Chiral separation of amino acids by ligand-exchange capillary electrochromatography using continuous beds

A chiral ligand-exchange phase for capillary electrochromatography based on continuous bed technology was developed. The chiral stationary phase is prepared by a one-step in situ copolymerization procedure using methacrylamide, piperazine diacrylamide, vinylsulfonic acid and N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline. These chiral continuous beds are inexpensive and easy to prepare. They also have several advantages over silica-based packed capillaries. Since the bed is covalently attached to the capillary wall, no frit is required. The applicability of this new approach to the chiral separation of underivatized amino acids is demonstrated.     

Enantioseparation of hydroxy acids on easy-to-prepare continuous beds for capillary electrochromatography

This paper deals with the enantioseparation of hydroxy acids by ligand-exchange capillary electrochromatography. A chiral continuous bed was easily prepared by in situ polymerization of monomers, including an L-4-hydroxyproline derivative. This phase showed chiral recognition for several hydroxy acids, in addition to amino acids.     

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography (CEC) is achieved with open-tubular capillaries (o-CEC), with packed capillaries (p-CEC) or with monolithic capillaries. In o-CEC, capillaries are coated with a thin film containing cyclodextrin derivatives, cellulose, proteins, poly-terguride or molecularly imprinted polymers as chiral selectors. In p-CEC, typical chiral HPLC stationary phases such as silica-bonded cyclodextrin or cellulose derivatives, proteins, glycoproteins, macrocyclic antibiotics, quinine-derived and 'Pirkle' selectors, polyacrylamides and molecularly imprinted polymers are used as chiral selectors. Chiral monolithic stationary phases prepared by in situ polymerization into the capillary were also developed for electrochromatographic enantiomer separation.     

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.     

Chiral separation of halogenated amino acids by ligand-exchange capillary electrophoresis

The chiral separation of halogenated amino acids by ligand-exchange CE is described. Halogenated amino acids attracted increasing interest in recent years because of their physiological activities. Different chiral selectors, as there are L-4-hydroxyproline, L-histidine, and N-alkyl derivatives of L-4-hydroxyproline in form of their copper(II) complexes, are compared for their chiral recognition ability for halogenated amino acids. The influence of various parameters, such as selector concentration, pH, organic modifier, and field strength, on the resolution was investigated. All halogenated amino acids investigated were baseline-separated under optimized conditions.     

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.     

Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography

This review summarizes recent developments in chiral separation in capillary zone electrophoresis (CZE), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC) covering literature published since the year 2000. New chiral selectors and innovative approaches for CE and CEC are introduced. Recent progress in column technology for CEC is highlighted and the development of new chiral stationary phases is discussed. This review is not dedicated to list applications but will focus on new developments.     

Polyrotaxane approach for synthesis of continuous beds for capillary electrochromatography

The polyrotaxane formation approach was evaluated for synthesis of continuous beds for capillary electrochromatography. This approach has the advantage of generating diverse electroosmotic and chromatographic properties without chemical reactions. The polyrotaxane derivatized continuous beds were formed adding the macrocyclic compounds to the solution of neutral acrylic monomers and crosslinker prior to the initiation of the polymerisation. Cationic and anionic derivatives of beta-cyclodextrin were used as macrocyclic compounds. Investigation of the electroosmotic properties indicated a template directed and enthalpy controlled self-assembly of the polyrotaxanes during the polymerisation of the continuous beds. This process was monomer-composition dependent and favored by the hydrophobicity of the polymeric skeleton. The morphology of the continuous beds was evaluated using high-resolution optical microscopy with CCD camera and atomic force microscopy. Reversed-phase capillary chromatography driven by electroosmosis, originating from the polyrotaxane structure, was performed using several test mixtures. Not primarily designed for the chiral chromatography the polyrotaxane derivatized continuous beds demonstrated enantioselective separation of D,L-metoprolol. The stability of the polyrotaxane derivatized continuous beds was tested. The beds demonstrated reproducible electroosmotic properties in the range from pH 4 to pH 9 (RSD=0.69%).     

Histamine-modified cationic beta-cyclodextrins as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids by capillary electrophoresis.

The enantiomeric separation of alpha-hydroxy acids and carboxylic acids was successfully performed by using 6-deoxy-6-N-histamino-beta-cyclodextrin (CD-hm), a monosubstituted positively charged beta-cyclodextrin (beta-CD) bearing a histamine moiety linked to the C6 of a glucose unit in the upper CD rim via the amino group. Good results were obtained at a low selector concentration (1 mM). The number of positive charges on the upper rim may be modulated as a function of pH, because of the different pKa of the amino and the imidazolyl groups, and was found to affect both the enantioselectivity and resolution factors. With the analogous 6-deoxy-[4-(2-aminoethyl)imidazolyl]-beta-cyclodextrin (CD-mh) bearing the histamine moiety linked to the C6 via the imidazolyl group, very poor results were obtained, showing that the proximity of the positive charge to the cavity plays an important role in the enantiomeric recognition. The complexation mode was studied by electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance (2-D NMR) ROESY experiments: the recognition model is consistent with an inclusion complexation of the aromatic ring of the analyte within the CD cavity coupled to electrostatic interactions between the carboxylate and the protonated amino group of the cyclodextrin.     

Continuous beds with vancomycin as chiral stationary phase for capillary electrochromatography

Enantiomeric separations in capillary electrochromatography (CEC) carried out using a continuous-bed chiral stationary phase (CSP) based on the macrocyclic antibiotic, vancomycin, is presented. The continuous beds were prepared from methacryloxypropyl modified fused silica capillaries (100 microm ID) by in situ copolymerization of N-(hydroxymethyl)acrylamide and piperazine diacrylamide with vinyl sulfonic acid comonomer used to introduce ionic functionality and thus a strong electroosmotic flow (EOF). The CSP was subsequently prepared by immobilizing the vancomycin stationary phase by reductive amination. Preliminary results have indicated that an extremely strong EOF is obtained in both the nonaqueous polar organic (15.2 x 10(-5) cm2 V(-1) s(-1) and the aqueous reversed-phase modes of operation (8.5 x 10(-5) cm2 V(-1) s(-1)). Enantioselectivity was obtained for four racemic compounds, the best of which was in the case of thalidomide which was separated in 10 minutes with high resolution (Rs = 2.5) and efficiency (120,000 plates meter(-1)) values.     

Strategy for the chiral separation of non-acidic pharmaceuticals using capillary electrochromatography

In completion of an earlier defined generic chiral screening approach, a generic separation strategy for basic, bifunctional, and neutral compounds was proposed and evaluated. This strategy adds to a previously defined strategy for acidic compounds. The screening experiment of the actual strategy used a mobile phase of 5 mM phosphate buffer pH 11.5/ACN (30/70 v/v), a temperature of 25 degrees C, and a voltage of 15 kV. The selected chiral stationary phases were Chiralpak AD-RH, Chiralcel OD-RH, Chiralcel OJ-RH, and Chiralpak AS-RH, all based on polysaccharide selectors. It was seen that 31 out of 48 test compounds were partially or baseline-resolved under screening conditions. After execution of the optimization steps of the strategy, this number increased to 41, with a total of 21 baseline-separated compounds. Combined with the results obtained from the acidic test set examined in the earlier defined strategy, of all tested compounds 82.5% showed enantioselectivity and 49.2% could be baseline-separated.     

手性整体柱在加压毛细管电色谱中拆分地匹福林对映体

采用一步原位键合法制备了环糊精衍生物毛细管整体柱,在加压毛细管电色谱模式下,对盐酸地匹福林对映体进行了手性分离,并考察了以NaH2PO4作为流动相时电解质溶液pH值、柱温及分离电压等因素对分离的影响.在优化的实验条件下,地匹福林对映体达到了基线分离.     

A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 num diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 num ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.     

Chemically L-prolinamide-modified monolithic silica column for enantiomeric separation of dansyl amino acids and hydroxy acids by capillary electrochromatography and mu-high performance liquid chromatography

A silica-based chiral monolithic column prepared by sol-gel process and chemical modification of chiral selector was used for enantioseparation of dansyl amino acids and hydroxy acids by capillary electrochromatography (CEC) and mu-high-performance liquid chromatography (mu-HPLC). L-Prolinamide was modified as a chiral selector. The chiral stationary phase (CSP), the chiral complex of Cu(II) with L-prolinamide, provides an anodic electroosmotic flow (EOF) in CEC. The EOF was found to be dependent on applied electric field strength, the pH, and the composition of mobile phases. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pore. D-Enantiomers migrated before L-enantiomers except for dansyl-(Dns)-DL-Ser. The separation efficiencies of up to 17600 (D) and 13,200 plates m(-1) (L) were achieved for the separation of DL-indole-3-lactic acid.     

分子印迹聚合物在毛细管电色谱拆分手性药物中的研究进展

手性药物通过与生物体内生物大分子之间的手性匹配与分子识别来发挥药理作用。两个对映体与体内手性环境相互作用的不同导致每个对映体表现出不同的药理活性、代谢过程、代谢速率及毒性等药代动力学特征。因此发展手性药物的拆分方法,对于手性药物的开发和生产过程的质量监控具有重要意义。分子印迹聚合物(MIPs)是以目标分子作为模板而制备的高分子聚合物,它具有特定的空间分子结构和官能团,对目标分子具有高度的特异性识别能力。基于该特点,MIPs非常适合于手性药物的拆分和纯化。毛细管电色谱(CEC)可同时基于毛细管电泳和液相色谱的分离机理对目标物进行分离,因此具有高分离效率和高选择性的特点。将MIPs材料作为CEC的固定相,可将这两种技术的优势结合,从而实现对手性药物的高效拆分。MIPs材料在1994年首次应用于CEC手性拆分,此后该研究领域开始获得关注和发展。MIPs材料主要通过4种模式在CEC中实现手性拆分,分别是作为开管柱、填充柱和整体柱的固定相以及分离介质中的准固定相。该综述以这4种模式作为分类基准,根据MIPs制备所需的材料和分离对象对其在CEC手性拆分中的应用进行了总结,揭示了MIPs在CEC手性...     

Protein separation by monolithic capillary electrochromatography

This work presents the separation of model proteins by capillary electrochromatography involving a monolithic stationary phase with C4 functionality. The monolith was fabricated in UV-transparent capillaries by employing a slight modification of a recently published photopolymerization procedure. With the number of theoretical plates per column ranging between 11000 and 33000, the separation efficiency proved to be lower than capillary zone electrophoresis where plate numbers ranged between 18000 and 66000. However, higher resolution was obtained due to the additional chromatographic separation mechanism. Inter- and intra-column reproducibility were evaluated, the latter could be significantly improved when using a rinsing procedure that contained 0.05% sodium dodecylsulfate in the mobile phase. Plate heights became nearly independent of mobile phase velocities higher than 0.5 mm/s indicating that high velocities can be applied without sacrificing efficiency. Furthermore, peak heights showed a dependence on injection times. For proteins, an increase in capacity factors was found when increasing the percentage of organic solvent in the mobile phase.     

Open tubular capillary columns with basic templates made by the generalized preparation protocol in capillary electrochromatography chiral separation and template structural effects on chiral separation capability

Some open tubular (OT) molecule imprinted polymer (MIP) silica capillary columns have been prepared using atenolol, sulpiride, methyl benzylamine (MBA) and (1-naphthyl)-ethylamine (NEA) as templates by the pre-established generalized preparation protocol. The four MIP thin layers of different templates showed quite different morphologies. The racemic selectivity of each MIP column for the template enantiomers was optimized by changing eluent composition and pH. The template structural effects on chiral separation performance have been examined. This work verifies the versatility of the generalized preparation protocol for OT-MIP silica capillary columns by extending its boundary toward templates with basic functional group moieties. This study is the very first report to demonstrate a generalized MIP preparation protocol that is valid for both acidic and basic templates. The chiral separation performances of atenolol and sulpiride by the MIPs of this study were found better than or comparable to those of atenolol and sulpiride obtained by non-MIP separation techniques and those of some basic template enantiomers obtained by MIP based techniques.     

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-μm diol-silica particles. The CSP mixed with 5-μm amino silica particles (3:1) was packed into 75-μm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1–3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor R_s=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)–water–acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)–water–methanol–acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12–20 min).     

Immobilization of phospholipid-avidin on fused-silica capillaries for chiral separation in open-tubular capillary electrochromatography

Phospholipid-coated fused-silica capillaries with immobilized avidin were applied in the chiral separation of D,L-tryptophan, D,L-PTH-serine, and D,L-PTH-threonine at pH 7.4 by open-tubular CEC. Liposomes prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap biotinyl), or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Biotinyl) with different amounts of phosphatidylserine were assessed as phospholipid coating materials. The stability of the coating and the success of the coating procedure were evaluated in terms of the repeatability of the enantiomer migration times and the resolution of enantiomers. The coating procedure itself significantly affected the migration times and resolution of the enantiomers. Reliable chiral separations with high separation efficiencies were achieved through careful choice of the coating method.     

Recent progress in enantiomeric separation by capillary electrochromatography

Recent progress in enantiomeric separations by capillary electrochromatography (CEC) is reviewed. The development of simple and robust CEC column technologies plays an important role for popularization of CEC. During the last several years, various approaches for the preparation of enantioselective columns have been reported. Currently, the monolithic column technology (continuous beds) represents the most advanced approach for the preparation of CEC columns. The development of new chiral stationary phase used for CEC is another important issue in this field. Fundamental investigations on electrochromatographic behaviors of various CSPs are necessary in order to understand the separation mechanism and thus improve the separation performance. Some chiral stationary phases performed better under nonaqueous CEC conditions than reversed-phase conditions. Coupling CEC with mass spectrometry (MS) provides a powerful tool for enantiomeric separation. Finally, some applications of enantiomeric separation by CEC are summarized.