超分子化合物毛细管电色谱固定相的研究进展

超分子化学是一门研究分子间特定识别能力的新兴学科,超分子化合物所具有的主-客体识别能力为高选择性的色谱分离提供了广阔的发展前景。毛细管电色谱是近年来发展起来的一种高效、高选择性的微分离技术,电色谱固定相是该技术的核心部分,一直是研究的热点。本文综述了1998年以来环糊精、杯芳烃、冠醚以及大环多胺等4种超分子化合物用作毛细管电色谱固定相的研究进展情况。     

Separation of enantiomers by capillary electrochromatography

The current popularity of capillary electrochromatography (CEC) has led to an increasing number of studies on the development and evaluation of enantioselective CEC systems. These studies clearly demonstrate that the most prominent advantage of electrically driven separation methods, the vastly increased column efficiency as compared to pressure-driven chromatography, can also be experimentally achieved for the separations of enantiomers. In analogy to high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), several approaches have been used. The addition of a chiral selector to the mobile phase is the simplest method. Less erroneous and more elegant approaches are those that use open-tubular, conventional packed, and monolithic columns containing chiral stationary phases that stereoselectively interact with enantiomers. This review evaluates the new techniques and compares them to enantioselective HPLC and CE. Further, it describes the various concepts of enantioselective CEC and focuses on the current ‘state-of-the-art' column technology.     

Chiral separation of lenalidomide by liquid chromatography on polysaccharide‐type stationary phases and by capillary electrophoresis using cyclodextrin selectors

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high‐performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide‐type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time‐dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile‐phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether‐β‐cyclodextrin, where quasi‐equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high‐performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether‐β‐cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)‐lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.     

Chiral separation of helical chromenes with chloromethyl phenylcarbamate polysaccharide‐based stationary phases

Two chloromethyl phenylcarbamate‐based chiral stationary phases, one containing an amylose‐type chiral selector (Lux Amylose 2, from Phenomenex) and the other a cellulose‐type one (Lux Cellulose‐4, from Phenomenex), were successfully used for the chiral resolution of three helical chromenes featuring a helicene‐like structure. The compound bearing a phenyl substituent on the helicene‐like structure was enantioresolved at 25°C with Lux Cellulose‐4 and a n‐hexane/1‐propanol 99:1 v/v eluent. With a n‐hexane/2‐propanol 99.8:0.2 v/v mobile phase, the same column (operated at 35°C) provided the separation of the four isomers of the compound having a hexyl residue on the helicene‐like motif and an additional asymmetric carbon. Lux Amylose‐2 was necessary for the enantioseparation of the compound having the sole hexyl residue on the helical scaffold. For the last compound a n‐hexane/2‐propanol 99.8:0.2 v/v eluent was used, and the column temperature was fixed at 5°C. The enantiomer elution order was appraised by using electronic circular dichroism and theoretical calculations. Notably, different thermodynamics of retention and enantioseparation were observed for molecules with pronounced structural similarity, that is, the enantiomer pairs of the compound containing the additional asymmetric carbon atom. Indeed, both entropically and enthalpically controlled adsorption and separation processes were observed.     

分子印迹技术在毛细管电色谱中的应用

分子印迹技术是制备具有分子识别功能聚合物，即分子印迹聚合物（MIPs)的一种新技术；毛细管电色谱（CEC）是一个具有发展前途的色谱新技术。将分子印迹技术和毛细管电色谱两种新技术相结合，优势互补，具有极大的发展潜力。本文对分子印迹技术在毛细管电色谱中的应用，以及各类MIPs-CEC毛细管柱的制备方法进行了较为全面的综述，引用文献52篇。     

The analysis of pharmaceutical bases on a silica stationary phase by capillary electrochromatography using aqueous mobile phases

Summary The capillary electrochromatographic (CEC) separation of a range of pharmaceutical bases was investigated on a commercially available silica stationary phase using aqueous mobile phases. The effects of mobile phase composition, buffer pH, applied voltage, and buffer anion on the retention behaviour of these bases were studied. Promising chromatography was obtained at pH 7.8 but was later found to be irreproducible. However, successful and reproducible chromatography of the bases was achieved at pH 2.3. We have previously demonstrated that the addition of mobile phase additives such as TEA-phosphate at low pH values has resulted in excellent CEC analysis of bases on reversed-phase packing materials. The same approach was applied to the analysis of bases on the silica phase in order to improve peak shape. Excellent chromatography was obtained for the analysis of strong pharmaceutical bases such as benzylamine, nortriptyline and diphenhydramine. The experimental investigations have shown that the CEC separation of a range of pharmaceutical bases can routinely be achieved with excellent peak shapes and peak efficiencies as high as 320,000 plates m⁻¹.     

3种金属有机骨架材料用于开管毛细管电色谱手性固定相分离外消旋体

金属有机骨架（MOFs）材料因具有丰富的拓扑结构、高比表面积、良好的稳定性、持久的孔结构以及可修饰的孔道表面等特点,在对映选择性催化和手性分离方面备受关注。该文通过水热法合成了3种具有手性的MOFs晶体,分别为Co₂（D-cam）₂（TMDPy）（简称为MOF 1）、[Cd（D-cam）（tmdpy）]·2H₂O（简称为MOF 2）和[Co_0.5Zn_0.5（L-Tyr）]_n（L-tyrCo/Zn）（简称为MOF 3）,并把它们用作固定相分别制成MOFs手性柱进行开管毛细管电色谱（OT-CEC）研究。在磷酸二氢钠-乙腈的流动相体系下,考察了3根MOFs手性柱对手性化合物的拆分性能。实验结果表明,这3种MOFs手性毛细管柱对部分外消旋体具有较好的拆分效果。目前将手性MOFs作为毛细管电色谱手性分离的研究正处在起步阶段并且具有良好的应用前景。     

Enantiomeric analysis of simendan on polysaccharide‐based stationary phases by polar organic solvent chromatography

Herein, the enantiomeric separation of simendan by high‐performance liquid chromatography with ultraviolet detection using polysaccharide‐based chiral stationary phases in polar organic mode is described. Three chiral columns (Chiralpak AD‐H, Chiralcel OD‐H, and Chiralpak AS) were screened using pure methanol and acetonitrile without additives under isocratic conditions. A reversed elution order was observed on the Chiralpak AD‐H column when the methanol content in the mobile phase (methanol–acetonitrile mixtures) was above 10%, whereby levosimendan eluted prior to dextrosimendan. Further, it was found that increasing temperature effectively improved the enantioresolution on the Chiralpak AD‐H column. Van't Hoff analysis was performed to evaluate the contribution of enthalpy and entropy to the chiral discrimination process. The best enantioseparation (α = 3.00, Rs = 12.85) was obtained on the Chiralpak AD‐H column with methanol as the mobile phase at 40°C. Thus, a quantitative method for the resolution of dextrosimendan was established and validated, which could be used as a reference for the determination of dextrosimendan in levosimendan products.     

Monolithic chiral stationary phases for liquid‐phase enantioseparation techniques

This short overview summarizes the development in the field of enantioselective monolithic chromatographic media and their application for pressure‐driven and electrokinetic separations. The major emphasis is put on the currently existing problems and the author's vision for their solution is provided. Due to the author's personal experience silica‐based monoliths are discussed in more detail although the key developments in the field of organic monolithic materials for separation of enantiomers are also discussed.     

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。     

Polymerised bicontinuous microemulsions as stationary phases for capillary electrochromatography

Summary Polymerisation of bicontinuous microemulsions yields porous monolithic structures with well defined pore sizes that are potentially suitable for use as stationary phases for capillary electrochromatography (CEC). A variety of pore sizes can be achieved by altering the composition of the microemulsion, which typically consists of butyl methacrylate (BMA) and ethylene glycol dimethacrylate (EGDMA) as the polymerisable oil phase. The aqueous phase consists of water, a surfactant (sodium dodecyl sulphate, SDS) and a co-surfactant (1-propanol). 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS) is also added to provide charges along the polymer backbone to allow electroosmotic flow (EOF) to occur. SEM analysis shows that in-situ polymerisation yields a monolithic structure with a porous topography. Investigations have shown that these monoliths are easy to prepare, robust and suitable for the separation of phthalates. They generate higher linear velocities than are achieved using the silica based HPLC packings normally used for CEC.     

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.     

β-环糊精键合手性固定相分离苯并恶嗪类对映体

研究了在反相高效液相色谱模式下,基于点击化学的β-环糊精手性固定相对苯并恶嗪类对映体的手性分离情况。考察了流动相中有机改性剂的类型和比例、缓冲盐的浓度和pH值对分离的影响。考察结果表明: 乙腈作为有机改性剂比甲醇更有利于苯并恶嗪对映体的分离;乙酸三乙胺缓冲盐体积分数从0.1%增大到1.0%时,苯并恶嗪对映体的保留时间和分离度都随之减小,在pH 4.1时苯并恶嗪对映体具有最大分离度。因此确定乙腈和体积分数为0.1%的乙酸三乙胺缓冲盐流动相(pH 4.1)为最佳分离条件。分离机理研究结果表明,固定相和样品之间的包容络合相互作用以及样品和固定相之间的氢键作用,是样品得以分离的基础。本研究为进一步深入研究β-环糊精固定相提供了实验基础,同时也证明了点击化学在手性环糊精固定相制备中具有极大潜力。     

Enantiomeric separation of amlodipine and its two chiral impurities by nano‐liquid chromatography and capillary electrochromatography using a chiral stationary phase based on cellulose tris(4‐chloro‐3‐methylphenylcarbamate)

In this work, a novel polysaccharide‐based chiral stationary phase, cellulose tris(4‐chloro‐3‐methylphenylcarbamate), also called Sepapak 4 has been evaluated for the chiral separation of amlodipine (AML) and its two impurities. AML is a powerful vasodilatator drug used for the treatment of hypertension. Capillary columns of 100 μm id packed with the chiral stationary phase were used for both nano‐LC and CEC experiments. The optimization of the mobile phase composed of ACN/water, (90:10, v/v) containing 15 mM ammonium borate pH 10.0 in nano‐LC allowed the chiral separation of AML and the two impurities, but not in a single run. With the purpose to obtain the separation of the three pairs of enantiomers simultaneously, CEC analyses were performed in the same conditions achieving better enantioresolution and higher separation efficiencies for each compound. To fully resolve the mixture of six enantiomers, parameters such as buffer pH and concentration sample injection have been then investigated. A mixture of ACN/water (90:10, v/v) containing 5 mM ammonium borate buffer pH 9.0 enabled the complete separation of the three couples of enantiomers in less than 30 min. The optimized CEC method was therefore validated and applied to the analysis of pharmaceutical formulation declared to contain only AML racemate.     

Enantioselective separation of twelve pairs of enantiomers on polysaccharide‐based chiral stationary phases and thermodynamic analysis of separation mechanism

The enantioseparation of twelve pairs of structurally related 1‐aryl‐1‐indanone derivatives was studied in the normal‐phase mode using three different polysaccharide‐type chiral stationary phases, namely Chiralpak IB, Chiralpak IC, and Chiralpak ID. n‐Hexane/2‐propanol and n‐hexane/ethanol were employed as mobile phases. Among all the investigated chiral columns, Chiralpak IC exhibited the most universal and the best enantioseparation ability toward all the racemates, particularly with the mobile phase composed of n‐hexane/2‐propanol (90/10, v/v). Then the effects of column temperature on retention and enantioselectivity were examined in the range of 25–40°C. Satisfactory enantioseparation was obtained at ambient temperature. The natural logarithm of retention and separation factors (ln k and ln α) versus the reciprocal of absolute temperature (1/T) (Van't Hoff plots) were found to be linear for all racemates, indicating that the retention and separation mechanisms were independent of temperature in the range investigated. Then, the thermodynamic parameters (ΔΔH°, ΔΔS°, and ΔΔG°) were calculated from Van't Hoff plots. These values indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. At last, the impact of small changes in molecular structures of the tested 1‐indanone derivatives on enantioseparation was also discussed.     

Triamine‐bonded stationary phase for open tubular capillary electrochromatography

A multi‐functional separation column modified with 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane was developed for open tubular capillary electrochromatography. This functional hydrophilic triamine‐bonded open tubular column could generate both anodic and cathodic EOF. When the pH of the running buffer was below 5.3 (30% 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane, v/v), the anodic EOF was exhibited, which greatly prevented the undesired adsorptions of basic proteins on the capillary inner wall. Favorable separation of four basic proteins (viz. trypsin, ribonuclease A, lysozyme and cytochrome c) was successfully achieved at pH 3.5 of 10 mmol/L phosphate buffer. The column efficiencies of proteins were in the range from 87 000 to 110 000 plates/m, and the RSD values for migration time of four proteins were less than 1.2% (run‐to‐run, n=5). The ionic analytes were also separated efficiently in the co‐electroosmotic mode. The average efficiencies ranged from 81 000 to 190 000 plates/m for seven aromatic acids and 186 000–245 000 plates/m for four nucleoside monophosphates, respectively, and good capillary column repeatability was gained with RSD of the migration time not more than 3.0%. The triamine‐bonded open tubular capillary column is favorable to be an alternative functional medium for the further analysis of basic proteins and anionic analytes.     

High‐performance liquid chromatography enantioseparation of polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases under multimodal elution

An investigation on the high‐performance liquid chromatography enantioseparation of 12 polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases is described. The overall study was directed toward the generation of efficient separations in order to obtain pure atropisomers that will serve as ligands for building homochiral metal organic frameworks. Four coated columns—namely, Lux Cellulose‐1, Lux Cellulose‐2, Lux Cellulose‐4, and Lux Amylose‐2—and two immobilized columns—namely, Chiralpak IC and IA—were used under normal, polar organic, and reversed‐phase elution modes. Moreover, Chiralcel OJ was considered under normal‐phase and polar organic conditions. The effect of the chiral selector and mobile phase composition on the enantioseparation, the enantiomer elution order and the beneficial effect of nonstandard solvents were studied. The effect of water in the mobile phase on the enantioselectivity and retention was investigated and retention profiles typical of hydrophilic interaction liquid chromatography were observed. Interesting phenomena of solvent‐induced enantiomer elution order reversal occurred under normal‐phase mode. All the considered 4,4′‐bipyridines were enantioseparated at the multimilligram level.     

Optimization of the HPLC enantioseparation of 3,3′‐dibromo‐5,5′‐disubstituted‐4,4′‐bipyridines using immobilized polysaccharide‐based chiral stationary phases

The HPLC enantioseparation of nine atropisomeric 3,3′,5,5′‐tetrasubstituted‐4,4′‐bipyridines was performed in normal and polar organic (PO) phase modes using two immobilized polysaccharide‐based chiral columns, namely, Chiralpak IA and Chiralpak IC. The separation of all racemic analytes, the effect of the chiral selector, and mobile phase (MP) composition on enantioseparation and the enantiomer elution order (EEO) were studied. The beneficial effect of nonstandard solvents, such as tetrahydrofuran (THF), dichloromethane (DCM), and methyl t‐butyl ether on enantioseparation was investigated. All selected 4,4′‐bipyridines were successfully enantioseparated on Chiralpak IA under normal or PO MPs with separation factors from 1.14 to 1.70 and resolutions from 1.3 to 6.5. Two bipyridines were enantioseparated at the multimilligram level on Chiralpak IA. Differently, Chiralpak IC was less versatile toward the considered class of compounds and only five bipyridines out of nine could be efficiently separated. In particular, on these columns, the ternary mixture n‐heptane/THF/DCM (90:5:5) as MP had a positive effect on enantioseparation. An interesting phenomenon of reversal of the EEO depending on the composition of the MP for the 3,3′‐dibromo‐5,5′‐bis‐(E)‐phenylethenyl‐4,4′‐bipyridine along with an exceptional enantioseparation for the 3,3′‐dibromo‐5,5′‐bis‐ferrocenylethynyl‐4,4′‐bipyridine (α = 8.33, R_s = 30.6) were observed on Chiralpak IC.     

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。     

Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Amylose tris(5-chloro-2-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSP) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the nature of silica, the particle size and pore diameter, the chiral selector loading onto silica, the mobile phase composition and pH, as well as separation variables such as a linear flow rate of the mobile phase, applied voltage in CEC, etc. on the separation of enantiomers was studied. It was found that CSPs based on amylose tris(5-chloro-2-methylphenylcarbamate) can be used for preparation of stable capillary columns for enantioseparations by nano-LC and CEC in combination with polar organic and aqueous–organic mobile phases. Higher peak efficiency was observed in CEC than in nano-LC.