Evaluation and application of liquid chromatographic columns coated with 'intelligent' ligands. II. Phospholipid column

The stationary phases of octadecylsilica (ODS) coated with phospholipid have been developed as a model of artificial lipid membranes for liquid chromatographic columns. An ODS column coated with phospholipid can be readily prepared by recycling a solution containing L-alpha-dipalmitoyl-phosphatidylcholine (DPPC) through an ODS column in a closed loop. DPPC becomes absorbed on the ODS surfaces by hydrophobic interaction between the acyl group of DPPC and the octadecyl group of the ODS surfaces. The DPPC column was usable when a mobile phase containing 30% (v/v) acetonitrile was delivered without detachment of the DPPC from the ODS surfaces. The retention behavior of ionic solutes on the DPPC column suggested that the retention was based on both ionic and electrostatic interactions between the solutes and the stationary phase. The retention factors on the DPPC column correlated well with the partition coefficients in liposome systems for alpha-adrenoceptor agonists and beta-blockers, indicating that the partition of solutes between the coated phase and buffer was similar to that in the liposome/water system. The DPPC column can be used in screening studies to predict the binding properties of drugs onto lipid membranes.     

反相-高效液相色谱手性流动相添加剂法测定酸奶及含乳饮料中乳酸对映异构体

选取2,3,6-三甲基-β-环糊精（TM-β-CD）作为流动相手性添加剂,建立了采用反相高效液相色谱法分离酸奶及含乳饮料中乳酸异构体的方法。实验采用hypersil ODS2-C18（250×5．0mm,5μm）色谱柱,以0．5mmol／L PH2．5的TM-β-CD（含4．5mmol／L H2SO4）作为流动相,流速为1．0mL／min,紫外检测波长为210nm。实验考察了不同色谱柱、柱平衡时间、手性流动相添加剂浓度及pH值对分离效果的影响,并进一步研究了方法的线性范围、检出限、精密度及回收率。     

Direct and indirect high-performance liquid chromatographic enantioseparation of beta-amino acids

Direct and indirect reversed-phase (RP) high-performance liquid chromatographic methods were developed for the separation of enantiomers of 18 unnatural beta-amino acids, including several beta-3-homo amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases (CSPs) containing macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column) and teicoplanin aglycone (Chirobiotic TAG column). The indirect method involved pre-column derivatization with a new chiral derivatizing agent (CDA), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester ((S)-NIFE), and subsequent separation of diastereomers on Discovery C18 and Hyperpep 300 C18 columns. The different methods were compared in systematic chromatographic examinations. The effects of organic modifier, mobile phase composition, pH and flow rate on the separation were investigated.     

Enantioseparation of Novel Chiral Tetrahedral Clusters on an Amylose Tris-(3,5-dimethylphenylcarbamate) Chiral Stationary Phase by Normal Phase HPLC

Introduction In recent years, chiral transition metal cluster has at-tracted a great deal of interests due to its potential ap-plication in asymmetric catalytic reaction.1-3 Producing catalysis for asymmetric induction using a rigid chiral framework would not only bring a basically conceptual breakthrough in the asymmetric catalysis, but also en-rich the methodology in the design of new chiral cata-lysts.4 So far, a lot of chiral clusters have been re-ported,5-9 but only a few of them have bee…     

Study of a monolithic silica capillary column coated with poly(octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds

The performance of a monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds was studied, and the results were compared to those obtained by using a monolithic silica capillary column modified with octadecylsilyl-(N,N-diethylamino)silane (ODS column). Benzene and naphthalene derivatives, polycyclic aromatic hydrocarbons (PAHs), steroids, alkyl phthalates, and tocopherol homologues were used as test samples. In general, compounds with aromatic character, rigid and planar structures, and lower length-to-breadth ratios (more compacted structures) seem to have more preference for the polymer coated stationary phase (ODM). Compounds with acidic character have also a higher retention on ODM columns because of the presence of ester groups in the stationary phase. The polymer coated column allowed the separation of some PAHs, alkyl phthalates, steroids, and of beta- and gamma-tocopherol isomers which cannot be separated under the same conditions on ODS columns, while keeping similar column efficiency. These results allowed to suggest ODM columns as a good alternative to conventional ODS columns for reversed-phase liquid chromatography.     

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.     

Application and comparison of derivatized cellulose and amylose chiral stationary phases for the separation of enantiomers of pharmaceutical compounds by high-performance liquid chromatography.

The direct high-performance liquid chromatographic separation of three pairs of structurally related enantiomers on derivatized cellulose and amylose chiral stationary phases (Chiralcel OD, Chiralpak AD and Chiralpak AS) was studied using hexane as the mobile phase with 2-propanol or ethanol as modifiers. The separation, retention and elution order of the enantiomers on the different columns using different alcohol modifiers were compared. The effect of structural variation of the solutes on their k' was noted. A reversal of elution order of one enantiomeric pair upon changing the mobile-phase modifier was observed. Chiralcel OD and Chiralpak AD columns provided different elution orders of the enantiomers, including a fourth pair of enantiomers that were not structurally related to the other three pairs.     

Enantioseparation of Novel Chiral Tetrahedral Clusters on an Amylosoe Tris-（3,5-dimethylphenylcarbamate） Chiral Stationary Phase by Normal Phase HPLC

Amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) coated on a kind of small particle silica gel was prepared. On this ADMPC chiral stationary phase (CSP), the direct enantiomeric separation of six novel chiral transition metal tetrahedral clusters has firstly been achieved using n-hexane as the mobile phase containing various alcohols as modifiers. The effect of mobile phase modifiers and the structural variation of the solutes on their retention factors (k‘) and resolutions (Rs) were investigated. The result suggests that not only the structure and concentration of alcohol in mobile phase, but also the structural differences in racemates can have a pronounced effect on enantiomeric separation. ADMPC-CSP is a suitable CSP for the optical resolution of chiral tetrahedral cluster by HPLC.     

直链淀粉手性固定相法分离利阿唑对映体

建立了利阿唑对映体的高效液相色谱拆分方法。采用Chiralpak AD-H手性柱在正相条件下直接拆分利阿唑对映体,考察了流动相中有机极性调节剂的种类和浓度、酸碱的种类和含量、柱温及流速等对利阿唑对映体分离的影响。确定了最佳的拆分条件:流动相为正己烷-乙醇(含0.3%二乙胺和0.1%冰醋酸)(体积比为80∶20),流速0.6 mL/min;检测波长254 nm;柱温20 ℃。在此条件下利阿唑对映体的分离度为3.4。该法简单快速,重现性好。     

Monolithic silica columns with chemically bonded tert-butylcarbamoylquinine chiral anion-exchanger selector as a stationary phase for enantiomer separations

An enantioselective silica rod type chiral stationary phase (CSP) is presented as a novel combination of the well-known enantiomer separation properties of immobilized tert-butyl-carbamoylquinine chiral anion-exchanger selector with the unique properties of monolithic silica material. The chromatographic behavior of the tert-butyl-carbamoylquinine silica rod was studied and compared with a similar prepared particulate material. Good selectivities were achieved for a spectrum of chiral test components like N-derivatized amino acids (DNB- Ac-, DNZ-, Bz-, Z-amino acids) and for Suprofen. The influence of mobile phase parameters, as well as the effect of serially coupling up to six 10 cm monolithic silica columns was studied and put in context to conventional columns of particulate 5 microm type CSP. Using that 60 cm long monolithic column it was possible to improve the enantiomer separation of Suprofen and achieve a baseline separation in less than 10 min of total separation time.     

卵类粘蛋白柱手性拆分酮基布洛芬

 在卵类粘蛋白柱上 ,考察了酮洛芬对映体在 3种流动相体系下的保留行为 ,建立了同时分离酮洛芬及其甲酯两对对映体的较佳色谱条件。方法可用于酶法立体选择性水解或酯化过程中生成的 4种化合物的同时测定     

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

 以稠环芳烃为探针,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶ZorbaxODS对稠环芳烃异构体的选择性,并对可能的保留机理进行了讨论。以甲醇-水(体积比为75∶25)为流动相,在烷基膦酸改性锆镁复合氧化物固定相上分离了8种稠环芳烃类化合物。     

去甲万古霉素键合手性固定相拆分β-受体阻滞剂类药物及其结构类似物的研究

以去甲万古霉素为手性选择剂制备了大环抗生素类手性固定相去甲万古霉素键合手性固定相(NVC-CSP)。在极性有机模式下对普萘洛尔、美托洛尔、阿替洛尔及烯丙洛尔等4种β-受体阻滞剂类药物及其结构类似物的外消旋体进行了手性拆分的研究,并考察了流动相组成、酸碱添加剂用量、温度以及流速对分离的影响。研究发现:在此模式下手性物质的保留均随着流动相中甲醇含量的增加而减弱,手性分离因子（α）随着流动相中甲醇含量的增加而升高;随着柱温的升高,大部分溶质在色谱柱上的保留减弱,α值降低;在一定范围内降低流速有利于对映体的分离;     

大环抗生素手性固定相拆分盐酸马布特罗对映体

基于三种大环抗生素类手性固定相Chirobiotic V,T和R,利用高效液相色谱法对盐酸马布特罗对映体进行了拆分;考察了洗脱模式、流动相组成、柱温等因素对分离的影响,对分离结果进行了比较,对分离机制进行了探讨.结果表明,盐酸马布特罗对映体在Chirobiotic R手性固定相上不能实现分离,在Chirobiotic V和T手性固定相上均可实现较好的基线分离.最佳色谱条件为:新极性有机相模式,流动相甲醇-冰醋酸-三乙胺(100∶0.01∶0.01,V/V/V),流速1.0mL/min,柱温20℃;相应的分离度分别可达3.08和3.73.与此同时,盐酸马布特罗对映体与大环抗生素类固定相之间的离子相互作用是实现对映体分离的最主要分离机制.     

C18柱串联冠醚手性柱高效分离3种芳香族氨基酸对映体

将C18柱与手性冠醚柱串联,建立了一种反相高效液相色谱法用于3种芳香族氨基酸对映体同时拆分的方法.考察了反相色谱流动相的组成、pH值、柱温、流速对对映体拆分的影响.实验结果表明,当流动相为HClO4-乙睛溶液(86:14,V/V,pH 2.0)、柱温20℃、流速0.4 mL/min时,3种氨基酸对映体可获得基线分离.进一步对比了C18柱、冠醚手性柱和串联顺序不同的4种分离模式,结果表明,C18柱不能拆分氨基酸对映体,仅能分离不同种类氨基酸;冠醚手性柱可分离氨基酸映体,但不同种类氨基酸色谱峰出现重叠;串联模式能实现3种氨基酸对映体的基线分离,实现双柱优势互补,而串联顺序对分离影响不大,仅影响色谱峰的峰形.     

Comparative LC Enantioseparation of Novel PPAR Agonists on Cellulose- and Amylose-Based Chiral Stationary Phases

The LC enantiomeric separation of several dual PPARα/γ agonists on the commercially available Chiralcel OD and Chiralpak AD columns has been evaluated in normal phase mode using a mobile phase consisting in a mixture of n-hexane, 2-propanol and trifluoroacetic acid at constant volume ratio. Most compounds were separated as underivatized acids without requiring time consuming analysis. Some complementary selectivity was evidenced on the two investigated chiral stationary phases related to the different accessibility of the active sites of the helical cavities. Additional information on the chiral recognition mechanism were deduced from the chromatographic behaviour of some selected methyl esters.     

高效液相色谱手性固定相法拆分阿折地平对映体

建立了阿折地平对映体的高效液相色谱拆分方法。采用Chiralpak AD-H (250 mm×4.6 mm, 5.0 μm, Daicel公司)手性色谱柱在正相条件下直接拆分阿折地平对映体,考察了固定相种类、流动相组成及柱温等对阿折地平对映体分离的影响。确定了最佳的拆分条件: 流动相为正己烷-异丙醇(90:10, v/v),流速为0.8 mL/min,检测波长为254 nm;柱温为20 ℃;在此条件下阿折地平对映体的分离度为3.3。该法简单快速,重现性好。     

高效液相色谱法手性拆分(2-戊基-3-苯基-2,3环氧丙烷基)二苯基磷酸酯对映体

采用多糖类手性色谱柱,建立了(2-戊基-3-苯基-2,3环氧丙烷基)二苯基磷酸酯对映体的高效液相色谱手性拆分方法。考察了手性柱类型、流动相组成、流速、柱温等对手性拆分的影响,并对分离机制进行了探讨。结果表明,采用Chiralpak AS-H柱(250×4.6mm,i.d.,5μm),以正己烷-异丙醇(85∶15,V/V)为流动相,在柱温25℃,流速1.0mL/min,检测波长210nm的条件下,(2-戊基-3-苯基-2,3环氧丙烷基)二苯基磷酸酯对映体能达到完全分离,且稳定性和重复性好。该方法也适用于(2-戊基-3-苯基-2,3环氧丙烷基)二苯基磷酸酯类似物的手性拆分。     

Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin

Summary A new HPLC stationary phase was synthesized by thein situ covalent immobilization of human serum albumin (HSA). The protein was immobilized on a commerically available diol column which had been activated with 1,1-carbonyldiimidazole. Initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes and that these separations may reflectin vitro binding to the HSA. The effects of mobile phase composition and temperature on the stereochemical resolutions are reported.     

一种手性中间体在键合型和涂覆型纤维素手性固定相上的拆分

制备了涂覆型和键合型纤维素-(3, 5-二甲基苯基氨基甲酸酯)固定相, 分别在制备的纤维素手性固定相上成功地拆分了一种手性中间体, 通过考察流动相中的改性剂(醇、四氢呋喃、三氯甲烷)对手性拆分的影响, 优化了手性中间体在两种手性固定相上的色谱分离条件, 并比较了手性中间体在涂覆和键合型纤维素手性固定相上的拆分. 结果表明, 涂覆型和键合型手性固定相对这种手性中间体均有较好的拆分效果, 在150 mm的色谱柱上, 这两种手性固定相对这种手性中间体的拆分能力相差不大, 但键合型固定相上可选择的流动相范围更广.