Separation of enantiomers of <Emphasis Type="Italic">N</Emphasis>-derivatives of amino acids by capillary electrophoresis using macrocyclic antibiotics

The potential for the application of macrocyclic antibiotic eremomycin as a chiral selector for the separation of enantiomers of N-derivatives of amino acids in capillary electrophoresis was estimated. The influence of several factors (the composition and pH of the running electrolyte, the concentration of the chiral selector, capillary geometry, the value of the applied voltage) on the migration of the derivatives of amino acids and enantioselectivity in the presence of eremomycin was studied in order to choose the separation conditions. Using the example of the dansyl derivatives of amino acids the enantioselectivities of vancomycin and eremomycin in capillary electrophoresis were compared.     

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.     

Enantiomeric separation by capillary electrochromatography. II. Chiral separation of dansyl amino acids and phenoxy acid herbicides on sulfonated silica having surface-bound hydroxypropyl-beta-cyclodextrin

A chiral silica-based stationary phase having surface-bound hydroxypropyl-beta-cyclodextrin (HP-beta-CD) with a relatively strong electroosmotic flow (EOF) was introduced for enantioseparation by capillary electrochromatography (CEC). The stationary phase contained a hydrophilic sulfonated sublayer to which a chiral top layer of HP-beta-CD was immobilized. While the sulfonated sublayer was to provide a relatively strong EOF, the top HP-beta-CD was to confer the desired chiral recognition towards enantiomeric solutes. This HP-beta-CD sulfonated silica (CDSS) stationary phase proved useful for the rapid separation of anionic enantiomers such as dansyl amino acids and phenoxy acid herbicides. The effects of the organic modifier content, pH, and ionic strength of the mobile phase on enantioseparation were investigated. Under the optimized separation conditions, ten dansyl amino acids and six phenoxy acid herbicides were enantioseparated with a resolution greater than unity.     

Displacement study on a vancomycin-based stationary phase using N-acetyl-D-alanine as a competing agent

The analysis of the binding data of D,L-dansyl amino acids on a vancomycin stationary phase is investigated in relation to the addition of N-acetyl-D-alanine in the mobile phase. This eluent additive acts as a specific competing agent for the aglycone pocket of the immobilized chiral selector. A model taking into account both stereoselective and nonstereoselective interactions between the solutes and the stationary phase is used to fit the experimental data. From the results, the theoretical approach is considered to be adequate to describe the competing agent dependence on solute retention. To the best of our knowledge, this report constitutes the first example of a displacement study on a macrocyclic antibiotic stationary phase. This work shows that dansyl amino acids bind to the active aglycone pocket of the selector and that this interaction is enantioselective. The results also demonstrate that additional enantioselective sites at the vancomycin surface are involved in the chiral discrimination of these solutes.     

Mono‐6A‐(4‐methoxybutylamino)‐6A‐β‐cyclodextrin as a chiral selector for enantiomeric separation

A new member of the family of methoxylalkylamino monosubstituted β‐cyclodextrins, mono‐6^A‐(4‐methoxybutylamino)‐6^A‐β‐cyclodextrin, has been developed as a chiral selector for enantioseparation in capillary electrophoresis. This amino cyclodextrin exhibited good enantioselectivities for 16 model acidic racemates including three dansyl amino acids at an optimum pH of 6.0. Excellent chiral resolutions over six were obtained for α‐hydroxy acids and 2‐phenoxypropionic acids with 3.0 mM chiral selector. The good chiral recognition for α‐hydroxyl acids was attributed to inclusion complexation, electrostatic interactions, and hydrogen bonding. The hydrogen‐bonding‐enhanced chiral recognition was revealed by NMR spectroscopy. The chiral separation of acidic racemates was further improved with the addition of methanol (≤10 vol%) as an organic additive.     

A Silica Monolithic Column with Chemically Bonded l-Pipecolic Acid as Chiral Stationary Phase for Enantiomeric Separation of Dansyl Amino Acids by CEC–MS

A silica monolithic column chemically modified with l-pipecolic acid as chiral stationary phase has been developed for chiral separation of dansyl amino acids by capillary electrochromatography–mass spectrometry (CEC–MS). The monolithic column was prepared by a sol–gel process and subsequent chemical modification by l-pipecolic acid as chiral selector with 3-glycidoxypropyltrimethoxysilane as spacer. Interestingly, it was found that the l-pipecolic acid-modified monolithic column can hold copper(II) ions tightly after loading Cu(II) ions during column preparation and conditioning and allows CEC separation to be conducted based on chiral ligand exchange with MS detection by a mobile phase without copper ions. It has been demonstrated that the chiral monolithic column operates well for enantioseparation of several dansyl amino acids by CEC–MS.

     

Enantiomeric separation by capillary electrochromatography. I. Chiral separation of dansyl amino acids and organochlorine pesticides on a diol-silica dynamically coated with hydroxypropyl-beta-cyclodextrin

In this work, a commercially available diol-silica stationary phase was converted in situ to a chiral stationary phase by dynamically coating it with hydroxypropyl-beta-cyclodextrin (HP-beta-CD). This stationary phase was shown useful for the capillary electrochromatography (CEC) separation of neutral and anionic enantiomers such as some organochlorine pesticides and dansyl amino acids, respectively. The inclusion of HP-beta-CD in the mobile phase to produce the in situ chiral stationary phase allowed the rapid separation of the anionic dansyl amino acid enantiomers at relatively low electroosmotic flow (EOF). The formation of host-guest complexes between the dansyl amino acids and the neutral HP-beta-CD in the mobile phase lowered the actual charge-to-mass ratios of the anionic solutes, thus speeding up their transport by the EOF across the packed capillary column. Several parameters affecting enantioseparation were investigated, including the concentration of HP-beta-CD, ionic strength, pH, and organic modifier content of the mobile phase.     

Effect of alkylimidazolium substituents on enantioseparation ability of single-isomer alkylimidazolium-beta-cyclodextrin derivatives in capillary electrophoresis

A family of 6-mono(3-alkylimidazolium)-β-cyclodextrins with one primary hydroxyl group replaced by an alkylimidazolium cation has been developed. The effect of alkyl substitutents on the enantioresolution ability of these single-isomer cyclodextrins towards dansyl amino acids has been studied by capillary electrophoresis. Systematical investigations on the effect of buffer pH and selector concentration on the enatioseparation show that chiral selectors with a shorter alkyl chain (R = C_nH_2n+1, n ≤ 4) presented more powerful chiral recognition ability. These newly introduced single-isomer β-cyclodextrin derivatives proved to be effective chiral selectors for most selected dansyl amino acids at low buffer pH (e.g. pH 5.0) with selector concentration no less than 3 mM. The apparent complex stability constants between alkylimidazolium β-CDs and dansyl amino acids were also theoretically determined by using the mobility difference model proposed by Wren and Rowe. The side alkyl chains from both dansyl amino acids and alkylimidazolium β-CDs displayed significant effect on the apparent complex stability constants. Both the optimum selector concentrations calculated according to the model, however, were much lower than the experimental values giving the maximum chiral resolution of enantiomers.     

Methodology for predicting the separation of proteins by hydrophobic interaction chromatography and its application to a cell extract

Ligand-exchange micellar electrokinetic capillary chromatography was used for the chiral resolution of underivatized and dansyl amino acid enantiomers simultaneously. The separation was achieved by chiral copper(II)-L-valine complexes incorporated in micelles of sodium dodecyl sulfate (SDS). The enantioresolution was strongly affected by SDS and a concentration of 20 mM SDS was shown to be necessary for the separation. Other impacting factors were investigated including pH, the molar ratio of copper(II) to L-valine and the total concentration of complex. Using the proposed method, 11 different dansyl amino acids and two underivatized amino acids were separated successfully with a running electrolyte of 20 mM NH4OAc, 4 mM CuSO4, 8 mM L-valine and 20 mM SDS at pH 9.0 in less than 25 min. Experiments were also performed with other amino acid ligands in order to vary the stability and the sterical arrangement of the copper(II) complexes and the possible chiral recognition mechanism was also discussed briefly.     

A novel chiral column for the HPLC separation of a series of dansyl amino and arylalkanoic acids

In a previous paper [C. Andre, M. Thomassin, A. Umrayami, L. Ismaili, B. Refouvelet, Y.C. Guillaume, Talanta 71 (2007) 1817] a novel cyclic hexapeptide molecule dissolved in the mobile phase was evaluated as a chiral selector (CS) for the enantiomer separation of a series of dansyl amino and arylalkanoic acids using high performance liquid chromatography (HPLC). In this paper, this CS was immobilized to the surface of a monolithic support and the enantioselectivity and the performance of this novel column are discussed.     

Stereoselective analysis of D and L dansyl amino acids as the mixed chelate copper(II) complexes by HPLC

This paper reviews the mixed chelation approach to resolution of the optical isomers of D and L dansyl amino acids by high performance liquid chromatography. The use of eluants containing Cu(II) complexes of L-proline, L-arginine, L-histidine, and L-histidine methyl ester effected the separation of many D and L amino acids, including those with aliphatic, polar, and aromatic substituents. The mechanism of separation, which is based on the preferential ternary complex formation of the analyte amino acid and the chiral chelate with Cu(II) in the mobile phase, is discussed. The stereoselectivity depends mainly on the different steric interactions between the alkyl side chains of the amino acid analytes and the chiral ligands coordinating around Cu(II), although such parameters as pH, temperature, organic modifier, and concentration of the chiral additive also affect the chromatographic separation. Among the chiral ligands studied, L-histidine methyl ester is unique in that it possesses both achiral selectivity for the dansyl amino acids and chiral selectivity for the respective D and L enantiomers. With a mobile phase gradient of acetonitrile in a buffer containing Cu(II) L-histidine methyl ester complex, a stereoselective procedure was devised for the analysis of D and L amino acid enantiomers, achieving the separation that the current amino acid analyzer could not perform. Finally, the use of the mixed chelation approach in two biomedical studies is described. In the first application, the histidine methyl ester gradient was adapted for analyzing amino acids in cerebrospinal fluid; in the second, an L-aspartame Cu(II) complex eluant was developed for measuring the urine concentration of D and L pipecolic acid (piperidine-2-carboxylic acid), a metabolite of lysine.     

Electrochromatographic enantioseparation using chiral ligand exchange monolithic sol-gel column

This paper describes the development of a monolithic sol-gel column modified with l-hydroxyproline as a ligand exchange chiral stationary phase. It has been demonstrated that the monolithic chiral stationary phase can be used for the enantioseparation of dansyl amino acids, free amino acids, hydroxy acids, and dipeptides by capillary electrochromatography and micro-liquid chromatography. The recommended mobile phase was acetonitrile/0.50 mM Cu(Ac)₂-50 mM NH₄Ac (7:3) adjusted to pH 6.5. The characteristics of the monolithic column using hydroxyproline as chiral selector in CEC have been discussed.     

Enantioseparation of dansyl amino acids by ligand‐exchange capillary electrophoresis with zinc(II)‐L‐phenylalaninamide complex

A novel method of chiral ligand‐exchange CE was developed with L ‐amino acylamides as a chiral ligand and zinc(II) as a central ion. It has been demonstrated that these chiral complexes, such as Zn(II)‐L ‐alaninamide, Zn(II)‐L ‐prolinamide, and Zn(II)‐L ‐phenylalaninamide, are suitable for use as chiral selectors for the enantioseparation of either individual pair of or mixed dansyl amino acids. The optimal separation running buffer consisted of 5 mM ammonium acetate, 100 mM boric acid, 4 mM ZnSO₄·7 H₂O, and 8 mM L ‐amino acylamides at pH 8.2. The experiments showed that apart from the effect of the concentration of the complexes on the resolution and the migration time, the buffer pH also had a sharp influence on resolution. The employed chiral ligands exhibited different enantioselectivities and enantiomer migration orders. D ‐Amino acids migrate faster than L ‐amino acids when Zn(II)‐L ‐alaninamide and Zn(II)‐L ‐phenylalaninamide are used as chiral selectors, but it was observed that the migration order is reversed when Zn(II)‐L ‐prolinamide is used as the chiral selector. Furthermore, the amount of dansylated amino acids is found to be highly dependent on the labeling temperature.     

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.     

Highlighting the role of the chlorine substituents in the glycopeptide antibiotic balhimycin for chiral recognition in capillary electrophoresis

The vancomycin-type glycopeptide antibiotic balhimycin (I) and its dehaloanalogue dechlorobalhimycin (III), which is characterized by the total substitution of the two chlorine atoms of I by hydrogen, were employed as chiral selectors for the enantioresolution of 11 racemic dansyl amino acids and six 2-arylpropionic acid nonsteroidal anti-inflammatory racemic drugs by CE. The observed enantioresolution capability of I for all test analytes is clearly higher than that observed for III. This result suggests that chlorine substituents of I played a major role in the enantioresolution of these test analytes. A dimerization-based mechanism is proposed in order to explain this phenomenon. The two chlorine substituents of each monomer, which mutually penetrate into the cavity of the adjacent molecule of the dimer, are assumed to promote dimerization and as a consequence also enantioresolution.     

二脲基桥联β-环糊精手性液相色谱键合相的制备与性能评价

采用六亚甲基二异氰酸酯与6-脱氧-6-羟乙基胺基-β-环糊精反应,合成二脲基桥联β-环糊精,并将其键合到硅胶表面,制备一种新型的二脲基桥联β-环糊精固定相(UBCDP)。通过红外光谱、质谱、元素分析等进行结构表征,以黄烷酮类、氨基酸类、三唑类手性药物和农药为探针,评价其手性色谱性能,探讨相关分离机理。进行色谱条件优化,并与单β-环糊精固定相(CDCSP)比较。试验了多种手性化合物,其中25个被拆分,2'-羟基黄烷酮、己唑醇、丹磺酰亮氨酸等分离度(R_s)达到1.52~4.35,且能拆分体积较大的橙皮甙。这与桥联β-环糊精的协同包结作用有关。而CDCSP仅能拆分少量的对映体,且分离度较低。UBCDP手性拆分能力更强,在手性药物和农药监测中有应用价值。     

Temperature and Solute Molecular Size Effects on the Retention and Enantioselectivity of a Series of D, L Dansyl Amino Acids on a Vancomycin-Based Chiral Stationary Phase

Summary The influence of column temperature (0–28 °C) and solute molecular size on the retention and enantioselectivity of a series of D, L dansyl amino acids with a non-polar side chain (valine, leucine, phenylalanine and tryptophan) were investigated using a vancomycin-based chiral stationary phase (CSP). The enthalpic and entropic terms for the solute-CSP association were determined from the linear vant Hoff plots. Two solute groups were distinguished in relation to these thermodynamic quantities: the solute group I (dansyl valine, dansyl leucine, dansyl phenylalanine) for which large negative values of enthalpic terms were obtained and the solute group II (dansyl tryptophan) for which H value was much less negative. The enthalpy-entropy compensation study revealed that the interaction mechanism was identical for the group I solute enantiomers but changed for D, L dansyl tryptophan. This was further exemplified as the group I compound enantiomers were resolved over the temperature range while the enantiomers of dansyl tryptophan were not separated in the operating conditions. Relationships between both the solute retention factors and apparent enantioselectivity, and the accessible surface area of the amino acid side chain indicated that when the solute molecular size increased (i) the retention was enhanced by the hydrophobic effect and (ii) the chiral discrimination decreased dependent, at least in part, on a steric hindrance phenomenon at the vancomycin aglycone pocket.     

Reversed phase planar chromatography of dansyl DL amino acids with bovine serum albumin in the mobile phase

Summary The chromatographic behavior of twelve dansyl DL amino acids, one D isomer and eleven L isomers on RP₁₈W/UV₂₅₄, RP₁₈W/F_254s, and Sil C₁₈–50 UV₂₅₄ plates developed with aqueous—organic solutions containing bovine serum albumin (BSA) as chiral complexing agent has been extensively investigated. Enantiomeric resolution is highly dependent on mobile phase pH and ionic strength, and on the concentration of both BSA and organic modifier. All the racemates have been resolved within a development time of 1 h 30 min. The selectivity factors () for the dansyl amino acids have been compared with those from planar chromatography for the corresponding DNP, DNPy, and Fmoc amino acids, and with those of the same dansyl derivatives on a column prepared from BSA bound to silica gel.     

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 capillary electrophoresis with cationic pyrrolidinium‐β‐cyclodextrin derivatives as chiral selectors

New single‐isomer, cationic β‐cyclodextrins, including mono‐6‐deoxy‐6‐pyrrolidine‐β‐cyclodextrin chloride (pyCDCl), mono‐6‐deoxy‐6‐(N‐methyl‐pyrrolidine)‐β‐cyclodextrin chloride (N‐CH₃‐pyCDCl), mono‐6‐deoxy‐6‐(N‐(2‐hydroxyethyl)‐pyrrolidine)‐β‐cyclodextrin chloride (N‐EtOH‐pyCDCl), mono‐6‐deoxy‐6‐(2‐hydroxymethyl‐pyrrolidine)‐β‐cyclodextrin chloride (2‐MeOH‐pyCDCl) were synthesized and used as chiral selectors in capillary electrophoresis for the enantioseparation of carboxylic and hydroxycarboxylic acids and dansyl amino acids. The unsubstituted pyCDCl exhibited the greatest resolving ability. Most analytes were resolved over a wide range of pH from 6.0 to 9.0 with this chiral selector. In general, increasing pH led to a decrease in resolution. The effective mobilities of all the analytes were found to decrease with increasing CD concentration. The optimal concentration for most carboxylic acids and dansyl amino acid was in the range 5–7.5 mM and >15 mM for hydroxycarboxylic acids. ¹H NMR experiments provided direct evidence of inclusion in the CD cavity.