色氨酸在自制人血清白蛋白手性柱上的拆分及手性识别机理研究

人血清白蛋白与三氯聚氰活化的氨丙基硅胶反应,制得人血清白蛋白键合手性固定相。反相模式下,色氨酸在该手性固定相上获得理想的拆分,分离因子可达3.51,分离度达5.49。探讨了流动相p H值、有机修饰剂、柱温等对手性拆分的影响。通过前沿分析法对色谱保留机理进行了探讨。     

前沿分析法测定色氨酸在牛血清白蛋白手性柱上的吸附等温线

利用三氯聚氰活化的氨丙基硅胶与牛血清白蛋白反应,快速而经济地制得牛血清白蛋白手性固定相。运用前沿分析法,反相模式下获得色氨酸对映体在牛血清白蛋白手性固定相上的平衡吸附量,并将实验数据与Langmuir、bi-Langmuir、Toth和Langmuir-Freundlich四种等温吸附模型进行了拟合,结果显示吸附数据与bi-Langmuir模型最为吻合,进一步说明了该手性固定相对色氨酸存在手性识别和非手性识别两种不同类型的吸附位点。     

无载体手性固定相拆分对映体

本文介绍了无载体纤维素三—(4—甲基苯甲酸酯)手性固定相对外消旋药物酮洛芬的拆分且就不同的柱尺寸及装填方式作了比较。为了反映无载体手性固定相的拆分能力,还将无载体柱与有载体柱、自制柱与日本柱作了比较。结果表明:无载体手性固定相对外消旋药物是有拆分能力的,只是其能力低于有载体的。若无载体手性固定相能与高新分离技术(如模拟移动床)相结合,将有望实现外消旋药物的低成本拆分。     

酰胺类手性固定相的研究进展

近年来手性拆分成为新的研究热点,手性固定相的研究发展异常迅速.立足于国内外手性固定相的研究成果,介绍了手性固定相的分类及其应用,并重点综述了基于酰胺的手性固定相的发展及其制备方法.同时简要介绍了作为手性固定相对不同异构体的识别机理,并对该领域未来的发展方向进行了展望.     

刷型手性固定相及其在手性化合物分离中的应用

对映体的分离在有机合成、动力学、药理学、药效学及农业化学等许多学科领域内具有重要的意义,液相色谱手性固定相法拆分对映体的方法被认为最准确、方便的方法之一,刷型手性固定相是液相色谱中非常重要的一类手性固定相。本文介绍了目前常用的几种刷型手性固定相及其在手性化合物分离中的应用情况。     

淀粉苯基氨基甲酸酯类手性固定相的制备及用于HPLC手性体分离性能的研究

合成了含有3,5-二甲基和3,5-二氯取代基团的混合型淀粉(苯基氨基甲酸酯)衍生物(CSP-2),并作为手性体分离材料涂敷在氨丙基化多孔硅胶表面,制得新型高效液相色谱(HPLC)用手性固定相;通过1H核磁共振(1H NMR)和红外光谱(IR)表征衍生物结构;以正己烷-异丙醇(9∶1,v/v)为流动相,对多种手性对映体进行了拆分;结果表明,CSP-2综合了单一取代基团淀粉(苯基氨基甲酸酯)衍生物的手性拆分性能,具有优越的手性分离能力,同时固定相的稳定性大大增强。     

有机相模式下华法令等在氯代淀粉衍生手性固定相上的拆分

利用涂敷型直链淀粉三-(5-氯-2-甲基苯基氨基甲酸酯)手性固定相Lux Amylose-2,有机相模式下考察了其对黄烷酮、氢化安息香、安息香及华法令等的拆分。乙腈作为流动相的主体,探讨了流动相中甲醇含量、醋酸添加剂、柱温等对手性拆分的影响。黄烷酮、氢化安息香及华法令得到了良好的分离,分离度分别可达11.39、12.15及6.33。在0.005 mg/mL-4.0 mg/mL区间,华法令峰面积与浓度之间呈良好的线性关系,检测限可达0.0001 mg/mL,可用于华法令异构体含量的分析。     

基于手性功能单体的L-色氨酸分子印迹复合膜的研制及性能研究

以L-色氨酸为印迹分子,S-2-巯基丙酸为手性功能单体,分别制备了25个L-色氨酸分子印迹复合膜(MICM_1~25)及相应的非印迹复合膜(NICM_1~25)。采用扫描电镜(SEM)表征最优印迹复合膜(MICM₅)及相应的非印迹复合膜(NICM₅)的外观形态,通过等温吸附模型对印迹复合膜的吸附性能进行评价,并结合高效液相色谱法(HPLC法)分析MICM₅对外消旋体DL-色氨酸的手性拆分能力。结果表明:以聚四氟乙烯膜为支撑膜,印迹分子、功能单体和交联剂的摩尔比为1∶8∶90,甲醇∶水(1∶1,体积比)为致孔剂可制备出较优的分子印迹复合膜,其对外消旋体DL-色氨酸有较好的手性拆分能力,拆分因子达到2.41。相对于常用功能单体制备的L-色氨酸分子印迹复合膜,采用S-2-巯基丙酸为手性功能单体制备的L-色氨酸分子印迹复合膜具有更高的亲和性和更好的手性拆分能力。     

万古霉素手性膜色谱的研究

以聚醚砜膜作为基膜,以万古霉素、1,6-己二异腈酸酯作为单体,利用界面聚合的方法制备了万古霉素-己二异腈酸酯聚醚砜手性高分子复合膜,使用自制的膜色谱装置结合高效液相色谱仪,对手性物质D,L-苯甘氨酸进行了手性膜色谱分离研究.详细研究了流速、流动相、进样量、膜层数、膜尺寸对拆分效果的影响.在优选分离条件下,该手性膜色谱对D,L-苯甘氨酸拆分的分离因子(α)和分离度(R_s)分别为5.66、0.66,该方法的最大优点是在水环境下能对手性化合物进行分离制备,这在色谱的手性分离中是少见的.     

HPLC用苯基氨基甲酸酯类手性固定相的制备及分离性能

合成了同时含有3,5-二甲基和3,5-二氯取代基团的纤维素(苯基氨基甲酸酯)衍生物(CSP-1),作为手性体分离材料涂敷在氨丙基化硅胶表面,制得新型高效液相色谱(HPLC)用手性固定相;利用1H核磁共振(1H-NMR)和红外光谱(IR)表征了衍生物结构;以正己烷-异丙醇(体积比9∶1)为流动相,对多种手性对映体进行了拆分。结果表明,CSP-1具有很好的手性体分离能力,综合了单一取代基团纤维素(苯基氨基甲酸酯)衍生物的手性拆分性能。     

R-1-苯基乙基氨基甲酸酯-β-环糊精键合手性固定相的制备与应用研究

利用R-1-苯基乙基异氰酸酯对β-环糊精键合固定相进行衍生,合成了R-1-苯基乙基氨基甲酸酯-β-环糊精手性固定相,填充后在反相条件下考察其对氢化安息香、安息香和α-苯乙醇的手性拆分,探讨了流动相中乙腈含量、缓冲盐类型等对手性拆分的影响。氢化安息香获得了基线分离,分离因子可达1.214,安息香得到了部分分离,α-苯乙醇未能拆开。结合线性溶剂强度(LSS)模型和计量置换理论(SDM-R)对色谱保留机理进行了探讨,认为水分子和乙腈分子一起参与了溶质的置换。     

Combining the enantioselectivities of L-valine diamide and permethylated beta-cyclodextrin in one gas chromatographic chiral stationary phase

An L-valine diamide chiral selector was attached to a polysiloxane through a long hydrocarbon spacer giving rise to a chiral stationary phase (CSP), Chirasil-Val-C11. The enantioselective properties of this readily accessible diamide CSP under gas chromatographic conditions were found to be similar to that of the commercially available Chirasil-Val CSP prepared by a polymer-analogous route. A new binary CSP, Chirasil-DexVal-C11, was synthesized by means of simultaneous attachment of both the L-valine diamide and permethylated beta-cyclodextrin selectors to a polysiloxane using platinum-catalyzed hydrosilylation, thereby overcoming the immiscibility problem known for Chirasil-Val and Chirasil-Dex. This binary CSP retained both the enantioselectivity of Chirasil-Val-C11 toward alpha-amino acid derivatives and the unsurpassed enantioselectivity of Chirasil-Dex toward underivatized chiral alcohols, ketones, and hydrocarbons. Furthermore, it was shown that the presence of the cyclodextrin selector in Chirasil-Val-C11 significantly improved the enantioseparation of proline, which represented a problematic amino acid on diamide CSPs.     

Characterization of a thermally induced irreversible conformational transition of amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase in enantioseparation of dihydropyrimidinone acid by quasi-equilibrated liquid chromatography and solid-state NMR

A thermally induced irreversible conformational transition of amylose tris(3,5-dimethylphenylcarbamate) (i.e., Chiralpak AD) chiral stationary phase (CSP) in the enantioseparation of dihydropyrimidinone (DHP) acid racemate was studied for the first time by quasi-equilibrated liquid chromatography with cyclic van't Hoff and step temperature programs and solid-state ((13)C CPMAS and (19)F MAS) NMR using ethanol and trifluoroacetic acid (TFA)-modified n-hexane as the mobile phase. The conformational transition was controlled by a single kinetically driven process, as evidenced by the chromatographic studies. Solid-state NMR was used to study the effect of the temperature on the conformational change of the solvated phase (with or without the DHP acid enantiomers and TFA) and provided some viable structural information about the CSP and the enantiomers.     

Combination of chiral capillary electrochromatography with electrospray ionization mass spectrometry: method development and assay of warfarin enantiomers in human plasma

The hyphenation of chiral capillary electrochromatography (CEC) with electrospray ionization mass spectrometry (ESI-MS) is very challenging but promising due to the fact that it combines sensitivity with high specificity and selectivity. In this work, CEC capillaries packed with (3R,4S)-Whelk-O1 chiral stationary phase were used for simultaneous enantioseparation of (+/-)-warfarin and its internal standard, (+/-)-coumachlor. Furthermore, both the chiral CEC separation and MS detection parameters were examined in detail. First, the influence of different column fabrication was investigated. Second, enantioseparation was optimized by varying CEC parameters, including acetonitrile concentration, buffer pH, and ionic strength. Under the optimum chiral CEC conditions, ESI-MS parameters such as sheath liquid pH and composition, sheath liquid flow rate, drying gas flow rate, drying gas temperature, nebulizer pressure, and fragmentor voltage were investigated to achieve maximum MS signals of the separated enantiomers. Finally, using solid-phase extraction as sample preparation method, (+/-)-warfarin spiked in 100-microL human plasma samples were analyzed. The calibration curves showed good linearity for both (R)-warfarin (R = 0.9979) and (S)-warfarin (R = 0.9978) enantiomers. The experimental limit of detection was approximately 25 ng/mL for both enantiomers. Even though the data are still preliminary, we can state with confidence that chiral CEC-ESI-MS has the potential to establish itself as a very powerful technique for the determination of enantiomeric ratios in human body fluid.     

Enantiomer separation by countercurrent chromatography using cinchona alkaloid derivatives as chiral selectors

Cinchona-derived anion-exchange-type chiral selectors have been adapted and employed in countercurrent chromatography (CCC) for the separation of enantiomers of N-derivatized amino acids and 2-aryloxypropionic acids. The accurate optimization of the enantioseparation in terms of solvent system composition, pH values, ionic strength, and CCC operating conditions was performed. A wide range of solvent mixtures was evaluated. Successful resolutions were achieved in systems such as ammonium acetate buffer/tert-amyl alcohol/methanol/heptane and especially ammonium acetate buffer/methyl isobutyl ketone or diisopropyl ether. Up to 300 mg (0.92 mmol) of N-(3,5-dinitrobenzoyl)-(+/-)-leucine was totally resolved in a single run using a 10 mM concentration of chiral selector in 122 mL of stationary phase. This amount could be increased up to 900 mg (2.77 mmol) when pH-zone-refining mode was applied. The results here presented account for the high potential of CCC as a preparative enantiomer separation technique.     

Synthesis, characterization, and application of chiral ionic liquids and their polymers in micellar electrokinetic chromatography

Two amino acid-derived (leucinol and N-methylpyrrolidinol) chiral ionic liquids are synthesized and characterized in both monomeric and polymeric forms. Leucinol-based chiral cationic surfactant is a room-temperature ionic liquid, and pyrrolidinol-based chiral cationic surfactant melts at 30-35 degrees C to form an ionic liquid (IL). The monomeric and polymeric ILs are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation, and partial specific volume. Herein, we present the first enantioseparation using chiral IL as a pseudostationary phase in capillary electrophoresis. Chiral separation of two acidic analytes, (+/-)-alpha-bromophenylacetic acid and (+/-)-2-(2-chlorophenoxy)propanoic acid (+/-)-(2-PPA) can be achieved with both monomers and polymers of undecenoxycarbonyl-L-pryrrolidinol bromide (L-UCPB) and undecenoxycarbonyl-L-leucinol bromide (L-UCLB) at 25 mM surfactant concentration using phosphate buffer at pH 7.50. The chiral recognition seems to be facilitated by the extent of interaction of the acidic analytes with the cationic headgroup of chiral selectors. Polysodium N-undecenoxycarbonyl-L-leucine sulfate (poly-L-SUCLS) and polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) were compared at high and low pH for the enantioseparation of (+/-)-(2-PPA). At pH 7.5, poly-L-SUCLS, poly-L-SUCL, and (+/-)-(2-PPA) are negatively charged resulting in no enantioseparation. However, chiral separation was observed for (+/-)-(2-PPA) using poly-L-SUCLS at low pH (pH 2.00) at which the analyte is neutral. The comparison of chiral separation of anionic and cationic surfactants demonstrates that the electrostatic interaction between the acidic analyte and cationic micelle plays a profound role in enantioseparation.     

Chemically L-phenylalaninamide-modified monolithic silica column prepared by a sol-gel process for enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography

A new type of chiral monolithic column was successfully developed for the enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography (LE-CEC) in this work. The monolithic column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and the chiral selector L-phenylalaninamide. After being conditioned with Cu(II) aqueous solution, the ligand exchange-chiral stationary phase (LE-CSP) possesses positive charges. When the external electric field was applied in CEC, electroosmotic flow (EOF) was generated on the surface of LE-CSP in the direction from the cathode to the anode. The EOF was found to be dependent on the applied electric field strength and the composition of the mobile phase. With the increase of pH of the mobile phase, the EOF showed a tendency to decrease. Scanning electron microscopy showed that the chiral monolithic column has a continuous skeleton and large through-pore structure. The separation efficiency (theoretic plate numbers) for the separation of Dns-DL-Leu reached up to 9.0 x 10(4) plates m(-1) for the D-enantiomer and 6.6 x 10(4) plates m(-1) for the L-enantiomer, by using pH 5.5, acetonitrile/0.50 mM Cu(Ac)2-50 mM NH4Ac (7:3) as mobile phase. The reproducibility and lifetime were satisfactory. CEC was carried out with conventional capillary electrophoresis equipment without pressurizing the ends of the capillary. No bubble was formed during the operation, after degassing the mobile phase and conditioning the column.     

High resolution of DL-tryptophan at high flow rates using a bovine serum albumin-multilayered porous hollow-fiber membrane

We describe a bovine serum albumin (BSA)-multilayer-adsorbed porous hollow-fiber membrane as a stationary phase that enables chiral separations at a high resolution and high rate. Epoxy-group-containing graft chains were uniformly immobilized on the surface of pores throughout a porous hollow-fiber membrane by radiation-induced graft polymerization. Subsequently, a diethylamino group as an anion-exchange moiety was introduced to the graft chains, which caused the chains to expand toward the interior of the pores due to mutual electrostatic repulsion. The expanding polymer chains provided multilayer binding sites for BSA as a chiral selector. BSA with a degree of multilayer binding of 4 specifically recognized L-tryptophan with a separation factor of 6.6 during permeation by a mobile phase (Tris-HC1 buffer) injected with a racemic solution of DL-tryptophan through the BSA-multilayered porous membrane. In addition, the separation factor was constant irrespective of flow rates of the mobile phase because of negligible diffusional mass-transfer resistance of tryptophan to BSA multilayered by the graft chains.