Anna Bielejewska

Comparative study on camphor enantiomers behavior under the conditions of gas-liquid chromatography and reversed-phase high-performance liquid chromatography systems modified with α- and β-cyclodextrins

The dependence of retention and selectivity parameters of camphor enantiomers on the concentration of alpha- and beta-cyclodextrins were studied under conditions of GLC (matrix solvent: Glycerol, 95 degrees C) and RP-HPLC (matrix solvent: Aqueous methanolic, 20 degrees C). It has been found that beta-cyclodextrin forms complex of 1:1 stoichiometry and does not recognize enantiomers of camphor. In contrast alpha-CD forming complexes of 1:2 stoichiometry appeared to be very efficient chiral selector of (+) and (-)-camphor. Relatively considerable differences have been observed between stability constants determined by GLC and RP-HPLC, what may be explained by the various natures of the matrix solvents and the various temperatures of the measurements. On the contrary, the enantioseparation factor alpha observed at higher concentrations of alpha-cyclodextrin stabilizes on the very similar value alpha+/-(GLC) approximately = alpha-/+(HPLC) approximately = 1.6. Simple theoretical considerations focusing on the differences in the mechanisms of the studied processes have been performed. According to them the enantiomer forming the more stable complex with the cyclodextrin should be eluted from the RP-HPLC column first and GLC column last. This fact has been confirmed experimentally.

Joint use of cyclodextrin additives in chiral discrimination by reversed-phase high-performance liquid chromatography: temperature effects

The temperature dependence of chiral separations was investigated in combined system of reversed-phase (RP) liquid chromatography using two chiral additives: single α or β native cyclodextrins and their permethylated derivatives. The model tested compounds of pharmaceutical interest were: methylphenobarbital, mephenytoin, morsuximide and camphor. Taking the localization of a complexation process as a criterion – the combined system with two selectors has been rationalized as occurring in three stages. The influence of temperature (in narrow range of 20°C) on retention and enantioselectivity was studied in; System I (complexation occurs in the mobile phase), in System II (complexation on the stationary phase) and in System III (complexation in both phases together). In System III (as for System I) it has been found that the model compounds could be classified into three groups based on their retention dependence on temperature: retention decrease with temperature decrease, retention increase with temperature decrease or no influence of temperature on retention. For all the compounds investigated, decrease in temperature increases the selectivity. Standard enthalpy (ΔH0) and entropy (ΔS0) changes of solute transfer between the mobile and the stationary phase and standard enthalpy (ΔH0CD) and entropy (ΔS0CD) changes of complex formation were also calculated. In Systems I and III, if the complexation in the mobile phase is favored process compared with interaction with the stationary phases (RP or covered by permethylated cyclodextrin), the shortest retention time and the best selectivity is observed at low temperature.

Comparison of chiral separation of basic drugs in capillary electrophoresis and liquid chromatography using neutral and negatively charged cyclodextrins

Liquid chromatography (LC) and capillary electrophoresis (CE) are very widely used as chiral separation methods. In this publication we try to find if the results obtained in CE and LC with the chiral selector added to the electrolyte and the mobile phase, respectively, can be used as tools for studying weak stereoselective interactions, and how this information can be useful for optimizing chiral separation processes. The manuscript presents a systematic comparison of chiral discrimination of model compounds in HPLC and CE using neutral and negatively charged cyclodextrins. The enantiomeric separation of basic chiral pharmaceuticals such as pheniramine, brompheniramine, metoxyphenamine, cyclopentolate, doxylamine and ketamine was investigated in capillary electrophoresis (CE) and liquid chromatography (HPLC) using negatively charged sulfated-beta-cyclodextrin (S-beta-CD) and neutral cyclodextrins (CDs). The apparent stability constants between the model compounds and cyclodextrins were estimated in both techniques. We discuss the influence of the stability constant and K1/K2 ratio of the investigated complexes on chiral separation obtained in both techniques.

Influence of organic solvent on the behaviour of camphor and α-pinene enantiomers in reversed-phase liquid chromatography systems with α-cyclodextrin as chiral additive

Reversed-phase liquid chromatography has been applied in order to gain insight into the alpha-cyclodextrin (alpha-CD)-solute complexation process, which occurs in the aqueous mobile phases containing a secondary achiral modifier. The model compounds tested were (+/-)-camphor and (+/-)-alpha-pinene. Methanol, ethanol, and 1 or 2-propanol were used as secondary modifiers. Retention factors and enantioseparation factors have been determined on a RP 18 stationary phase as a function of the alpha-CD concentration, secondary modifier content, and temperature changes. The shortest retention and the best separation of studied compounds were achieved for aqueous-methanol eluents. Apparent stability constants in various binary aqueous-organic solvent mixtures have been evaluated for alpha-CD complexes of camphor enantiomers. Using the competition concept, values for the stability constants in pure water have been calculated. It has been found that: (1) the quotient of the stability constants for both enantiomers, denoted as absolute enantioselectivity E, always remains constant at a fixed value (E approximately 1.9), which may indicate that the complex composition does not change, (2) only the first step in the complexation process is altered by changing the solvent, which does not seem to affect the separation of the enantiomers, (3) the remarkable enantioselectivity that is observed results from the second step in the complexation process, (4) enthalpy changes are much more favourable for camphor-alpha-cyclodextrin complex formation than for the transfer of camphor to the stationary phase, which means that complexation dominates over adsorption and retention is shorter at lower temperatures, (5) the difference in free energy changes of complexation (AAG) between the enantiomers of camphor is about 1.5 kJ/mol at 20 degrees C.

Evaluation of Ligand-Selector Interaction from Effective Diffusion Coefficient

We present an analytical technique for determination of ligand-selector equilibrium binding constants. The method is based on the measurements of effective molecular diffusion coefficient of the ligand during Poiseuille flow through a long (approximately 25 m), thin (0.254 mm +/- 0.05 mm ID) capillary with and without the selector. The data are analyzed using the Taylor dispersion theory. Bovine Serum Albumin (BSA) and cyclodextrin (CD) were taken as model selectors. We have tested our method on the following selector-ligand complexes: BSA with warfarin, propranolol, noscapine, salicylic acid, and riboflavin, and cyclodextrin with 4-nitrophenol. The results are in good agreement with data from the literature and with our own results obtained within classical chromatography. This method works equally well for uncharged and charged compounds.

Improved chiral recognition of some compounds via the simultaneous use of β-cyclodextrin and its permethylated derivative in a reversed-phase high-performance liquid chromatographic system

Two chiral additives, β-cyclodextrin and (2,3,6-tri-O-methyl)-β-cyclodextrin, were applied to improve the enantioselectivity of reversed-phase high-performance liquid chromatography for methylphenobarbital, glutethimide, mephenytoin and morsuximide. It was found that the joint use of these two additives leads to an improved enantioselectivity, except for mephenytoin.

Studies on the behaviour of α-, β- and γ-cyclodextrins and some derivatives under reversed-phase liquid Chromatographic conditions

Abstract The separation processes of α-, β- and γ -cyclodextrins and their various methyl derivatives have been investigated with Knauer polarimetric (Chiralyser) and refractive index (RI) detectors. RP18 and RP8 hydrocarbon packings and an NH 2 bonded phase were applied as stationary phases. Aqueous methanolic or ethanolic solutions were used as mobile phases. It has been found that the Chiralyser detector response is approximately linear at low concentrations of solutes and that its detection capabilities are about 40 times better than those of the RI detector. Differences in the order of elution of α-, β- and γ -cyclodextrins have been observed for various stationary phases as well as for various mobile phase compositions. The optimal conditions for analytical determinations of cyclodextrins and their derivatives have been discussed.

Chromatographic studies on the inclusion of isomeric dimethylnaphthalenes by β- and γ-cyclodextrin

SummaryReversed-phase high performance liquid chromatography and gas-liquid chromatography have been applied to studies on the complexation of cyclodextrins with isomeric dimethylnaphthalenes. Both β and γ-cyclodextrins have been found to form inclusion complexes with dimethylnaphthalenes. With the exception of the complex of 1,8-dimethylnaphthalene with β-cyclodextrin, all the other complexes under investigation are of moderate stability. The remarkable affinity of β-cyclodextrin for 1,8-dimethylnaphthalene has been observed by both GC and HPLC. The stability constant of this complex was always about 1.5–2.0 orders of magnitude greater than the corresponding value for the other isomers. The chromatographic data suggest that there is an especially good fit of the 1,8-dimethylnaphthalene molecule into the β-cyclodextrin cavity. The importance of studying the complicated phenomena of complexation by cyclodextrin using various experimental and theoretical methods is stressed.

Chiral discrimination by high-performance liquid chromatography with joint use of two cyclodextrin additives

Abstract The diversity of adsorption properties of native and permethylated cyclodextrins enabled us to propose a new advantageous chromatographic system with two chiral selectors working jointly (system III). Fundamental information regarding the retention mechanism in such a system with two chiral additives is presented. Depending on the localisation of the chiral complexation process three chromatographic systems have been designed and experimentally compared under the same chromatographic conditions (solvent composition, temperature). Phenomenological and theoretical models of the above mentioned systems, where complexation occurs in the mobile phase (system I), in the stationary phase (system II) or in both phases together (system III), have been studied and experimentally confirmed. Species of biological interest were used as model compounds. It has been found that almost always system III offers the best results, although all the investigated substances display different chromatographic behaviour, i.e., adsorption on RP18 phase and complexation by native and permethylated cyclodextrin. The final enantioselectivity coefficient ( α 3 ) may be predicted as a product of partial coefficients ( α 1 , α 2 ) according to the equation α 3 = α 1 · ga 2 .

A Study of Multiple Complexation of α-, β- and γ-Cyclodextrins: Surprisingly Differing Stoichiometries of β- and γ-Cyclodextrin Complexes

Chromatographic, NMR and molecular modelling studies indicate that benzene-sym-tris-N,N,N-carbonyltriglycylglycine N′-1-adamantylamide forms the strongest complex with β-cyclodextrin (β-CD), encapsulating the terminal adamantyl group. The analogous complex with γ-CD is weaker with a deeper penetration of the guest into the host cavity, while the complex with α-CD is very weak. Remarkably, longitudinal relaxation times T 1 for the complexes with β- and γ-CD exhibit considerable differences in the signals for the adamantyl protons while no differences in the corresponding signals for the branch protons were found. Chromatographic measurements for the complexes with β- and γ-CD with the dendrimer revealed interesting differences in the stoichiometry of the dendrimer complexes in mixed solvents chosen because of the low solubility of the dendrimer under study and its CD complexes. Namely, in a 60:40 (v/v) methanol:water mixture the latter complex was undoubtedly of 1:1 stoichiometry while for the former the most plausible stoichiometry was 1:3. The binding constants of the multiple dendrimer complexes with β-CD (in 20:80(v/v) ethanol:water) of 1:1, 1:2 and 1:3 stoichiometry were estimated to be equal to ca 4 × 102 M-1, 1 × 102 M-1 and 25 × 103 M-1 while only a 1:1 complex with a binding constant of ca 6 × 102 M-1 (correlation factor R=0.97) was found for the dendrimer complex with the larger γ-CD (in 60:40 (v/v) methanol:water). (Attempts to fit the experimental curve assuming a mixture of complexes of 1:1 and 1:2 stoichiometry resulted in a value for the constant K 2 three orders of magnitude less than that for K 1.) The considerably larger value for K 3 compared with K 1 and K 2 seems to indicate that the dendrimer complex formation is cooperative. Such behaviour may be due to the more hydrophilic environment of the third adamantyl group in the 1:2 complex favouring its complexation. Chromatographic measurements for the compound mimicking one dendrimer branch yielded a 1:1 complex with γ-CD with a binding constant of while it was shown to complex at both adamantyl and benzene ends by β-CD with the respective constants of and