Type Chiral Stationary Phases Research Articles

Synthesis of covalently bonded cellulose derivative chiral stationary phases with a bifunctional reagent of 3-(triethoxysilyl)propyl isocyanate

A bifunctional reagent of 3-(triethoxysilyl)propyl isocyanate (TEPI) was initially adopted as a spacer reagent to prepare the bonded types of chiral stationary phases (CSPs) with cellulose derivatives. The silica-based CSPs were chemically prepared with non-regioselective and regioselective approaches and their chiral resolving capabilities were evaluated in terms of HPLC resolution of test enantiomers. It was observed that the chiral recognition capabilities of the non-regioselectively prepared CSPs were influenced by the amount of TEPI used. And also, the regioselectively prepared CSP generally showed a slightly higher resolution power than the non-regioselectively prepared CSP, while the non-regioselective procedures were highly advantageous to rapid preparation. In addition, chiral recognition of the prepared CSPs was affected by the properties of the used silica matrices.

Monochloro-Substituted Phenyl Carbamoylated β-Cyclodextrins as π-Acid Chiral Stationary Phases for High-Performance Liquid Chromatography

Three π-acid type chiral stationary phases based on monochloro-substituted phenyl carbamates of β-cyclodextrin have been synthesized. First, β-cyclodextrin was reacted with 5-bromo-1-pentene in dry DMF at room temperature to give 6A-O-(4′-pentenyl)-β-cyclodextrin. The remaining twenty hydroxyl groups of this derivatized β-cyclodextrin were blocked by further reaction with 2-chlorophenyl isocyanate, 3-chlorophenyl isocyanate, and 4-chlorophenyl isocyanate in dry pyridine, at 85 °C, to yield the three corresponding carbamates. After hydrosilylation with triethoxysilane at 80 °C, catalysed by tetra(triphenylphosphine)platinum(0), these carbamates were anchored on to silica gel by heating under reflux in toluene. All the β-cyclodextrin derivatives and CSPs prepared were characterized by IR and 1H NMR spectroscopy and elemental analysis. The surface concentrations of 2-chlorophenyl carbamoylated β-cyclodextrin, 3-chlorophenyl carbamoylated β-cyclodextrin, and 4-chlorophenyl carbamoylated β-cyclodextrin on silica gel were 0.27, 0.28 and 0.30 μmol m–2, respectively. Under normal-phase conditions the separation ability of CSPs containing 3- and 4-chlorophenyl carbamate was better than that of the CSPs containing 2-chlorophenyl carbamate.

Direct separation and quantitative determination of clenbuterol enantiomers by high performance liquid chromatography using an amide type chiral stationary phase

Enantiomers of clenbuterol were directly separated by a new high performance chromatographic method on Chirex 3005 column. Several parameters such as mobile phase composition, column temperature and flow rate were studied. Baseline enantioseparation was achieved, using the optimized mobile phase of n-hexane–1,2-dicholoethane–methanol (54:38:8, v/v/v) at 17 °C and 1.0 ml/min, with the separation factor ( α) 1.43 and the resolution factor ( R S) 1.81. The mechanism of separation was also discussed. Standard linear calibration cures were established for the R- and S-enantiomers, over the range of 26.1–1045.8 and 5.7–229.6 nmol/ml, with the correlation coefficient of 0.9999 for both. The limits of detection were 0.47 and 1.04 nmol/ml for R- and S-enantiomers, respectively. Recovery and precision of the method were also evaluated, which had been successfully used to monitor and identify quantitatively the profile of the clenbuterol enantiomers in human serum.

Synthesis and characteristics of composite chiral stationary phases based on cellulose derivatives

AbstractComposite chiral stationary phases (CSPs) were prepared on the basis of cellulose derivatives coated or bonded onto silica. “Molecular exterior” type CSPs were prepared by mixing together two different cellulose tris‐derivatives before or after being coated or bonded onto silica, and the “molecular interior” type was obtained by synthesizing non‐regioselectively heterosubstituted cellulose derivatives coated or bonded onto silica. For the sake of comparison, the individual phases were also prepared with corresponding cellulose derivatives by coating or bonding approaches, respectively. All of the prepared CSPs were characterized and their chiral recognition properties were evaluated by HPLC with several test racemates. The experimental results demonstrated that the “molecular exterior” CSPs generally exhibit chiral recognition capacities intermediate between those of the two individual phases. However, in the separation of some racemates higher enantioselectivity may be achieved on the “molecular interior” phases than on individual phases, thus broadening the application range of a single cellulose‐based CSP.

Micro-HPLC and standard-size HPLC for the separation of peptide stereoisomers employing an ion-exchange principle

Standard-size (4 mm ID) and micro-HPLC columns (0.5 mm ID) packed with a quinine-based ion-exchange type chiral stationary phase are comparatively evaluated for the separation of peptide enantiomers with up to six amino acid residues. The results show that downscaling the separation system in order to gain the advantages of miniaturized HPLC is possible without sacrificing separation power. Further, five different N-terminal protections (3,5-dinitrobenzoyl, 2,4-dinitrophenyl, 3,5-dinitrobenzyloxycarbonyl, carbazole-9-carbonyl, and 9-fluorenylmethoxycarbonyl) of the analytes are investigated regarding their effect on enantioselectivity. A comparison between a hydro-organic and a polar-organic mobile phase is also reported. The enantiomers of the peptides containing one to four amino acid residues were baseline resolved, while for the penta- and hexamers only partial separations were possible. In addition, all four stereoisomers of alanylalanine could be baseline separated.

Central composite design as a powerful optimisation technique for enantioresolution of the rac-11-dihydrooracin—the principal metabolite of the potential cytostatic drug oracin

Three types of chiral stationary phase were used to achieve chromatographic resolution of enantiomers of rac-11-dihydrooracin (DHO), the principal metabolite of a potential cytostatic drug oracin. Chiralcel OD-R as a chiral stationary phase with mobile phase comprising acetonitrile (modifier) and sodium perchlorate (buffering component) proved to be the most suitable system. Chemometric optimisation based on the Box–Wilson central composite design was employed to find the optimum resolution. The optimum factor space was defined by three parameters: temperature, modifier concentration and buffer concentration. Newly designed chromatographic response functions based on a combination of resolution R S and retention time of the last component eluted t RL were employed to evaluate the resolution with regard to quality and quantity. Optimum values predicted from those models of response surfaces were in excellent agreement with the experimental results. The chromatographic resolution of DHO enantiomers is suitable for xenobiochemical studies on stereoselectivity and stereospecificity of biotransformation enzymes.

Preparative chiral chromatographic resolution of enantiomers in drug discovery

A brief account is given of the role of preparative chromatography for the direct separation of enantiomers in the drug discovery process. Although it is not yet possible to predict the outcome of a chromatographic resolution attempt, and the optimisation procedure sometimes might be time-consuming, the technique is still indispensable as a means to obtain both enantiomers in pure form from a drug racemate for biological testing. The most suitable types of chiral stationary phases (CSPs) available for this purpose are discussed with special reference to loadability and compatibility with different mobile phase systems.

Strong versus weak chiral cation exchangers: Comparative evaluation for enantiomer separation of chiral bases by non-aqueous CEC

Novel enantioselective silica-supported strong and weak cation exchange (SCX and WCX) materials (3.5 μm particles) based on enantiomerically pure N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutanesulfonic acid and corresponding phosphonic acid as well as carboxylic acid structural analogs as chiral selectors have been evaluated for enantiomer separation of chiral bases by non-aqueous capillary electrochromatography (CEC). Capillary columns packed with these chiral stationary phases (CSPs) showed enantioselectivity in non-aqueous CEC towards a variety of chiral bases including amino alcohols such as β-sympathomimetics and β-blockers. Chromatographic and electrokinetic properties of the strong and weak chiral cation exchangers were evaluated comparatively in terms of their pH* profile, i.e. in terms of their dependence on the base-to-acid ratio of the background electrolyte. It turned out that the SCX type CSPs, and in particular the one based on the β-amino sulfonic acid show a broader window of applicable and suitable experimental conditions for CEC. For example, a strong and constant EOF was obtained on the sulfonic acid based CSP over the entire pH* range studied, while the EOF velocity of the carboxylic acid based CSP was slow under acidic conditions. In the separation of chiral bases, the ion-exchange retention mechanism dominated over electrophoretic migration under most conditions, especially on the SCX type CSPs. The SCX phases exhibited reasonable enantioselectivity over a wider pH* range, while the weak chiral cation exchanger (WCX type CSP) showed enantiomer separation capabilities for primary, secondary, and tertiary chiral amines only in the alkaline pH* range. Sulfonic and phosphonic acid based CSPs possess broad spectrum of applicability. For example, clenbuterol enantiomers were well baseline resolved both on suifonic acid based CSP (a = 1.33, R s = 14.2) as well as phosphonic acid based CSP (a = 1.13, R s = 4.9). In contrast, under the same conditions the corresponding carboxylic acid CSP exhibited enantioselectivity a of 1.08 and resolution R s of 1.3 only.

Copolymeric (lR-trans)-N,N'-1,2-cyclohexylene-bisbenzamide oligodimethylsiloxane chiral stationary phase for gas chromatography.

A copolymeric stationary phase, consisting of a chiral selective part, i.e. (1R-trans)-N,N'-1,2-cyclohexylenebisbenzamide, and an efficient siloxane oligomeric part, was successfully applied to open tubular column GC analysis. The efficiency and the chiral selectivity of this stationary phase were studied in detail, and high capacity and efficiency at elevated GC temperatures were especially noted. Several drugs and other enantiomeric pairs were separated. The shown examples demonstrate a broad application range for this type of chiral stationary phase in GC analysis.

Enantioseparation of tetramisole by capillary electrophoresis and high performance liquid chromatography and application of these techniques to enantiomeric purity determination of a veterinary drug formulation of L-levamisole

The enantioseparation of the chiral anthelmintic drug tetramisole was studied by capillary electrophoresis using various cyclodextrins as chiral selectors. In particular, the following aspects have been addressed: (1) the enantiomer affinity pattern of tetramisole towards various cyclodextrins; (2) development of a capillary electrophoretic method for quantitative determination of the enantiomeric impurity, i.e. of the R-(+) enantiomer, in S-(–)-tetramisole (levamisole); (3) application of the capillary electrophoretic method for the determination of the enantiomeric impurity of R-(+)-tetramisole in commercial samples and in a veterinary drug formulation of S-(–)-tetramisole; and (4) comparison of capillary electrophoretic and traditional pharmacopoeial polarimetric methods for the enantiomeric impurity determination in S-(–)-tetramisole. Opposite affinity order of tetramisole enantiomers was observed towards different cyclodextrins, depending on the nature of the substituents and the substitution pattern. In addition, an enantioseparation of tetramisole was developed by high performance liquid chromatography on polysaccharide type chiral stationary phase. This method was compared to the aforementioned capillary electrophoretic method. Preliminary validation results indicated that capillary electrophoresis represents a flexible, fast, and inexpensive technique for determination of the enantiomeric impurity of R-(+)-tetramisole in the commercial samples and drug formulations of S-(–)-tetramisole (levamisole). Both of the commercial samples as well as the veterinary drug formulation of levamisole contained R-(+)-tetramisole as enantiomeric impurity. Capillary electrophoresis represents a superior method for enantiomeric impurity determinations compared to the traditional polarimetric method still used in the major pharmacopoeias. CE offers the advantages of miniaturised techniques, as well as higher peak efficiency, flexibility, and shorter method development time compared to HPLC.

Low-molecular-weight chiral cation exchangers: novel chiral stationary phases and their application for enantioseparation of chiral bases by nonaqueous capillary electrochromatography.

Cation exchange type chiral stationary phases (CSPs) based on 3,5-dichlorobenzoyl amino acid and amino phosphonic acid derivatives as chiral selectors (SOs) and silica as chromatographic support were developed and applied to enantiomer separations of chiral bases by nonaqueous capillary electrochromatography (NA-CEC). As a rationale for efficient CSP development we adopted the combined use of the "reciprocity principle of chiral recognition" and nonaqueous ion-pair CE as screening assay. Thus, (S)-atenolol was employed as chiral counter-ion added to the BGE in CE and a series of N-derivatized amino acids and amino phosphonic acids were screened to derive reciprocally information on their chiral recognition abilities for atenolol enantiomers. Two SO candidates, namely N-(3,5-dichlorobenzoyl)-O-allyl-tyrosine and N-(4-allyloxy-3,5-dichlorobenzoyl)-1-amino-3-methylbutane phosphonic acid that have been identified as potential SOs in the CE screening were, after immobilization on thiol-modified silica, evaluated in cation-exchange NA-CEC. The strong chiral cation exchanger with the free phosphonic acid group exhibited enhanced enantioselectivity compared to the weak chiral cation exchanger with the carboxylic acid group. A wide variety of chiral bases could be successfully resolved on the strong chiral cation exchanger with alpha-values up to 2.2 and efficiencies up to 375000 m-1 including beta-blockers and other amino alcohols, local anesthetics like etidocaine, antimalarial agents like mefloquine, Tröger's base, phenothiazines like promethazine, and antihistaminics. The influence of several experimental parameters (electrolyte concentration, acid-base ratio and acetonitrile-methanol ratio) was evaluated.

Comparative enantioseparation of selected chiral drugs on four different polysaccharide-type chiral stationary phases using polar organic mobile phases

The enantiomers of randomly selected chiral drugs and drug analogs of various structural and pharmacological groups were resolved on four different polysaccharide-type chiral stationary phases (CSP) using pure methanol and acetonitrile as mobile phases. Polysaccharide phenylester type CSP, Chiralcel-OJ although resolving the enantiomers of some chiral drugs was less universal in the combination with methanol and acetonitrile as mobile phases. Among polysaccharide phenylcarabamates amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak-AD) was superior over the corresponding cellulose derivative, cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel-OD). However, another derivative of cellulose, namely, cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) exhibited higher chiral recognition ability compared to Chiralpak-AD material. This study confirms previous findings about the applicability of polysaccharide type CSPs in so called polar organic mode as well as shows high potential of CDCPC as a practically useful CSP for High performance liquid chromatography (HPLC) enantioseparations.

Comparative HPLC enantioseparation of new chiral hydantoin derivatives on three different polysaccharide type chiral stationary phases

The enantioseparation of eighteen new chiral hydantoin derivatives was studied on three different polysaccharide type chiral stationary phases (CSP) Chiralpak AD, Chiralcel OD and Chiralcel OJ in the normal-phase HPLC mode. Chiralpak AD material exhibited the most universal chiral resolving ability and allowed the enantioseparation of 17 out of 18 compounds followed by Chiralcel OD (10 enantioseparations of 12 tested compounds) and Chiralcel OJ (eight enantioseparation from 13 tested analytes). Some complementary separations were observed and all of 18 compounds could be resolved at least with one of the three chiral CSP under the conditions of this study. With regard to the structure of the analytes, bulky electron rich substituents at C5 of the hydantoin nucleus appear to favor stereoselective interactions.

Evaluation of enantioselective nonaqueous ion-pair capillary electrophoresis as screening assay in the development of new ion exchange type chiral stationary phases

This study evaluates how enantioselectivity values for the separation of chiral N-derivatized amino acids achieved in non-aqueous ion-pair CE with cinchona alkaloid derived chiral counter-ions are related quantitatively to those obtained in enantioselective anion-exchange chromatography with the same selectors immobilized on silica supports. Three different setups of CE experiments, viz. a conventional total filling technique (TFT), a countercurrent technique (CCT), and a partial filling technique (PFT), were investigated and were tested in terms of the goodness of fit of correlations between apparent electrophoretic and chromatographic a values. Both selectands and selectors have been varied. Correlation coefficients higher than 0.95 are commonly obtained with all three CE techniques, yet even better fits are found for TFT-CE and CCT-CE. The common premise for the good correlations is the equal molecular recognition mechanism in ion-pair and ion-exchange modes, both involving ionic interactions as driving force for selectand-selector association, and the fact that separation selectivity in both techniques is primarily determined by the difference of the binding constants of (R) and (S) enantiomers with the chiral selector, i.e. the intrinsic or thermodynamic enantioselectivity. Factors related to the separation technique, which also influence apparent enantioselectivity and possibly contribute to a deterioration of the fit, are discussed. The good correlations prove that enantioselective ion-pair CE may be used as screening method in the development and optimization of ion-exchange type chiral stationary phases.

Quantitative structure–enantioselective retention relationships for chromatographic separation of arylalkylcarbinols on Pirkle type chiral stationary phases

Quantitative structure–retention (QSRR, retention factors log k 1 and log k 2 for the first and second eluted enantiomer) as well as enantioselective retention relationships (QSERR, separation factor log α) for a series of 42 chiral arylalkylcarbinols on four brush-type chiral stationary phases are derived by multiple linear regression analyses and artificial neuronal network calculations using 2D and 3D molecular descriptors including those obtained by quantum chemical calculations. Separation factors are in addition modeled by the 3D-QSAR method of comparative molecular field analysis (CoMFA). For the retention factors the LUMO energy turns out to be the most important descriptor, whereas for log α it is the hydrophobicity of the analytes. With CoMFA both the steric and electrostatic field are found to be of almost comparable significance.

Influence of the structure of organic phosphonate compounds on chiral separation on a pirkle type chiral stationary phase

The enantiomers of eightO,O-dialkyl-2-benzyloxycarbonyl-aminoarylmethyl-phosphonates are directly separated on anN-(3,5-dinitrobenzoyl)leucine (DNBleu) column. The influence of the mobile phase composition and the column temperature on the retention and the enantioselectivity are investigated. The influence of the length and steric hindrance of alkoxyl groups of the phosphonate ester and the nature of the substituentp-Cl and pH on the benzene ring which is attached to the chiral carbon atom on chiral separation are discussed also.

On-column deracemization of an atropisomeric biphenyl by quinine-based stationary phase and determination of rotational energy barrier by enantioselective stopped-flow HPLC and CEC.

The reversible enantiomerization of axially chiral 2'-dodecyloxy-6-nitrobiphenyl-2-carboxylic acid was studied in the presence of a brush type chiral stationary phase based on O-(tert-butylcarbamoyl) quinine as chiral selector unit by stopped-flow high-performance liquid chromatography (sfHPLC) and capillary electrochromatography (sfCEC). After initial separation of the enantiomers in the first section of the column, the flow was stopped and the resolved species allowed to enantiomerize on-column. From this conversion, which could be determined from the enantiomeric ratios at different enantiomerization times, kinetic rate constants were calculated. By sfHPLC at a constant temperature of 15 degrees C, kinetic rate constants in the presence of the CSP were found to be 4.1 x 10(-5) s(-1) and 2.2 x 10(-5) s(-1) for the (-) and (+)-enantiomers, respectively, corresponding to half-lives of 279 and 530 min. Thus, apparent activation energies of enantiomerization were calculated to be 93.0 and 94.6 kJ mol(-1) for the (-) and (+)-enantiomers. On the macroscopic level, the apparent difference of rotational energy barriers and kinetic rate constants for both enantiomers is reflected as deracemization. For example, starting from a racemic mixture, an enantiomeric excess (ee) of 14% was seen in the stopped-flow HPLC experiment described after an enantiomerization time of 220 min at 15 degrees C, and a maximal ee of 17% can be approximated after infinite enantiomerization time. There is good agreement between HPLC and CEC results as well as their experimental errors, confirming that the new sfCEC technique may be a valuable and convenient tool to study interconversion processes.

Investigation of mandelic acid bonding on Pirkle type chromatographic stationary phases by Raman spectroscopy

The bonding of mandelic acid enantiomers has been studied on benzene–leucine, dinitrobenzene–leucine and dinitrobenzene–phenylalanine type chiral stationary phases connected to zeolite A supports. The π-donor, π-acceptor and H-bonding interactions responsible for diastereomer pair formations can be studied under quasi in situ chromatographic conditions by Fourier transform Raman and surface enhanced Raman spectroscopic techniques. Structural differences between diastereomer pairs result in observable spectral differences at a phase load of approx. 50%. It was shown that the decreasing π-acceptor character of the phase is associated with its increasing capability of H-bond formation. Correlating spectral data to chromatographic results it can be concluded that, in addition to H-bonding as well as to π-donor–π-acceptor interactions, steric hindrances due to bulky moieties of either the stationary phase or the analyte molecules are of importance in successful separations.

Chiral anion exchangers applied to capillary electrochromatography enantioseparation of oppositely charged chiral analytes: investigation of stationary and mobile phase parameters

Weak anion-exchange (WAX) type chiral stationary phases (CSPs) based on tert.-butyl carbamoyl quinine as chiral selector (SO) and different types of silica particles (porous and non-porous) as chromatographic support are evaluated in packed capillary electrochromatography (CEC). Their ability to resolve the enantiomers of negatively charged chiral analytes, e.g., N-derivatized amino acids, in the anion-exchange mode and their electrochromatographic characteristics are described in dependence of several mobile phase parameters (pH, buffer type and concentration, organic modifier type and concentration) and other experimental variables (electric field strength, capillary temperature). The inherent “zwitterionic” surface character of such silica-based WAX type CSPs (positively charged SO and negatively charged residual silanols) allows the reversal of the electroosmotic flow (EOF) towards the anode at pH values below the isoelectric point (p I) of the modified surface, whereas a cathodic EOF results at pH values above the p I. Since for negatively charged analytes also an electrophoretic transport increment has to be considered, which can be either in or against the EOF direction, several distinct modes of elution have been observed under different stationary phase and mobile phase conditions: (i) co-electrophoretic elution of the negatively charged solutes with the anodic EOF in the negative polarity mode, (ii) counter-electrophoretic elution with the cathodic EOF in the positive polarity mode, and (iii) electrophoretically dominated elution in the negative polarity mode with a cathodic EOF directed to the injection end of the capillary. Useful enantioseparations of chiral acids have been obtained with all three modes. Enantioselectivity values as high as under pressure-driven conditions and theoretical plate numbers up to 120 000 per meter could be achieved under electrically driven conditions. A repeatability study yielded RSD values below 2% for retention times and RSD values in the range of 5–10% for theoretical plate numbers and resolution, thus clearly establishing the reliability of the investigated anion-exchange type CEC enantioseparation methods.

Detection of 7- N, N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) carbamoyl amino acids generated by post-column desulfuration in the simultaneous determination of the sequence and d/ l-configuration of peptides using a fluorogenic Edman reagent, 7- N, N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate

A modified Edman sequencing method using 7- N, N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for amino acid sequence and d/ l-configuration determination has been developed. DBD-thiazolinone (TZ)-amino acids generated by Edman sequencing method were hydrolyzed and then oxidized to DBD-carbamoyl (CA)-amino acids, instead of the conversion reaction to thiohydantoin (TH)-amino acid in the conventional Edman method. The oxidative desulfuration step operated manually with formation of DBD-CA-amino acids was difficult to control because of the production of by-products by the side-chain oxidation of amino acid and inapplicability to the conventional automated protein sequencer. Therefore, post-column desulfuration method in which the reaction was carried out after the separation of analyte on a column was designed. DBD-thiocarbamoyl (TC)-amino acids generated by the modified sequencing method were separated on the reversed phase or Pirkle type chiral stationary phase column and then oxidized to DBD-CA-amino acids with hydrogen peroxide as a post-column reagent and detected fluorimetrically. Using the method, the determination of the amino acid sequence and d/ l-configuration of a d-amino acid containing peptide was achieved and gave us the recognition to the utility of the method.