Chiral separation with ligand-exchange micellar electrokinetic chromatography using a D-penicillamine-copper(II) ternary complex as chiral selector

D-Penicillamine is demonstrated for the first time as a chiral ligand for the enantioseparation of dansyl amino acids based on ligand-exchange micellar electrokinetic chromatography (LE-MEKC). Copper(II) was used as the central ion in the ternary complex. The effect of surfactant on the resolution was significant. A concentration of 20 mM sodium dodecyl sulfate (SDS) was shown to be necessary for the separation. Other important parameters, such as the concentration ratio of D-penicillamine (D-PEN) to Cu2+, the kind of metal central ion, the type and pH value of buffer, were also investigated. N-Acetyl-D-penicillamine and L-valine (Val), with similar structure to D-penicillamine, were applied as their copper(II) complexes as chiral selector and the chiral recognition mechanism is briefly discussed. Under optimum experimental conditions, i.e., 20 mM NH4OAc, pH 6.5, a 2:1 concentration ratio of D-penicillamine to Cu(II), 4 mM CuSO4 and 8 mM D-penicillamine, the chiral separation of eight pairs of different dansyl amino acid enantiomers was accomplished with resolution ranging from 1.1 to 5.9. When L-PEN was used instead of D-PEN, reversal of the migration order was observed.     

Chiral separation of amino acids and glycyl dipeptides by chiral ligand-exchange capillary electrophoresis comparing Cu(II), Co(II), Ni(II) and Zn(II) complexes of three different sugar acids

This paper deals with comparative studies on the use of copper(II), cobalt(II), nickel(II) and zinc(II) complexes of d-gluconic acid, d-saccharic acid and l-threonic acid as chiral selectors for the enantioseparation of aromatic amino acids and glycyl dipeptides using the principle of ligand-exchange capillary electrophoresis. Although copper(II) is the most frequently used central ion in ligand-exchange capillary electrophoresis, in the case of d-gluconic acid cobalt(II) was shown to be an alternative for the enantioseparation of amino acids. Glycyl dipeptides, however, were resolved only with copper(II) complexes. Zn(II) as a central ion was not effective in all cases and with Ni(II) only some partial separations were achieved.     

Chiral resolution of tryptophan derivatives by CE using canine serum albumin and bovine serum albumin as chiral selectors

This work deals with the application of BSA and canine serum albumin (CSA) for enantioseparation of tryptophan derivatives with CE. The aim of this work was the investigation of the influence of different functional groups of tryptophan derivatives on enantioseparation. CSA as a chiral selector was tested to compare its selector properties with those of BSA. The enantiomers of the tryptophan derivatives were separated by adding BSA or CSA to the BGE. The influence of pH, temperature, BSA and CSA concentration and organic modifiers was investigated. It was found that the stereoselectivity for the different tryptophan derivatives is dependent on the albumin species. It turned out that the different functional groups of the derivatives showed a significant influence on stereoselectivity.     

Comparison of enantioseparations using Cu(II) complexes with L-amino acid amides as chiral selectors or chiral stationary phases by capillary electrophoresis,capillary electrochromatography and micro liquid chromatography

In this paper we report that Cu(II) complexes with L-amino acid amides were used as chiral selectors for enantioseparation by capillary electrophoresis, capillary electrochromatography (CEC) and micro liquid chromatography using chemically modified monolithic columns. The enantioselectivity, enantiomer migration order, and the performance have been compared when different chiral selectors were used in these modes. L-Enantiomers showed longer retention times than D-forms in both CEC and LC modes. However, it has interestingly been observed that the migration order of Dns-DL-Ser showed an exception in CEC using L-prolinamide-modified column that Dns-L-Ser was eluted as the first peak. On the basis of proposed structures of complexes in the chiral recognition, differences in migration orders and recognition mechanism were discussed.     

Chiral CE of aromatic amino acids by ligand-exchange with zinc(II)-L-lysine complex

A novel method of chiral ligand-exchange CE was developed with either L- or D-lysine (Lys) as a chiral ligand and zinc(II) as a central ion. This type of chiral complexes was explored for the first time to efficiently separate either individual pairs of or mixed aromatic amino acid enantiomers. Using a running buffer of 5 mM ammonium acetate, 100 mM boric acid, 3 mM ZnSO(4) x 7H(2)O and 6 mM L-Lys at pH 7.6, unlabeled D,L-tryptophan, D,L-phenylalanine, and D,L-tyrosine were well separated, giving a chiral resolution of up to 7.09. The best separation was obtained at a Lys-to-zinc ratio of 2:1, zinc concentration of 2-4 mM and running buffer pH 7.6. The buffer pH was determined to have a strong influence on resolution, while buffer composition and concentration impacted on both the resolution and peak shape. Boric acid with some ammonium acetate was an adoptable buffer system, and some additives like ethylene diamine tetraacetic acid capable of destroying the complex should be avoided. Fine-tuning of the chiral resolution and elution order was achieved by regulating the ratio of L-Lys to D-Lys; i.e. the resolution increased from zero to its highest value as the ratio ascended from 1:0 to 1:infinitive, and L-isomers eluted before or after D-isomers in excessive D- or L-Lys, respectively.     

Separation of dansylated amino acid enantiomers by chiral ligand-exchange CE with a zinc(II) L-arginine complex as the selecting system

A facile chiral ligand-exchange capillary electrophoretic method has been explored for the enantioseparation and UV detection of dansyl-amino acids with Zn(II) L-arginine complex as a chiral selecting system. Successful enantioseparation of 17 pairs of amino acid enantiomers has been achieved with a buffer of 100 mM boric acid, 5 mM ammonium acetate, 3 mM ZnSO4 and 6 mM L-Arg at pH 8.0, of which 10 pairs were fully resolved with resolution in between 1.59 and 4.21. This new method was shown to be applicable to the separation of some mixed pairs of amino acids and to the quantitative analysis of some real samples such as rice vinegars, with a linear range between 0.8 and 150 microg/mL, correlation coefficient above 0.99 and recovery in between 90.1 and 112.4%. It was found that amino acids with low resistance side chain(s), low tendency to form intramolecular hydrogen bond or high tendency to form intermolecular hydrogen bonds are more easily enantioseparated than those with extra carboxyl and/or phenyl groups. By the use of the suggested buffer, the running pH should be selected at 7.4-9.0 to compromise the resolution and elution speed.     

Chiral separation of underivatized amino acids by ligand-exchange capillary electrophoresis using a copper(II)-L-lysine complex as selector

A method based on capillary zone electrophoresis is presented for the determination of the purity of commercial dimeric cyanine dyes (TOTO, YOYO, BOBO, all -1 and -3 species, LOLO-1, POPO-1) that are common as fluorescent probes for nucleic acid staining. These dyes are tetracharged cations, and have a strong tendency to interact with negatively charged centres, where they are rapidly adsorbed, especially from aqueous solutions. Thus anionic sites at the capillary wall must be avoided, and aqueous buffers are not suitable. The method introduced here avoids both complications, using non-aqueous N,N-dimethylacetamide as solvent, and suppressing the dissociation of silanol groups at the capillary surface due to selection of acidic separation conditions (20 mmol/l perchloric acid as background electrolyte). The present method enables the determination of the purity of all 10 dyes in less than 15 min. The selectivity of the method allows separation of at least five main and differentiating a number of unresolved minor contaminants as demonstrated in detail for TOTO-3 as an example. Quantitation (with 100% normalisation of the peak areas) of nine lots of this dye results in a purity between 33 and 87%.     

Chiral separation of hydroxyflavanones in cyclodextrin-modified capillary zone electrophoresis using sulfated cyclodextrins as chiral selectors

Chiral separations of three hydroxyflavanone aglycones, including 2'-, 3'-, and 4'-hydroxyflavanone, in capillary zone electrophoresis (CZE) using randomly sulfate-substituted beta-cyclodextrin (S-beta-CD) or dual cyclodextrin (CD) systems consisting of S-beta-CD and a neutral CD at low pH were investigated. The results indicate that S-beta-CD is an excellent chiral selector for enantioseparation of 2'-hydroxyflavanone and is a good chiral selector for 3'-hydroxyflavanone. Depending on the concentration of S-beta-CD ranging from 2.0 to 0.75% (w/v), the enantioresolution values were 10.5-19.5 and 1.8-3.4 for 2'- and 3'-hydroxyflavanone, respectively. The enantiomers of 4'-hydroxyflavanone could be effectively separated with S-beta-CD at a concentration of 2.0% (w/v) within 20 min. The enantioselectivity and enantioresolution follow the order 2'-hydroxyflavanone>3'-hydroxyflavanone>4'-hydroxyflavanone. Alternatively, with the addition of sodium dodecyl sulfate (SDS) monomers at low concentrations in the electrophoretic system, enantioselectivity of these hydroxyflavanone aglycones could be enhanced with dual CD systems. In this case, SDS monomer acted as a complexing agent probably first with S-beta-CD and then subsequently with the analytes for increasing the effective electrophoretic mobility of the analytes towards the anode and as a selectivity controller for affecting the selectivity of hydroxyflavanones. Better enantioseparation between 2'-hydroxyflavanone and 3'-hydroxyflavanone could be achieved with a dual CD system consisting of S-beta-CD and gamma-CD than that with S-beta-CD and beta-CD. In addition, possible chiral recognition mechanisms of hydroxyflavanones are discussed.     

Enantioseparation by chromatographic and electromigration techniques using ligand-exchange as chiral separation principle

This article gives a short overview of the application of the principle of chiral ligand-exchange in HPLC, CE, and CEC. Since its introduction by Davankov, more than thousand articles have appeared in this field. Citing all these papers would extend the scope of this review—it would fill several books. Therefore only some milestones are mentioned in this article and it will focus on our own activities in this field. Some new developments are mentioned, and selected biochemical and biomedical application are presented.     

Chiral separation based on ligand-exchange capillary electrophoresis using a copper(II)-L-ornithine ternary complex as selector

A ligand-exchange capillary electrophoresis was explored, with L-ornithine as the ligand and copper(II) as the central ion. Its applicability was demonstrated with underivatized and dansyl amino acids, a dipeptide, and drugs with amino alcohol structure. The enantioselectivity was found to be strongly dependent on pH and copper(II)-L-Orn complex concentration. Due to the adsorption of the positively charged species onto the capillary inner walls, the chiral separation selectivity is very high while the efficiency is relatively low. Permanent 1,3-propanediamine-coated capillaries show an improved separation efficiency and theoretical plate numbers increasing from 10(4) to 10(5). Similar phenomena were observed when sodium dodecyl sulfate (SDS) micelles were added to the copper(II) complex solution. The poor separation efficiency of chiral compounds in uncoated capillaries may result from the low rate of the ligand-exchange reactions, and the high enantioselectivity may derive from the complexing process in the adsorbed phase.     

Direct chiral resolution of malic acid in apple juice by ligand-exchange capillary electrophoresis using copper(II)-L-tartaric acid as a chiral selector

Chiral resolution of native DL-malic acid was achieved by ligand-exchange capillary electrophoresis using copper(II)-L-tartrate as a chiral selector. Factors affecting chiral resolution, migration time, and peak area of malic acid were studied. The running conditions for optimum separation of malic acid were found to be 1 mM copper(II) sulfate-1 mM L-tartrate (pH 5.1) with an effective voltage of -20 kV at 30 degrees C, using direct detection at 280 nm, and resolution (Rs) of racemic malic acid was approximately 4. With this system, D- and L-malic acids in apple juice were analyzed successfully.     

Enantiomer separation of drugs by capillary electrophoresis using proteins as chiral selectors

The separation of drug enantiomers using proteins as the chiral selectors in capillary electrophoresis (CE) is considered in this review. The proteins used include albumins such as bovine serum albumin, human serum albumin and serum albumins from other species, glycoproteins such as alpha1-acid glycoprotein, crude ovomucoid, ovoglycoprotein, avidin and riboflavin binding protein, enzymes such as fungal cellulase, cellobiohydrolase I, pepsin and lysozyme and other proteins such as casein, human serum transferrin and ovotransferrin. Protein-based CE is carried out in two modes: in one proteins are immobilized or adsorbed within the capillary, or protein-immobilized silica gels are packed into the capillary (affinity capillary electrochromatography mode), and in the other proteins are dissolved in the running buffer (affinity CE mode). Furthermore, the advantages and limitations of the two modes and the factors affecting the chiral separations of various drugs by protein-based CE are discussed.     

Chiral separation of newly synthesized arylpropionic acids by capillary electrophoresis using cyclodextrins or a glycopeptide antibiotic as chiral selectors

Summary The chiral separation of two newly synthesized arylpropionic acids of pharmaceutical interest, namely 2-[(5′-benzoil-2′-hydroxy)phenyl]-propionic acid (DF-1738y) and 2-[(4′-benzoiloxy-2′-hydroxy)phenyl]-propionic acid (DF-1770y), was performed by Capillary Zone Electrophoresis (CZE) using either cyclodextrins or antibiotics as chiral selectors in coated capillary. In order to optimize the separation, the effect on the migration time and resolution of type and concentration of the chiral selector, the buffer pH and the capillary temperature were studied. Several cyclodextrins, namely the charged 6^A-monomethylamino-β-cyclodextrin (MeNH-β-CD) and the neutral methyl-β-cyclodextrins (M-β-CD) and heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD), were tested for the enantiomeric separation of aryl propionic acids (APAs) compounds. Of these TM-β-CD provided the highest enantiomeric resolution at pH 5, however only DF-1738y optical isomers were baseline resolved. Using background electrolytes (BGEs) at higher pHs (pH=6–7) supported with the above listed CDs, an enantioresolution increase was recognized only for compound DF-1738y. In contrast DF-1770y exhibited the highest resolution at the lowest pH value studied (pH 4). The macrocyclic antibiotic vancomycin was therefore added to the BGE and tested as chiral selector using the partial filling counter current mode in order to obtain a sensitive analysis, high resolution and reduced antibiotic adsorption on the capillary wall. 5 mM vancomycin dissolved in the BGE at pH 5 and 25°C provided relatively high enantiomeric resolution (R _DF-1738y=3.4,R _DF-1770y=2.22) of both compounds.     

Application of ligand-exchange capillary electrophoresis to the chiral separation of alpha-hydroxy acids and beta-blockers

The application of the principle of ligand-exchange capillary electrophoresis to two substance classes is described. As chiral selector N-(2-hydroxyoctyl)-L-4-hydroxyproline-copper(II) complex was used. This principle was applied to the chiral separation of alpha-hydroxy acids and drugs containing amino alcohol structure such as beta-blockers. The enantioselectivity was found to be strongly dependent on pH corresponding to the optimal conditions for complex formation for each structure class.     

Use of chiral amino acid ester‐based ionic liquids as chiral selectors in CE

In this study, the applicability of a chiral ionic liquid (CIL) as the sole chiral selector in CE was investigated for the first time. In particular, five amino acid ester‐based CILs were synthesized and used as additives in the BGE in order to evaluate their chiral recognition ability. The performance of these CILs as the sole chiral selectors was evaluated by using 1,1′‐binaphthyl‐2,2‐diylhydrogenphosphate (BNP) as the analyte and by comparing the resolution values. Different parameters were examined, such as the alkyl group bulkiness and the configuration of the cation, the anion type of the CIL and its concentration, and the pH of the BGE, in order to optimize the separation of the enantiomers and to demonstrate the effect that each parameter has on the chiral‐recognition ability of the CIL. Baseline separation of BNP within 13 min was achieved by using a BGE of 100 mM Tris/10 mM sodium tetraboratedecahydrate (pH 8) and a chiral selector of 60 mM l ‐alanine tert butyl ester lactate. The run‐to‐run and batch‐to‐batch reproducibilities were also evaluated by computing the %RSD values of the EOF and the two enantiomer peaks. In both cases, very good reproducibilities were observed, since all %RSD values were below 1%.     

Copper(II) complexes of lipophilic aminoglycoside derivatives for the amino acid enantiomeric separation by ligand-exchange liquid chromatography

In this paper, a new class of ligand-exchange chiral stationary phase (LE-CSP) based on the copper complexes of lipophilic aminoglycoside derivatives was reported. Different stationary phases were developed by coating reversed-phase liquid chromatography supports with three neamine derivatives carrying a lipophilic octadecyl chain at the 4', 5 and 6 positions, respectively. The enantioselective ability of these LE neamine-based CSPs was evaluated and the 4'-derivative coated column was found to be the most interesting one for the amino acid resolution. The effects of the variation of several chromatographic parameters on the enantioseparation were evaluated in order to identify the analysis optimal conditions.     

Assay of aromatic amino acid enantiomers in rice-brewed suspensions by chiral ligand-exchange CE

The aim of this work was to assay seasoning D- or L-aromatic amino acids (AAs) in rice-brewed suspensions, Laozao in Chinese, by chiral ligand-exchange CE with UV detection and Zn(II) complex as a chiral selecting system. Resolution and peak retention were found to be parallel to the basicity of the AA chiral ligands, and basic L-Arg was known to work the best at pH 8.20 compared with L-Lys and other AA ligands. Baseline separation of DL-aromatic AAs and partially separation of some FMOC-labeled nonaromatic AAs have been achieved using a running buffer of 5 mM ammonium acetate, 100 mM boric acid, 3 mM ZnSO(4), and 6 mM L-Arg at pH 8.20. The aromatic amino acids in four brands of Laozao were measured in a range of 0.25-20 microg/mL for Typ, 1.00-120 microg/mL for Phe, and 2.50-200 microg/mL for Tyr, with linear regression coefficient all over 0.999. The LOD (S/N=3) was 0.15 microg/mL for Typ, 0.50 microg/mL for Phe, and 1.25 microg/mL for Tyr. The recovery of the method determined by spiking with the supernates of Laozao as background was 94.0-112.9%. The RSDs of migration time and peak area measured from six injections of tyrosine were 0.2 and 2.7%, respectively, for run-to-run, and 1.6 and 3.2%, respectively for day-to-day. Interestingly, there were only L-Trp, D-Tyr, and L-Tyr found in the assayed four brands of Laozao. They may serve as an index to recognize the brand of Laozao.     

Chiral separation and determination of the optical purity of thiamphenicol-related chiral compound by capillary zone electrophoresis with cyclodextrins as chiral selectors

Summary One classical method for quantitation of amino acids in proteins is hydrolysis of the proteins and determination of the free amino acids. Although the drastic experimental conditions necessary for complete hydrolysis always cause degradation of some of the amino acids, if mild hydrolysis conditions are used, a mixture of amino acids and oligopeptides is obtained. If these conditions are adequately tuned, the oligopeptides are almost exclusively dipeptides. For this reason we have initiated a study to find a derivatizing agent suitable for the analysis of amino acids and dipeptides by an absolute method of quantitation already tested for amino acids. FMOC-Cl was found to be a suitable derivatizing agent for this purpose.     

Chiral resolution of monosaccharides as 1-phenyl-3-methyl-5-pyrazolone derivatives by ligand-exchange CE using borate anion as a central ion of the chiral selector

Six reducing monosaccharides (mannose, galactose, fucose, glucose, xylose, and arabinose) were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) and chiral resolution of these racemic PMP-monosaccharides was studied by ligand-exchange CE using borate anion as a central ion of the chiral selector and (S)-3-amino-1,2-propanediol (SAP) as a chiral selector ligand. PMP-mannose, PMP-galactose and PMP-fucose were successfully enantioseparated. Lowering the capillary temperature increased the resolution of PMP-mannose system, but decreased that of PMP-galactose and PMP-fucose systems. Whereas the maximum resolution was obtained at pH 8.9 in the PMP-mannose system, resolution increased gradually with pH in the PMP-galactose and PMP-fucose systems. Expecting the formation of the ternary borate complexes with SAP and PMP-monosaccharide in the CE experiments, the optimized structures of the borate diastereomers were obtained by semiempirical molecular orbital calculations to discuss the structural difference of the diastereomers in connection with the enantioseparation behaviors.     

以L-酒石酸正己酯-硼酸配合物作为反相液相色谱手性流动相添加剂拆分5种β-受体阻滞剂

本文以L-酒石酸正己酯-硼酸配合物为手性流动相添加剂,建立了普萘洛尔、艾司洛尔、美托洛尔、比索洛尔、索他洛尔和阿替洛尔六种β-受体阻滞剂的反相高效液相色谱手性分离方法。对影响对映体分离的主要因素：L-酒石酸正己酯、硼酸浓度,缓冲溶液种类、浓度、pH值和有机改性剂-甲醇含量等进行了详细考察。最佳色谱条件为：Venusil MP-C18色谱柱（4.6 mm × 250 mm,5 μm）,流动相为15 mmol/L乙酸铵-甲醇（体积比为20: 80或30: 70,含60 mmol/L硼酸,70 mmol/L L-酒石酸正己酯,醋酸调节pH值6.00）,检测波长214 nm。在最佳分离条件下,五对对映体（普萘洛尔、艾司洛尔、美托洛尔、比索洛尔、索他洛尔）可以分别获得基线分离。