temperature on the elution order of enantiomers. The interesting phenomena of reversal of enantiomer elution order function of the polysaccharide backbone (cellulose or amylose), type of derivative (carbamate or benzoate), nature and position of the substituent(s) in the phenylcarbamate moiety, the polar modifier of the mobile phase (ethanol or 2-propanol), its content in the mobile phase, and separation temperature were investigated. In addition, an unusual increase in retention with increasing temperature was observed for some arylpropionic acid derivatives.

Comparative enantioseparation of talinolol in aqueous and non-aqueous capillary electrophoresis and study of related selector-selectand interactions by nuclear magnetic resonance spectroscopy.

The enantiomers of the chiral ß-blocker drug talinolol were separated with two single component sulfated ß-cyclodextrin (CD) derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-ß-CD) (HDMS-ß-CD) and heptakis (2,3-di-O-acetyl-6-sulfo)-ß-CD) (HDAS-ß-CD), in aqueous and non-aqueous capillary electrophoresis (CE). The enantiomer affinity pattern of talinolol toward these two CDs was opposite in both aqueous and non-aqueous CE. However, the enantiomer affinity pattern for a given CD derivative did not change when aqueous buffer was replaced with non-aqueous background electrolyte. The structures of the analyte-selector complexes in both, aqueous and non-aqueous electrolytes were studied using rotating frame nuclear Overhauser effect (ROESY) NMR spectroscopy. Inclusion complex formation between the enantiomers of talinolol and HDAS-ß-CD was confirmed in aqueous buffer, while the complex between the enantiomers of talinolol and HDMS-ß-CD was of the external type. The complex of the talinolol enantiomers with HDAS-ß-CD in non-aqueous electrolyte was also of the external type. In spite of external complex formation excellent separation of the enantiomers was observed in non-aqueous CE.

HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide-based chiral columns.

The separation of enantiomers of five chiral dihydropyridine derivatives was studied on five different polysaccharide-based chiral HPLC columns with various normal-phase (NP), polar organic, and reversed-phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on enantiomer elution order (EEO) with various columns and mobile phase composition. The interesting phenomenon of reversal of EEO, recently reported in the case of amlodipine (AML) depending on the concentration of formic acid in acetonitrile, was also confirmed with NP eluents. Under RP conditions at relatively low water content, the EEO of AML could also be reverted by varying the concentration of formic acid in the mobile phase. However, at higher water content the same parameter did not affect the EEO, but only induced gradual decrease in resolution up to complete co-elution of enantiomers. Additionally, in organic-aqueous mobile phases retention factors decreased with increasing water content but only up to 20% (v/v), while above this concentration the expected typical RP behavior was observed. The presence of the commonly used additive diethylamine in the mobile phase seems important for observing a reversal in EEO with increasing concentration of formic acid. The reversal of the EEO was characteristic of AML only and was not observed for any of other dihydropyridines included in this study.

Separation of enantiomers of ephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE, NMR and high resolution MS studies.

The enantiomer migration order (EMO) of ephedrine was investigated in the presence of various CDs in CE. The molecular mechanisms of chiral recognition were followed for the ephedrine complexes with native α- and ß-CD and heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD) by CE, NMR spectroscopy and high-resolution MS. Minor structural differences were observed between the complexes of ephedrine with α- and ß-CD although the migration order of enantiomers was opposite when these two CDs were applied as chiral selectors in CE. The EMO was also opposite between ß-CD and HDAS-ß-CD. Significant structural differences were observed between ephedrine complexes with the native CDs and HDAS-ß-CD. The latter CD was advantageous as chiral CE selector not only due to its opposite electrophoretic mobility compared with that of the cationic chiral analyte, but also primarily due to its enhanced chiral recognition ability towards the enantiomers of ephedrine.

About the role of enantioselective selector-selectand interactions and the mobilities of diastereomeric associates in enantiomer separations using CE.

It is generally accepted that the selective binding of enantiomers of the chiral analyte to a chiral selector is necessary for enantioseparations in CE, whereas the role of mobility differences between the temporary diastereomeric associates formed between the enantiomers and the chiral selector has been commonly neglected. One of the authors of this study suggested in 1997 that the mobility difference between the diastereomeric associates of two enantiomers with the chiral selector may be solely responsible for a separation of enantiomers in CE and enantioselective selector-selectand binding may be not necessarily required. Several indirect confirmations of this hypothesis have been described in the literature within the last few years but a dedicated study proving this concept has not been published yet. The present data obtained for the two chiral antimycotic drugs ketoconazole and terconazole by CE and NMR spectroscopy unequivocally support this concept.

Enantioseparations in nonaqueous capillary liquid chromatography and capillary electrochromatography using cellulose tris(3,5-dimethylphenylcarbamate) as chiral stationary phase.

The potential of the widely used chiral stationary phase for high-performance liquid chromatography (HPLC) enantioseparations, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC, sold under the trade name Chiralcel OD) was evaluated under the conditions of nonaqueous capillary electrochromatography (CEC). The effect of the particle size of the silica gel, the loading of CDMPC on the silica gel and nature of the organic solvent, as well as electrolyte salts on the separation characteristics were investigated. This study illustrates the applicability of CDMPC for obtaining highly efficient enantioseparations under the conditions of nonaqueous CEC. Comparative study of enantioseparations in capillary liquid chromatography (CLC) and CEC indicated the significant advantages of CEC such as higher plate number at the similar linear flow rates of the mobile phase as well as better tolerance of higher linear flow rates.