Enantioseparation of anionic analytes by non-aqueous capillary electrophoresis using quinine and quinidine derivatives as chiral counter-ions

A non-aqueous capillary electrophoretic method developed for the enantioseparation of N-protected amino acids has been applied to the investigation of five new quinine and quinidine derivatives as chiral selectors: 1-adamantyl carbamoylated quinine, 3,4-dichlorophenyl carbamoylated quinidine, allyl carbamoylated dihydroquinine, allyl carbamoylated dihydroquinidine and 1-methyl quininium iodide. The composition of the background electrolyte was 12.5 mM ammonia, 100 mM octanoic acid in an ethanol-methanol (60:40 v/v) mixture containing a 10 mM concentration of the chiral selector. Under these conditions, the enantioseparation of a series of various benzoyl, 3,5-dinitrobenzoyl and 3,5-dinitrobenzyloxycarbonyl amino acid derivatives was studied with respect to selectand-selector relationship and enantioselectivity.     

Enantiomer separation of N-protected amino acids by non-aqueous capillary electrophoresis and high-performance liquid chromatography with tert.-butyl carbamoylated quinine in either the background electrolyte or the stationary phase

A non-aqueous CE method was developed for evaluating the chiral discrimination potential of cinchona alkaloids and different kinds of carbamoylated derivatives of quinine and quinidine type chiral selectors towards acidic analytes, in particular a series of various Bz (benzoyl), DNB (3,5-dinitrobenzoyl) and DNZ (3,5-dinitrobenzyloxycarbonyl) amino acid derivatives. In this study, the enantioselectivity values obtained in non-aqueous CE with tert.-butyl carbamoylated quinine as chiral additive have been compared with the values found for the same series of selectands in HPLC using the same selector immobilized onto silica as chiral stationary phase. Similarly to the background electrolyte used in CE an ethanol-methanol mixture (60:40, v/v) containing 100 mM octanoic acid and 12.5 mM ammonia has been selected as HPLC mobile phase. Under these conditions, a good correlation (r = 0.954) between the enantioselectivities observed with the two techniques has been obtained. Thus the non-aqueous CE method can be applied as a screening tool for the rapid evaluation of the chiral discrimination potential of a large set of newly developed chiral selectors derived from quinine and related alkaloids.     

Impact of amines as co-modifiers on the enantioseparation of various amino acid derivatives on a tert-butyl carbamoylated quinine-based chiral stationary phase

A tert-butyl carbamoylated quinine-based chiral stationary phase (CSP) for direct enantiomer separation of various natural and unnatural amino acid derivatives was studied. The influence of functional groups in the amino acid side chains upon the enantioseparation is discussed with the aim of realizing contributions to their overall chiral recognition. The effects of various amines as co-modifiers upon retention and overall enantioselectivity of amino acid derivatives in polar organic solvents was systematically investigated. In general, retention times decreased with increasing amine concentrations without a distinct alteration of enantioselectivity. All analytes were rapidly resolved on the CSP with the methanol-based mobile phase containing 87 mM acetic acid and 7 mM triethylamine.     

Simultaneous separation of the stereoisomers of 1-amino-2-hydroxy and 2-amino-1-hydroxypropane phosphonic acids by stereoselective capillary electrophoresis employing a quinine carbamate type chiral selector

A stereoselective nonaqueous capillary electrophoresis (CE) method utilizing O-(tert-butylcarbamoyl) quinine as chiral ion-pair agent and additive to the non aqueous background electrolyte was evaluated for the simultaneous separation of the enantiomers and diastereomers of 1 -amino-2-hydroxypropane phosphonic acid besides the corresponding beta-aminophosphonic acid analogs, the stereoisomers of 2-amino-1-hydroxypropane phosphonic acid, in a single run. The separations have been carried out using the partial filling technique to avoid strong background signal from the quinine selector. It conveniently allowed the baseline separation of all eight components of interest (alpha- as well as beta-aminophosphonic acids) as N-2,4-dinitrophenyl derivatives in a single run. Moreover, the absolute configurations of all eight peaks were identified. Compared to the quinine carbamate selector, the corresponding 'pseudo-enantiomeric' O-(tert-butylcarbamoyl) quinidine selector exhibited reserved elution order and nearly identical resolutions. The proposed CE method turned out to be advantageous over stereoselective high-performance liquid chromatography (HPLC) with a quinine carbamate type stationary phase, which showed high enantioselectivity, but failed to simultaneously separate all eight components.     

A comprehensive chemoselective and enantioselective 2D-HPLC set-up for fast enantiomer analysis of a multicomponent mixture of derivatized amino acids

A feasibility study on the fast enantioselective two-dimensional HPLC separation of racemic amino acid derivatives is presented. The method involves the on-line coupling of a narrow-bore C18 RP column in the first dimension to a short enantioselective column based on nonporous 1.5 μm particles modified with quinidine carbamate as chiral selector in the second dimension. Conceptually, the system was designed to enable both time-controlled repeated transfer of fractions of the eluate and detector-controlled transfer of selected fractions from column 1 to column 2. To avoid volume overloading of the second chiral column, a narrow-bore reversed phase column was installed in the first dimension. Due to the fast (less than 1.5 minutes) enantiomer separation that occurs in the second dimension, the overall analysis time for the two-dimensional separation of a mixture of nine racemic 3,5-dinitrobenzoyl amino acids was optimized at 16 minutes.     

Improvement of chiral stationary phases based on cinchona alkaloids bonded to crown ethers by chiral modification

To improve the chiral recognition capability of a cinchona alkaloid crown ether chiral stationary phase, the crown ether moiety was modified by the chiral group of (1S, 2S)‐2‐aminocyclohexyl phenylcarbamate. Both quinine and quinidine‐based stationary phases were evaluated by chiral acids, chiral primary amines and amino acids. The quinine/quinidine and crown ether provided ion‐exchange sites and complex interaction site for carboxyl group and primary amine group in amino acids, respectively, which were necessary for the chiral discrimination of amino acid enantiomers. The introduction of the chiral group greatly improved the chiral recognition for chiral primary amines. The structure of crown ether moiety was proved to play a dominant role in the chiral recognitions for chiral primary amines and amino acids.     

Investigation of an enantioselective non-aqueous capillary electrochromatography system applied to the separation of chiral acids

A weak anion-exchange type chiral stationary phase (CSP) based on tert.-butylcarbamoylquinine as chiral selector and silica as chromatographic support was applied to non-aqueous capillary electrochromatography. The mobile phases used consisted of acetonitrile and methanol as organic solvents, and acetic acid and triethylamine were added as background electrolytes. The influence of several experimental parameters (electrolyte concentration, acetic acid-triethylamine ratio, acetonitrile-methanol ratio and temperature) was evaluated in order to obtain improved enantioselectivity and efficiency as well as short run times for the enantiomeric separation of negatively charged chiral analytes including benzyloxycarbonyl, N-(3,5-dinitrobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzoyl, acetyl and N-(2,4-dinitrophenyl) derivatized amino acids and profens. Solvent composition of acetonitrile-methanol (80:20) and enhanced electrolyte concentrations up to 600 mM acetic acid at a constant acid-base ratio of 100:1 with high applied voltages of -25 kV proved to be optimum regarding short retention times and improved efficiencies. For example, the enantiomers of Fmoc-Leu could be separated in less than 10 min with a resolution factor of 6.9 and about 100000 theoretical plates per meter.     

Comparative Enantiomer-Resolving Ability of Coated and Covalently Immobilized Versions of Two Polysaccharide-Based Chiral Selectors in High-Performance Liquid Chromatography

The separation of enantiomers of over 175 randomly selected chiral acidic, basic, and neutral compounds was studied on 4 polysaccharide-based chiral columns made by coating or covalent attachment of cellulose 3,5-dichlorophenylcarbamate or amylose 3,5-dimethylphenylcarbamate on the surface of silica. Triscarbamate derivatives of cellulose or amylose were used for the preparation of coated-type columns, while in the case of covalently immobilized chiral stationary phases, the respective polysaccharides were not completely carbamoylated but only close to triscarbamates. It was found that this minimal difference in the chemical composition of the polysaccharide derivatives resulted in significantly different enantiomer-resolving ability for certain groups of chiral compounds while only marginally different for other chiral analytes. This potential difference between coated- and covalently immobilized versions of the “same” chiral selector must be considered in method development with these columns, as well as in method transfer between them.     

Novel Pirkle-type quinine 3,5-dinitrophenylcarbamate chiral stationary phase implementing click chemistry

A new Pirkle-anion exchange hybrid-type chiral stationary phase (CSP-1) has been synthesized by immobilizing 10,11-didehydroquinine 3,5-dinitrophenylcarbamate onto 3-azidopropyl silica gel using click chemistry (1,3-dipolar Huisgen cycloaddition). This chiral selector and CSP contain a strong π-accepting 3,5-dinitrophenyl residue besides the π-basic quinoline group and an ionizable tertiary amino group. In concert with ion pairing it offers π-donor-π-acceptor interactions resulting in an enhancement of the selectivity toward specific π-donating analytes such as aryloxypropionic acids and profens. A representative set of these analytes has been investigated under various chromatographic conditions (polar-organic, reversed- and normal-phase) leading to base-line enantioseparations with selectivity (α) values up to 1.8. Control experiments with related quinine tert-butylcarbamate phase grafted onto the surface either by thioether (Chiralpak QN-AX) or 1,2,3-triazole linker revealed the impact of the additional aromatic moiety in the chiral selector motif.     

Enantioseparation of Three β-Agonists Using Di-n-butyl d-Tartrate-Boric Acid Complex as Chiral Selector by Means of MEEKC

In this study, a self-prepared complex chiral selector, di-n-butyl d-tartrate-boric acid complex, by the reaction of di-n-butyl d-tartrate with boric acid in a running buffer was used as a chiral selector for the enantioseparation of three β-agonists including clenbuterol, cycloclenbuterol and tulobuterol by means of microemulsion electrokinetic chromatography (MEEKC). Three β-agonists were successfully enantioseparated using the chiral system, indicating that the di-n-butyl d-tartrate-boric acid complex was a useful chiral selector. The effects of di-n-butyl d-tartrate and sodium tetraborate concentration, surfactant concentration, cosurfactant, phosphate, buffer pH and composition, as well as applied voltage were extensively investigated to achieve a good enantioseparation. The di-n-butyl d-tartrate and sodium tetraborate concentration in the running buffer had great influence on the chiral resolution (R _s). Three β-agonists which could not be separated with only di-n-butyl d-tartrate, obtained good chiral separation using the complex chiral selector; among them, two pairs including clenbuterol and cycloclenbuterol could be baseline resolved in 7 min under optimized experimental conditions of 0.8% (w/v) di-n-butyl d-tartrate, 40 mM sodium tetraborate, 3.0% (w/v) Tween-20 and 60 mM sodium dihydrogen phosphate with 25 kV as running voltage. The results indicated that the method could be used for the enantioseparation of three β-agonists.

     

Capillary electrophoretic chiral separation of Cinchona alkaloids using a cyclodextrin selector

A new capillary electrophoretic method for the chiral separation of four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) was developed using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin as the chiral selector. The inner walls of the separation capillary were modified with a thin polyacrylamide layer, which substantially reduced the electroosmotic flow and improved the chiral resolution and the reproducibility of the migration time of the analytes. Various operation parameters were optimised, including the pH, the capillary temperature, the concentration of the background electrolyte, and the concentration of the chiral selector. Baseline separation of the two diastereomer pairs was achieved in 12 minutes in ammonium acetate background electrolyte pH 5.0 with addition of cyclodextrin in a concentration of 3 mM or higher.     

The carboxyl derivatives of 6,8-di-(tert.-butyl)phenoxazine: Synthesis,oxidation reactions and fluorescence

New carboxyl-containing o-aminophenols and phenoxazines were synthesized by condensation of 3,5-di-(tert.-butyl)-quinone with p-aminobenzoic and anthranilic acids. Oxidative transformations of the o-aminophenols and intermediate o-iminoquinones occur with the formation of the ESR detected phenoxazinyl radicals, which furthermore transform to phenoxazines or the dimeric products emerged through the radical attack at the C1 carbon of a formed phenoxazine. Molecular structure of the dimer obtained by oxidation of methyl ester of 4-[3,5-di-(tert.-butyl)-1-(2′-hydroxyphenyl)amino]benzoic acid was X-ray determined. Reaction of 4-[3,5-di-(tert.-butyl)-1-(2′-hydroxyphenyl)amino]benzoic acid with thionyl chloride gives rise to the formation of a derivative of 2-oxido-3H-benzo[d,j][1,2,3]oxathiazol system, the structure of which was established using X-ray crystallography. Solutions of methyl-6,8-di-(tert.-butyl)-10H-phenoxazine-3-carboxylate solvents display intense fluorescence covering a broad spectral region in the range of 400–600?nm.     

Effect of Steric Hindrance on the Resolution of the Enantiomers of Alkyl Isothiocyanate Derivatives of Amino Acids on a Teicoplanin CSP Using a Methanol-Based Mobile Phase

The effect of steric hindrance on the resolution of the enantiomers of alkyl (i.e. methyl, ethyl, propyl, butyl, and tert-butyl) isothiocyanate derivatives of amino acids on a teicoplanin chiral stationary phase (CSP), with a methanol-based mobile phase, has been studied. Resolution was found to depend on the size of the alkyl group attached to the isothiocyanate reagent and deteriorated as the size of the group increased from methyl to tert-butyl under the same chromatographic conditions. This indicates that interaction between the isothiocyanate group and the chiral selector is important in chiral recognition. Better-than-baseline resolution was achieved for many amino acids with a basic amino or an amide group, for example histidine, lysine, arginine, and asparagine, because of increased solubility in the mobile phase after chemical derivatization. Revised: 22 September and 7 October 2005     

Enantioseparation of chiral sulfonates by liquid chromatography and subcritical fluid chromatography

Tert‐butylcarbamoyl‐quinine and ‐quinidine weak anion‐exchange chiral stationary phases (Chiralpak® QN‐AX and QD‐AX) have been applied for the separation of sodium β‐ketosulfonates, such as sodium chalconesulfonates and derivatives thereof. The influence of type and amount of co‐ and counterions on retention and enantioresolution was investigated using polar organic mobile phases. Both columns exhibited remarkable enantiodiscrimination properties for the investigated test solutes, in which the quinidine‐based column showed better enantioselectivity and slightly stronger retention for all analytes compared to the quinine‐derived chiral stationary phase. With an optimized mobile phase (MeOH, 50 mM HOAc, 25 mM NH₃), 12 of 13 chiral sulfonates could be baseline separated within 8 min using the quinidine‐derivatized column. Furthermore, subcritical fluid chromatography (SubFC) mode with a CO₂‐based mobile phase using a buffered methanolic modifier was compared to HPLC. Generally, SubFC exhibited slightly inferior enantioselectivities and lower elution power but also provided unique baseline resolution for one compound.     

Direct High‐Performance Liquid Chromatographic Enantioseparation of β‐Methyl‐Substituted Unusual Amino Acids on a Quinine‐Derived Chiral Anion‐Exchange Stationary Phase

A quinine‐derived chiral anion‐exchange stationary phase was used for the direct high‐performance liquid chromatographic separation of the enantiomers of the N‐protected unusual β‐substituted α‐amino acids, β‐methylphenylalanine, β‐methyltyrosine, β‐methyltryptophan, and β‐methyl‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid. The readily prepared 2,4‐dinitrophenyl and tert‐butyloxycarbonyl derivatives were well separated, and in most cases the separation of all four stereoisomers of these β‐methyl‐α‐amino acids could be obtained in one chromatographic run. The elution sequences of the enantiomers of the different derivatives were determined and revealed a dependence on the type of the N‐protecting group. In this context, the effects of different protecting groups (acetyl, tert‐butyloxycarbonyl, benzoyl, 3,5‐dinitrobenzoyl, benzyloxycarbonyl, 3,5‐dinitrobenzyloxycarbonyl, 2,4‐dinitrophenyl, and 9‐fluorenylmethoxycarbonyl) on the chromatographic behavior were investigated.     

Preparation and evaluation of a chiral HPLC stationary phase based on cone calix[4]arene functionalized at the upper rim with l‐alanine units

Here we report a new chiral stationary phase (CSP) immobilized on silica gel based on cone calix[4]arene functionalized at the upper rim with two l ‐alanine units as new chiral selector that has been used in high‐performance liquid chromatography. The CSP was prepared by covalently bonding the allyl groups at the lower rim of calix[4]arene to silica gel by thiol‐ene click chemistry reaction. Elemental analysis of the CSP showed that 120 μmol of chiral selector bonded per gram of silica gel. 1‐Hexene was used for end‐capping of unreacted mercapto groups on silica gel. Since the CSP is chemically bonded to the silica, it can be used in the normal‐phase and reversed‐phase mode and with halogenated solvents as mobile phases, if desired. The chromatographic performance of the CSP was evaluated in the enantioseparation of the 3,5‐dinitrobenzoyl derivatives of some amino acids, diclofop‐methyl and dl ‐mandelic acid.     

Chiral separation of NBD-amino acids by ligand-exchange micro-channel electrophoresis.

The chiral separation of amino acid derivatives by ligand-exchange electrophoresis in a microchannel chip was performed for the first time. A Cu(II) complex with L-prolinamide was used as a chiral selector. The migration behaviors of eleven NBD-DL-amino acids were investigated by ligand-exchange capillary electrophoresis (LE-CE). The enantiomer of five NBD-amino acids (Ser, Thr, Val, Phe and His) could be separated by LE-CE using a 20 mM ammonium acetate buffer (pH 9.0) containing 10 mM copper acetate, 20 mM L-prolinamide and 1 mM SDS. NBD-His was eluted in the order D-form and L-form, while the elution order of another enantiomers was L-form and D-form. Under this condition, the enantioseparation of these five NBD-amino acids by ligand-exchange microchip electrophoresis (LE-ME) was investigated using a glass microchip. The enantioseparation of NBD-Ser, -Thr and -His could be successfully accomplished by LE-ME. LE-ME was superior to LE-CE in terms of the short migration time and a good enantiomeric separation.     

Chiral recognition: design and preparation of chiral stationary phases using selectors derived from ugi multicomponent condensation reactions and a combinatorial approach

Combinatorial approaches together with high-throughput screening have been used to develop highly selective stationary phases for chiral recognition. Libraries of potential chiral selectors have been prepared by the Ugi multicomponent condensation reactions and screened for their enantioselectivity using the reciprocal approach involving a chiral stationary phase with immobilized model target compound N-(3,5-dinitrobenzoyl)-alpha-l-leucine. The best candidates were identified from the library of phenyl amides of 2-oxo-azetidineacetic acid derivatives. This screening also enabled specification of the functionalities of the selector desired to achieve the highest level of chiral recognition. The substituents of the phenyl ring adjacent to the chiral center of the selector candidates exhibited the most profound effect on the chiral recognition. The best candidate was then synthesized on a larger scale, resolved into single enantiomers using preparative enantioselective HPLC, and attached to porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) beads via an ester linkage to afford the desired stationary phase. Selectivities alpha as high as 3.2 were found for the separation of a variety of amino acid derivatives.     

Chiral separation of dansyl amino acids in capillary electrophoresis using mono-(3-methyl-imidazolium)-beta-cyclodextrin chloride as selector

Enantioseparations of fourteen dansyl amino acids were achieved by using a positively-charged single-isomer beta-cyclodextrin, mono-(3-methyl-imidazolium)-beta-cyclodextrin chloride, as a chiral selector. Separation parameters such as buffer pH, selector concentration, separation temperature, and organic modifier were investigated for the enantioseparation in order to achieve the maximum possible resolution. Chiral separation of dansyl amino acids was found to be highly dependent on pH since the degree of protonation of these amino acids can alter the strength of electrostatic interaction and/or inclusion complexation between each enantiomer and chiral selector. In general, the chiral resolution of dansyl amino acids was enhanced at higher pH, which indicates that the carboxylate group on the analytes may interact with the imidazolium group of cationic cyclodextrin. For most analytes, a distinct maximum in enantioresolution was obtained at pH 8.0. Moreover, the chiral separation can be further improved by careful tuning of the separation parameters such as higher selector concentration (e.g. 10 mM), lower temperature, and addition of methanol. Enantioseparation of a standard mixture of these dansyl amino acids was further achieved in a single run within 30 min.     

Novel cinchona alkaloid O9-hydrazide derivatives as chiral anion-exchange-type selectors: Synthesis and chromatographic evaluation

Summary Six new quinine (QN) O₉-hydrazide derivatives with different substituents have been synthesized and immobilized on porous silica gel for HPLC. The chiral resolving power of these anion-exchange-type chiral stationary phases (CSPs) has been investigated and compared with that of four carbamate QN derivatives with analogous substitution. The unsubstituted QN-hydrazide derivative was usually the best chiral selector of the hydrazide series. Among the substituted hydrazide derivatives the introduction of a tritylcarbonyl or atert-butylcarbonyl group at the β position of the hydrazide function improved chiral recognition by the resulting CSPs. Although carbamate functionality seemed to favour the resolution of the enantiomers of many of the racemic compounds tested, the hydrazide series resulted in better separations of the enantiomers of the DNP derivatives of amino acids and of certain acidic drugs of therapeutic interest, such as the profens. The selectivity factors of these types of compounds on these QN-hydrazide derivatives are the best yet obtained on QN-derived chiral selectors.