Characterization of complexes between phenethylamine enantiomers and β‐cyclodextrin derivatives by capillary electrophoresis—Determination of binding constants and complex mobilities

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte‐cyclodextrin‐complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β‐cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x ‐reciprocal, y ‐reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β‐cyclodextrin, (2‐hydroxypropyl)‐β‐cyclodextrin, methyl‐β‐cyclodextrin and 6‐O‐α‐maltosyl‐β‐cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer‐cyclodextrin‐complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β‐cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes.     

Permethylated cyclodextrins in the GC separation of racemic mixtures of volatiles: Part 1

The chromatographic separation of racemic mixtures of volatile compounds by 2,3,6-trimethyl-α-, β- and γ-cyclodextrins is discussed. Columns were prepared by mixing the derivatized cyclodextrin with OV-1701 or hydroxy-terminated OV-1701 (OV-1701-OH) following Schurig's method [1]. About 130 racemates with widely differing structures were used to test the performances of 2,3,6-permethylated-α, β- and, γ-cyclodextrins mixed with the polysiloxane polymers in different ratios. The influence of the different types of cyclodextrin on racemate separation is shown, and some phenomena which might be helpful in the elucidation of the chromatographic behavior involved are also described. The influence both of the percentage of cyclodextrin in the polysiloxane, and of the operating conditions (carrier gas, flow rate, and temperature) in the separation of flavor and fragrance racemates is also evaluated.     

Comparison of Different Di‐tert‐butyldimethyl‐Silylated Cyclodextrins as Chiral Stationary Phases in Capillary Gas Chromatography

Separation factors and thermodynamic data for the separation of various chiral analytes on different di‐O‐tert‐butyldimethyl‐silylated cyclodextrin derivatives are collected and described. Modifying the substitution pattern of the tert‐butyldimethylsilyl group in position 2 and 3 or changing from β‐ to γ‐cyclodextrin significantly affects the separation properties of the cyclodextrin derivatives.     

Migration order of dipeptide and tripeptide enantiomers in the presence of single isomer and randomly sulfated cyclodextrins as a function of pH

The present study was conducted in order to evaluate the cyclodextrin (CD)-mediated chiral separation of peptide enantiomers as uncharged analytes at pH 5.3 using randomly sulfated beta-cyclodextrin, heptakis-6-sulfato-beta-CD and heptakis-(2,3-diacetyl-6-sulfato)-beta-CD as chiral selectors. Although less effective compared to stronger acidic conditions, the CDs proved to be suitable chiral selectors for the present set of peptides at pH 5.3. The carrier ability of the negatively charged CDs upon reversal of the applied voltage may also be exploited leading to a reversal of the migration order. In addition, reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 5.3 was also observed for Ala-Tyr in the presence of randomly sulfated beta-CD, for Ala-Phe, Ala-Tyr, Phe-Phe, Asp-PheNH(2) and Gly-Ala-Phe in the presence of heptakis-6-sulfato-beta-CD, and for Phe-Phe and Ala-Leu in the presence of heptakis-(2,3-diacetyl-6-sulfato)-beta-CD. The migration behavior could be explained on the basis of the complexation constants and the mobilities of the peptide-CD complexes. While a change in the affinity pattern of the CDs upon increasing the pH was observed for some peptides, complex mobility was the primary factor for other peptide-CD combinations affecting the enantiomer migration order at the two pH values studied.     

Chiral separation of brompheniramine enantiomers by recycling high‐speed countercurrent chromatography using carboxymethyl‐β‐cyclodextrin as a chiral selector

A recycling high‐speed countercurrent chromatography protocol was proposed for the enantioseparation of brompheniramine by employing β‐cyclodextrin derivatives as a chiral selector. The two‐phase solvent system of n‐hexane/isobutyl acetate/0.10 mol/L phosphate buffer solution with a volume ratio of 2:4:6 was selected by a series of extraction experiments. Factors that affected the distribution of the enantiomers over the two‐phase system (e.g., the type and concentration of β‐cyclodextrin derivatives = pH value of the aqueous solution, and the separation temperature) were also investigated. In addition, the theory of thermodynamics is applied to verify the feasibility of the enantioseparation process and the corresponding results demonstrate that this separation process is feasible. The optimized conditions include carboxymethyl‐β‐cyclodextrin concentration of 0.010 mol/L, pH of 7.5, and temperature of 5°C. Under the optimal conditions, the purities of both monomer molecules were over 99%, and the recovery yields were 88% for (+)‐brompheniramine and 85% for (–)‐brompheniramine, respectively.     

卡替诺尔和氟西汀对映体的高效毛细管电泳分离

考察了以羧甲基－β－（环糊精－β－CD）、β－环糊清（β－CD）、羟丙基－β－环糊精（HP－β－CD）、二甲基－β－环糊精（DM－β－CD)为手性选择剂,在50mmol/L醋酸三乙胺缓冲溶液中分离卡替诺尔和氟西汀对映体。该文还通过考察手性选择剂的浓度、背景电解质的酸度、背景电解质的类型等因素对映体手性分离的影响,对分离条件进行了优化,初步探讨了手性识别机理。实验结果表明：用约4mmol/L的CM－β－CD分离氟西汀和卡替诺尔对映体,能使对映体达到良好分离,不仅节约了分析成本,也简化了分析过程。     

Multi-chamber apparatus for preparative isoelectric focusing

A multi-chamber apparatus for preparative isoelectric focusing is described. The apparatus is constructed of 32 separation chambers and 2 electrode chambers, all separated by uncharged porous membranes. The total volume of the 32 separation chambers is 660 mL. A cooling system and a stirring system are built in. Human serum proteins were separated by isoelectric focusing in a natural pH gradient. The fractionation was monitored by fused rocket immunoelectrophoresis. The number of proteins in each fraction was monitored by crossed immunoelectrophoresis. The apparent pI values of IgG, transferrin and alpha-1-antitrypsin are as found in the literature. Orosomucoid (alpha-1-acid glycoprotein) (pI = 1.8) is concentrated at the acid end of the pH gradient.     

Determination of fenfluramine enantiomers in pharmaceutical formulations by capillary zone electrophoresis

Summary Capillary zone electrophoresis has been used for the enantiomeric separation of racemic ortho-fenfluramine and meta-fenfluramine employing a phosphate buffer at pH 2.5 added with cyclodextrins. The cyclodextrin type and concentration strongly influenced the chiral resolution. The uncharged β-cyclodextrin polymer gave enantiomeric resolution of both ortho and meta isomers, while γ-cyclodextrin was a good chiral selector for only ortho-fenfluramine; heptakis-2,3,6-tri-O-methyl-β-cyclodextrin permitted base line separation of meta-fenfluramine enantiomers but only partial resolution of racemic ortho-fenfluramine. The optimized electrophoretic method was applied to the quantitative analysis of 1-meta-fenfluramine (minor component in the mixture) and d-meta-fenfluramine in a commercial pharmaceutical formulation. Good reproducibility for migration time and corrected peak areas (R.S.D. <0.8 % and <1.2 %, respectively) was achieved and the presence of the minor component of the mixture was found to be in accord to previous determinations performed by other analytical methods.     

Determination of fenfluramine enantiomers in pharmaceutical formulations by capillary zone electrophoresis

Summary Capillary zone electrophoresis has been used for the enantiomeric separation of racemic ortho-fenfluramine and meta-fenfluramine employing a phosphate buffer at pH 2.5 added with cyclodextrins. The cyclodextrin type and concentration strongly influenced the chiral resolution. The uncharged β-cyclodextrin polymer gave enantiomeric resolution of both ortho and meta isomers, while γ-cyclodextrin was a good chiral selector for only ortho-fenfluramine; heptakis-2,3,6-tri-O-methyl-β-cyclodextrin permitted base line separation of meta-fenfluramine enantiomers but only partial resolution of racemic ortho-fenfluramine. The optimized electrophoretic method was applied to the quantitative analysis of 1-meta-fenfluramine (minor component in the mixture) and d-meta-fenfluramine in a commercial pharmaceutical formulation. Good reproducibility for migration time and corrected peak areas (R.S.D. <0.8% and <1.2%, respectively) was achieved and the presence of the minor component of the mixture was found to be in accord to previous determinations performed by other analytical methods.     

Stereoisomer separation of flavanones and flavanone‐7‐O‐glycosides by means of nanoliquid chromatography employing derivatized β‐cyclodextrins as mobile‐phase additive

A nanoliquid chromatographic method for the stereoisomer separation of some flavanone aglycones and 7‐O‐glycosides has been proposed employing a C18 capillary column and a chiral mobile‐phase additive such as cyclodextrin. The chiral separation of eriodictyol, naringenin, and hesperitin was obtained by addition of carboxymethyl‐β‐cyclodextrin to the mobile phase, whereas eriocitrin, naringin, narirutin, and hesperidin diastereoisomers were resolved by using sulfobutyl ether‐β‐cyclodextrin. The influence of the composition of the mobile phase, the length of the capillary column, and the flow rate on the chiral recognition were investigated. At optimum conditions, baseline separation for the selected aglycones and glycosylated forms were achieved with a mobile phase consisting of 50 mM sodium acetate buffer pH 3 and 30% methanol containing 20 mM of carboxymethyl‐β‐cyclodextrin and 10 mM of sulfobutyl ether‐β‐cyclodextrin, respectively. Precision, linearity, and sensitivity of the method were tested. Limits of detection and quantification for the studied flavanone glycosides were in the range 1.3‐2.5 and 7.5‐12.5 µg/mL, respectively. The method was used for the determination of the diastereomeric composition of the flavanone‐7‐O‐glycosides in Citrus juices after solid‐phase extraction procedure.     

Capillary electrophoresis separation of peptide diastereomers that contain methionine sulfoxide by dual cyclodextrin‐crown ether systems†

A dual‐selector system employing achiral crown ethers in combination with cyclodextrins has been developed for the separation of peptide diastereomers that contain methionine sulfoxide. The combinations of the crown ethers 15‐crown‐5, 18‐crown‐6, Kryptofix® 21 and Kryptofix® 22 and β‐cyclodextrin, carboxymethyl‐β‐cyclodextrin, and sulfated β‐cyclodextrin were screened at pH 2.5 and pH 8.0 using a 40/50.2 cm, 50 μm id fused‐silica capillary and a separation voltage of 25 kV. No diastereomer separation was observed in the sole presence of crown ethers, while only sulfated β‐cyclodextrin was able to resolve some peptide diastereomers at pH 8.0. Depending on the amino acid sequence of the peptide and the applied cyclodextrin, the addition of crown ethers, especially the Krpytofix® diaza‐crown ethers, resulted in significantly enhanced chiral recognition. Keeping one selector of the dual system constant, increasing concentrations of the second selector resulted in increased peak resolution and analyte migration time for peptide‐crown ether‐cyclodextrin combinations. The simultaneous diastereomer separation of three structurally related peptides was achieved using the dual selector system.     

Enantioseparation of esbiothrin by cyclodextrin‐modified microemulsion and micellar electrokinetic chromatography

A comparison between chiral cyclodextrin‐modified microemulsion electrokinetic chromatography (CD‐MEEKC) and cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC) for the enantiomeric separation of esbiothrin was carried out. For both methods, the separation conditions were optimized by varying CD types and concentration, running buffer pH and compositions, organic modifiers, and temperature. The optimal CD‐MEEKC conditions were 0.8% n‐heptane, 2.3% SDS, 6.6% n‐butanol, 90.3% 10 mM sodium tetraborate containing 3% (w/v, the ratio of CD mass to microemulsion volume) methyl‐β‐cyclodextrin, pH 10, 25°C. The optimized CD‐MEKC conditions were 3.3% SDS, 96.7% 10 mM sodium tetraborate containing 5% (w/v) β‐CD, pH 10, 25°C. The difference in physicochemical properties of the buffer and CDs resulted in different optimal CD type. The competitive distribution between the microemulsion (or micelle) and chiral CD contributed to the chiral separation. Both methods provided excellent separation (R_s ～? 3) with similar migration time (ca. 15 min). CD‐MEEKC provided higher separation efficiencies (>300000) than CD‐MEKC (>200000). The LODs for CD‐MEEKC and CD‐MEKC were 4.7 μg/mL and 3.2 μg/mL, respectively. The RSDs of migration time and peak area for CD‐MEEKC were slightly higher than for CD‐MEKC. Both the demonstrated CD‐MEEKC and CD‐MEKC methods provided high efficiencies, low LODs, and reproducible enantioseparations of esbiothrin.     

Separation of conjugated trienoic fatty acid isomers by capillary electrophoresis

A method for direct resolution of conjugated trienoic fatty acid isomers by capillary electrophoresis has been developed. To obtain complete separation a dual cyclodextrin system was used. This contained heptakis-(6-sulfo)-beta-cyclodextrin (charged). Beta-cyclodextrin (uncharged) and sodium dodecylsulfate. Under optimized conditions, all seven isomers were well separated. On average, separation efficiency was 2.9 x 10(5) plates/m.     

Enantioselective Partitioning of Racemic Ibuprofen in a Biphasic Recognition Chiral Extraction System

Enantioselective partitioning of ibuprofen enantiomers in a biphasic recognition chiral extraction system was studied. A combination of hydrophobic L‐isobutyl tartrate in organic phase and hydrophilic β‐cyclodextrin derivative in aqueous phase is necessary to establish a biphasic recognition chiral extraction system. The studies performed involve an enantioselective extraction in a biphasic system, where ibuprofen enantiomers form four complexes with the β‐cyclodextrin derivative in aqueous phase and the D(L)‐isobutyl tartrate in organic phase, respectively. In these biphasic resolutions, the types and the concentrations of the extractants, pH and temperature all exert a considerable influence on the biphasic recognition process. Good enantioselectivities for ibuprofen enantiomers were obtained at pH≦2.5 and a ratio of 2:1 of [L‐isobutyl tartrate] to [HP‐β‐CD]. Biphasic recognition chiral extraction is of strong chiral separation ability, and may be very helpful to optimize the extraction systems and realize the large‐scale production of enantiomers.     

Photoirradiation surface molecularly imprinted polymers for the separation of 6‐O‐α‐d‐maltosyl‐β‐cyclodextrin

Photoirradiation surface molecularly imprinted polymers for the separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin were synthesized using functionalized silica as a matrix, 4‐(phenyldiazenyl)phenol as a light‐sensitive monomer, and 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin as a template. Fourier transform infrared spectroscopy results indicated that 4‐(phenyldiazenyl)phenol was grafted onto the surface of functionalized silica. The obtained imprinted polymers exhibited specific recognition toward 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin. Equilibrium binding experiments showed that the photoirradiation surface molecularly imprinted polymers obtained the maximum adsorption amount of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin at 20.5 mg/g. In binding kinetic experiments, the adsorption reached saturation within 2 h with binding capacity of 72.8%. The experimental results showed that the adsorption capacity and selectivity of imprinted polymers were effective for the separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin, indicating that imprinted polymers could be used to isolate 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin from a conversion mixture containing β‐cyclodextrin and maltose. The results showed that the imprinted polymers prepared by this method were very promising for the selective separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin.     

Surfactant‐assisted electromembrane extraction combined with cyclodextrin‐modified capillary electrophoresis for the separation and quantification of Tranylcypromine enantiomers in biological samples

Surfactant‐assisted electromembrane extraction coupled with cyclodextrin‐modified capillary electrophoresis was developed for the separation and determination of Tranylcypromine enantiomers in biological samples. This combination would provide a new strategy for selective and sensitive determination of target analytes. The addition of surfactant in the donor solution improved the analyte transport into the lumen of hollow fiber that resulted in an enhancement in the analytes migration into acceptor solution. Optimization of the variables, affecting proposed method, was carried out and best results were achieved with a 175 V potential as driving force of the electromembrane extraction, 2‐nitrophenyloctylether as the supported liquid membrane, donor solution containing 0.2 mM Triton X‐100 with pH 3 and 0.1 M HCl for acceptor solution. Then, the extract was analyzed using cyclodextrin‐modified capillary electrophoresis method for separation of Tranylcypromine enantiomers. The best results were obtained with a phosphate running buffer (100 mM, pH 2.0) containing 7% w/v hydroxypropyl‐α‐cyclodextrin. Under the optimum conditions, a low limit of detection (3.03 ng/mL), good linearity (R² > 0.9953), and relative standard deviations below 4.0% (n = 5) were obtained. Finally, this procedure was applied to determine the concentration of Tranylcypromine enantiomers in urine samples with satisfactory results.     

Equilibrium Studies on Reactive Extraction of α-Cyclohexylmandelic Enantiomers Using Hydrophilic β-Cyclodextrin Derivatives Extractants

β‐Cyclodextrin (β‐CD) is negligibly soluble in organic liquids and can be modified to achieve a higher solubility in water. In this paper, racemic α‐cyclohexyl‐mandelic acid (α‐CHMA) was separated by chiral reactive extraction with aqueous β‐cyclodextrin derivatives. Hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD), hydroxyethyl‐β‐cyclodextrin (HE‐β‐CD), and methyl‐β‐cyclodextrin (Me‐β‐CD) were selected as chiral selectors for reactive extraction of α‐CHMA enantiomers from organic phase to aqueous phase. Factors affecting the extraction efficiency were investigated, including the types of organic solvents and β‐CD derivatives, the concentrations of the chiral selector and α‐CHMA enantiomers, pH and temperature. The experimental results demonstrate that HP‐β‐CD, HE‐β‐CD, and Me‐β‐CD have stronger recognition abilities for S‐α‐CHMA than for R‐α‐CHMA. Among the three derivatives, HP‐β‐CD shows the strongest separation factor for α‐CHMA enantiomers. A high enantioseparation efficiency with a maximum separation factor (α) of 2.02 is observed at pH 2.5 and 5°C.     

New cyclodextrin derivatives as chiral selectors in capillary electrophoresis

The separation of three pairs of enantiomeric herbicides has been successfully achieved by capillary electrophoresis at two different pH values in the presence of cyclodextrin derivatives previously synthesized in our laboratory. Two of these derivatives constitute a new class of receptor, the hemispherodextrins, in which a trehalose capping moiety is bonded to beta-cyclodextrin. Because of their peculiar structure hemispherodextrins have very promising characteristics and the low receptor concentration required to achieve separation deserves particular interest.     

New cyclodextrin derivatives as chiral selectors in capillary electrophoresis

The separation of three pairs of enantiomeric herbicides has been successfully achieved by capillary electrophoresis at two different pH values in the presence of cyclodextrin derivatives previously synthesized in our laboratory. Two of these derivatives constitute a new class of receptor, the hemispherodextrins, in which a trehalose capping moiety is bonded to β-cyclodextrin. Because of their peculiar structure hemispherodextrins have very promising characteristics and the low receptor concentration required to achieve separation deserves particular interest.     

Separation of neutral and basic enantiomers by cyclodextrin electrokinetic chromatography using anionic cyclodextrin derivatives as chiral pseudo-stationary phases

Separations of neutral and basic racemates were performed using five different anionic cyclodextrin (CD) derivatives as chiral selectors, viz. carboxymethylated β-CD, β-CD phosphate sodium salt, sulfobutyl ether β-CD sodium salt, carboxymethylated γ-CD, and γ-CD phosphate sodium salt. For the separation of neutral racemates, an untreated fused silica capillary was employed and various neutral racemates were successfully separated. Since the pH of the buffer affected the electroosmotic flow (EOF), the resolution was improved by changing the buffer pH. A polyacrylamide coated capillary was employed for the separation of basic racemates to suppress EOF and to prevent adsorption of cationic analyte on the capillary surface. By choosing an appropriate type and concentration of anionic CD, about 40 basic racemates were successfully separated. Some rough binding constants of basic analytes with an anionic β-CD were measured to discuss the optimum concentration of the CD. The migration direction was dependent on the binding constants and the concentration of the CD. The analyte strongly bound to the anionic CD migrated towards the anode but the weakly bound one moved towards the cathode. Anionic γ-CDs were also very useful for the separation of basic enantiomers. Five neutral CDs were employed as chiral selectors to compare selectivity between charged and neutral CDs, and eleven racemates could only be resolved using anionic CDs. The separation of some basic racemates in human plasma was also described. The direct injection of plasma samples was possible for some enantiomers that did not interact strongly with plasma proteins.