奎宁-冠醚组合型手性固定相直接拆分氨基酸的机理

合成了一种新型奎宁-冠醚组合型手性固定相(QN-CR CSP)并用于氨基酸手性对映体的直接拆分,该固定相对12种氨基酸对映体有良好的手性拆分能力。基于氨基酸手性识别中离子交换和络合的协同作用,建立了一种新型的等温吸附模型。通过迎头特殊点洗脱法(FACP)测定色氨酸(Trp)在不同金属离子添加剂条件下的等温吸附线,验证了模型的合理性。流动相中的Li⁺、Na⁺、K⁺等金属离子与氨基酸竞争固定相中的冠醚络合位点,随着金属离子与冠醚的络合作用力和络合吸附平衡常数增大,固定相对Trp的手性拆分能力下降。该模型的建立对理解氨基酸在此类固定相中的手性保留行为以及固定相结构的进一步优化具有重要意义。     

伊瑞霉素键合手性毛细管整体柱的制备与对映体分离

以具有22个不同种类手性中心的新型大环抗生素伊瑞霉素为手性选择器,基于环氧基团高反应活性的特征,将伊瑞霉素用一步法键合到甲基丙烯酸酯整体柱表面制备伊瑞霉素键合手性毛细管整体柱。通过对制备条件进行优化,证实该制备方法可在较宽的pH范围(6.0～9.0)内进行,方法简单易行,反应条件温和。应用制备的手性毛细管整体柱在毛细管电色谱模式下,对5种手性氨基酸对映体和手性药物罗格列酮对映体进行拆分,均得到了基线分离,说明伊瑞霉素手性固定相具有较强的手性拆分能力。在优化的色谱条件下,6种对映体的分析时间均小于4 min,分析速度快。通过对有机调节剂、缓冲液pH值和缓冲盐浓度等分离条件进行系统考察,初步探讨了该手性毛细管整体柱对不同溶质的手性识别机理。     

Evaluation and Comparison of a 3,5‐Dimethylphenyl Isocyanate Teicoplanin with Phenyl Isocyanate Teicoplanin Chiral Stationary Phase Using RP‐HPLC

HPLC enantiomeric separations of 8 α‐amino acids were achieved using two self‐made chiral stationary phases (CSP)–phenyl isocyanate teicoplanin (Phe‐TE) and 3,5‐dimethylphenyl isocyanate teicoplanin (DMP‐TE), using reversed phase mobile phases. The Phe‐TE or the DMP‐TE CSP was prepared from the TE using derivative agents, phenyl isocyanate or 3,5‐dimethylphenyl isocyanate, respectively. The chromatographic results were given as the retention, selectivity, resolution factor and the enantioselective free energy difference corresponding to the separation of the two enantiomers. The effect of pH, organic modifier type and amount were discussed, and the stereoselectivities for two TE‐based CSPs were compared. The chiral selectivity factor for six α‐amino acids on DMP‐TE is somewhat bigger than that on Phe‐TE CSP under reversed phase (RP) mode. Comparison of the enantiomeric separations using self‐made Phe‐TE and DMP‐TE was conducted to gain a better understanding of the chiral recognition mechanism of the macrocyclic glycopeptide CSP.     

Enantiomeric and diastereomeric high-performance liquid chromatographic separation of cyclic beta-substituted alpha-amino acids on a teicoplanin chiral stationary phase

High-performance liquid chromatographic (HPLC) separation of stereomeric cyclic beta-substituted or-quaternary alpha-amino acids was performed on a chiral stationary phase based on the glycopeptide antibiotic teicoplanin. The investigated amino acids are the 1-amino-2-methylcyclohexanecarboxylic acids, the 1-amino-2-hydroxycyclohexanecarboxylic acids, Ala, Cha, Phe and Tle. The effects of the mobile phase composition (type and content of organic modifier, pH) and of the temperature on the enantio- and diastereoselectivity were studied and the conditions were optimised to resolve the four stereomers of one amino acid in a single chromatographic run. The influence of the modifier concentration and the pH of the mobile phase reveal two enantiomeric and diastereomeric discrimination mechanisms based on different interactions with the stationary phase. For optimal separation of diastereomers the column has to be conditioned with an acidic eluent.     

Influence of preferential adsorption of mobile phase on retention behavior of amino acids on the teicoplanin chiral selector

The adsorption behavior of two amino acids, i.e., l,d-threonine and l,d-methionine has been investigated on the chiral stationary phase (CSP)column packed with teicoplanin bonded to a silica support. The study has been performed under non-linear conditions of adsorption isotherm for various types of organic modifiers (methanol, ethanol, propan-2-ol and acetonitrile) in the reversed-phase mode. A heterogeneous adsorption mechanism of amino acids has been identified that was strongly affected by the nature of organic modifier. Generally, isotherm non-linearity and retention decreased with decrease of the modifier content in the mobile phase exhibiting a minimum at water-rich mobile phases. These trends were suggested to result from a combined effect of the mobile as well as the adsorbed phase composition. To determine the composition of the adsorbed phase the excess adsorption of modifiers in aqueous solutions has been measured and their binary adsorption equilibria have been quantified and compared. Strongly non-ideal behavior of solvents in the mobile phase and the adsorbed phase has been accounted for by activity coefficients. The fraction of the modifiers in the adsorbed phase decreased in the sequence: methanol, ethanol, propan-2-ol and acetonitrile.     

金纳米粒子修饰的手性毛细管电色谱固定相的制备及性能表征

制备了粒径为15 nm的金纳米粒子(GNPs)并将其修饰到氨基衍生化的硅胶整体柱内,通过化学键合法将牛血清白蛋白(BSA)固载到GNPs的表面作为手性固定相。通过透射电子显微镜、扫描电子显微镜等方法进行表征,结果表明,GNPs分散性良好,并被成功地修饰到毛细管柱内,含量高达17.18%。优化了BSA手性柱的制备条件,最终确定了体积分数为10%的3-氨丙基三乙氧基硅烷(APTES)和15 g/L BSA为最佳反应条件。在毛细管电色谱分离模式下,对缓冲液pH值、电压等分离条件进行了考察,最终选择了10 mmol/L pH 7.4的磷酸缓冲液和15 kV运行电压作为最佳分离条件。手性柱对3种手性化合物(色氨酸、阿替洛尔和麻黄碱)有拆分效果,对色氨酸能实现基线分离。与物理吸附法相比,化学键合法制备的手性柱拆分效果好,分析物无需柱前衍生化,且色谱柱稳定性良好。该文的制备方法也为其他类型手性选择剂的引入提供了良好的思路。     

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.     

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).     

Enantioseparation of Amino Acids on a Chiral Stationary Phase Derived from L‐α‐Norleucinyl‐ and Pyrrolidinyl‐disubstituted Cyanuric Chloride by High‐Performance Liquid Chromatography

A silica‐based chiral stationary phase (CSP) derived from L‐α‐norleucinyl‐ and pyrrolidinyl‐disubstituted cyanuric chloride was prepared for the enantioseparation of methyl esters of N‐(3,5‐dinitrobenzoyl) amino acids by high‐performance liquid chromatography. The chromatographic results show that effective enantioseparation of methyl esters of N‐(3,5‐dinitrobenzoyl)amino acids, except for proline, was achieved on this chiral stationary phase. The chromatographic resolution of racemic n‐propyl ester of N‐(3,5‐dinitrobenzoyl)valine on CSP‐B is better than that of racemic methyl ester of N‐(3,5‐dinitrobenzoyl)valine on CSP‐B or CSP‐A reported previously (J. Chromatogr. A, 676 (1994) 303). The comparison of the chromatographic results obtained in this study with those on CSP‐A reported previously reveals that steric effect, instead of hydrophobic interaction, between the n‐butyl group attached to the chiral center of the chiral selector and the alkyl group attached to the chiral center of the chiral selectand plays a significant role in chiral discrimination. The increase in the selectivity factor of methyl esters of N‐(3,5‐dinitrobenzoyl)amino acids with bulky alkyl groups was examined on CSP‐B.     

Separation of racemic 2,4-dinitrophenyl amino acids on carboxymethyl-β-cyclodextrin coated zirconia in RPLC

We report preparation and use of carboxymethyl-β-cyclodextrin-coated zirconia as a chiral stationary phase (CSP) for separation of enantiomers of 2,4-dinitrophenyl (DNP) amino acids in reversed-phase high performance liquid chromatography. The CSP showed good enantioselectivity for some of the amino acids studied. Effects of pH and amount of methanol in mobile phases on retention and enantioselectivity for the analytes were examined.     

Resolution of beta-amino acids on a high performance liquid chromatographic doubly tethered chiral stationary phase containing N-CH3 amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) containing N-CH(3 )amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied to the resolution of various beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were consistent with those on the corresponding singly tethered CSP while the chiral recognition ability of the doubly tethered CSP was generally greater in terms of both the separation (alpha) and the resolution factors (R(S)) than that of the corresponding singly tethered CSP. From these results, it was concluded that attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the CSP improves the chiral recognition ability for the resolution of beta-amino acids without any change in the chiral recognition mode. The retention factors (k(1)) on the doubly tethered CSP were larger than those on the corresponding singly tethered CSP and these retention factors were found to be controllable with the variation of the type and the content of the organic and/or acidic modifier in the aqueous mobile phase without significant change in the separation and the resolution factors.     

Enantioseparation of a novel “click” chemistry derived native β-cyclodextrin chiral stationary phase for high-performance liquid chromatography

A novel native β-cyclodextrin chiral stationary phase was prepared via “click” chemistry with cuprous iodide–triphenylphosphine complex as the catalyst and applied for enantioseparation of Dns-amino acids, substituted phenyl and phenoxy group modified propionic acids, flavonoids, and some pharmaceutical compounds such as nimodipine, propranolol, brompheniramine and bendroflumethiazide in reversed-phase high-performance liquid chromatography. The studied analytes could be resolved under different separation conditions. The resolution of Dns-DL-Leu could reach 5.08 using a mobile phase consisting of 1% (w/w) triethylammonium acetate buffer (pH 4.11) and methanol (50:50 v/v). The effects of buffer pH and the content of organic modifier on enantioseparation of Dns-amino acids by this novel chiral phase were being investigated. The separation results demonstrate that click chemistry, a versatile reaction, affords a facile approach towards the preparation of stable chiral stationary phases.     

Effect of the residual silanol group protection on the liquid chromatographic resolution of racemic primary amino compounds on a chiral stationary phase based on optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6

A liquid chromatographic chiral stationary phase (CSP) based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6, which has been utilized in the resolution of alpha-amino acids, amines and amino alcohols, was treated with excess of n-octyltriethoxysilane to prepare a new improved CSP. The residual silanol groups of the original CSP were protected by n-octyl groups in the new CSP. The chiral recognition ability of the new CSP was superior to that of the original CSP in the resolution of alpha-amino acids, amines and amino alcohols. Retention factors (k1) for the resolution of alpha-amino acids were lower on the new CSP than on the original CSP while those for the resolution of amines and amino alcohols were higher on the new CSP than on the original CSP. The improved chiral recognition ability of the new CSP and the retention behaviors of the two enantiomers on the new CSP have been rationalized to stem from the removal of the non-enantioselective interactions between the analytes and the residual silanol groups of the original CSP and the improved lipophilicity of the CSP.     

Unusual resolution of N-(3,5-dinitrobenzoyl)-alpha-amino acids on a chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

While HPLC chiral stationary phases (CSPs) based on chiral crown ethers have been known useful for the resolution of only racemic primary amino compounds or some secondary amino compounds, in this study, we first demonstrated that the CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid is also useful for the resolution of N-benzoyl-alpha-amino acids, which do not contain a primary or secondary amino group. Especially, N-(3,5-dinitrobenzoyl)-alpha-amino acids were resolved better than corresponding N-(3-nitrobenzoyl)- or N-benzoyl-alpha-amino acids, the separation (alpha) and the resolution factors (R(S)) for the resolution of eight N-(3,5-dinitrobenzoyl)-alpha-amino acids being in the range of 1.06-1.81 and 0.54-2.81, respectively. The optimum mobile phase condition was the mixture of acetic acid-triethylamine-acetonitrile with the ratio of 0.05/0.25/100 (v/v/v).     

Liquid chromatographic resolution of 1-aryl-1,2,3,4-tetrahydroisoquinolines on a chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A liquid chromatographic chiral stationary phase (CSP) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied for the first time to the resolution of biologically important 1-aryl-1,2,3,4-tetrahydroisoquinolines. The unusual resolution of cyclic secondary amino compounds on a chiral crown ether-based CSP was quite successful with the use of a mixture of methanol-acetonitrile-triethylamine at a ratio of 30/70/0.5 (v/v/v) as a mobile phase. From the chromatographic behaviours for the resolution of seven 1-aryl-1,2,3,4-tetrahydroisoquinolines, the steric bulkiness of the 1-phenyl ring at the chiral center of analytes was concluded to play an important role in the chiral recognition.     

Chromatographic enantioseparation of amino acids using a new chiral stationary phase based on a macrocyclic glycopeptide antibiotic

The separation of the enantiomers of several a-amino acids was studied on a new chiral stationary phase (CSP) which is based on the macrocyclic glycopeptide antibiotic eremomycin attached to silica particles. Retention and separation factors were determined under analytical conditions at ambient temperature for different mobile phase compositions. In order to evaluate the potential with respect to preparative separations the adsorption isotherms of D- and L-methionine were determined for one mobile phase composition applying the elution by characteristic point method. The isotherms were validated by comparing experimentally determined elution profiles with predictions based on the equilibrium dispersive model. Finally, the performance of the eremomycin CSP was compared with a commercially available CSP based on the macrocyclic antibiotic teicoplanin. After determining the isotherms of D- and L-methionine also for the teicoplanin phase, the equilibrium dispersive model was used for both CSP to identify optimal operating conditions. For the separation and conditions considered the new eremomycin CSP revealed a better performance compared to the teicoplanin CSP.     

Effect of the residual silanol group protection on the liquid chromatographic resolution of α‐amino acids and proton pump inhibitors on a ligand exchange chiral stationary phase

A new ligand exchange chiral stationary phase (new CSP) containing residual silanol group‐protecting n‐octyl groups on the silica surface was prepared by treating a ligand exchange CSP (original CSP) based on sodium N‐[(R)‐2‐hydroxy‐1‐phenylethyl]‐N‐undecylaminoacetate bonded to silica gel with excess n‐octyltriethoxysilane. The new and original CSPs containing an identical amount of chiral selector were applied to the resolution of α‐amino acids and proton pump inhibitors (PPIs) including omeprazole, pantoprazole, lansoprazole, and rabeprazole. The separation factors (α) and resolutions (R_S) were greater on the new CSP than on the original CSP except for the resolution of asparagine. The trends of the retention factors (k₁) for the resolution of α‐amino acids on the new and original CSPs with the variation of the organic modifier content in aqueous mobile phase were opposite to those for the resolution of PPIs. Removal of the nonenantioselective interactions between the residual silanol groups and the analytes and the improved lipophilicity of the new CSP were proposed to be responsible for the improved chiral recognition ability of the new CSP and the different retention behaviors of the enantiomers between the new and original CSPs.     

Preparation of a new doubly tethered chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and its application

A new doubly tethered chiral stationary phase (CSP) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was prepared by attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the corresponding singly tethered CSP, which was previously developed in our laboratory. The new doubly tethered CSP was applied successfully to the resolution of various racemic alpha-amino acids, amines and amino alcohols containing a primary amino group. In most cases, the chiral recognition efficiency of the new doubly tethered CSP was superior to that of the corresponding singly tethered one in the resolution of alpha-amino acids, amines and amino alcohols. In the resolution of some racemic primary amino compounds, the new doubly tethered and the corresponding singly tethered CSPs were complementary with each other. The chiral resolution behaviors on the new doubly tethered CSP were examined with the variation of the type and content of organic and acidic modifiers in aqueous mobile phase and the column temperature. The chiral resolution behaviors on the new doubly tethered CSP were generally quite similar to those on the corresponding singly tethered CSP. The stability of the new doubly tethered CSP was greater than that of the corresponding singly tethered CSP.     

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.     

One-pot synthesis of a novel chiral Zr-based metal-organic framework for capillary electrochromatographic enantioseparation

A novel chiral stationary phase (CSP) of Zr-based metal-organic framework, l -Cys-PCN-224, was prepared by one-pot method and applied for the enantioseparation by capillary electrochromatography. The CSP was characterized by X-ray diffraction, thermogravimetric analysis, X-ray photoelectron spectroscopy, Fourier-transform infrared spectra, nitrogen adsorption/desorption, circular dichroism spectrum, zeta-potential, and so on. The results revealed that the CSP had good crystallinity, high specific surface area (2580 m²/g), and good thermal stability. Meanwhile, the cross-section of l -Cys-PCN-224-bonded open-tubular (OT) column was observed by a scanning electron microscope, which proved the successful bonding of l -Cys-PCN-224 particles to the inner wall. Relative standard deviations of the column efficiencies were 3.87%–9.14%, and not obviously changed after 200 runs, which indicated that l -Cys-PCN-224-bonded OT column had the better stability and reproducibility. Excellent chiral separation performance was verified with nine kinds of natural amino acids including acidic, neutral, and basic as the analytes. All amino acids studied achieved good separation with the resolution of 1.38–13.9 and selector factor of 1.11–3.71. These results demonstrated that the CSP had an excellent potential in the chiral separation field.