Chiral separation strategy in polar organic solvent chromatography and performance comparison with normal-phase liquid and supercritical-fluid chromatography

A strategy, including a rapid screening and several optimisation steps, for the separation of chiral molecules of pharmaceutical interest by polar organic solvent chromatography (POSC), using four polysaccharide-based stationary phases, is proposed and compared with previously reported strategies in normal-phase (NPLC) and supercritical fluid chromatography (SFC). In a first part of this paper, different examples demonstrate the effectiveness of the POSC strategy for fast method development. Optimisation is based on the use of experimental design to map the experimental domain in an efficient way. In the second part, the best screening results, obtained after performance of earlier defined chromatographic screening strategies in NPLC and SFC, are compared to those obtained in POSC. The three techniques show complementary separation results and allowed baseline separation of 23 of 25 compounds. POSC is found to be a very interesting separation mode compared to NPLC, because of the many fast (< 10 min) baseline separations obtained.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

Updating a chiral separation strategy for non-acidic drugs with capillary electrochromatography applicable for both chlorinated and non-chlorinated polysaccharide selectors

A generic strategy for the chiral separation of non-acidic pharmaceuticals was updated to complete an approach defined earlier. The selected chiral stationary phases are all polysaccharide selectors, chlorinated, and non-chlorinated, namely Lux(?) Amylose 2, Chiralcel(?) OD-RH, Lux(?) Cellulose 4, and Chiralpak(?) AD-RH. In this study, the screening step of a strategy defined earlier was updated and the optimization steps were re-evaluated for the applied chiral stationary phases. These screening and optimization conditions were studied by analyzing 20 pharmaceuticals at different organic modifier contents, temperatures, or applied voltages. The proposed chiral separation strategy was then evaluated with a test set of 19 non-acidic drugs. Seventeen compounds (89.5%) of the latter set could be resolved of which eight (42%) were baseline separated. The strategy thus proved to be applicable on compounds different from those used for its development.     

Screening approach for chiral separation of pharmaceuticals IV. Polar organic solvent chromatography

The aim of this work is to determine generic screening conditions and an initial simple separation strategy allowing the rapid separation of drug enantiomers in polar organic solvent chromatography (POSC). Four cellulose/amylose-based stationary phases were investigated in detail using two mobile phase basis solvents commonly applied in this mode, i.e. acetonitrile and methanol. Polar mode is interesting for use in purification of enantiomers. In a first step, the parameters potentially influencing the separation, such as addition of an alcohol to the polar organic solvent or the type of mobile phase additive(s), were examined by means of experimental designs. Afterwards, the factors found most important are investigated in more detail. Results showed that the cellulose- and amylose-based stationary phases have very broad and complementary enantiorecognition abilities in the POSC mode. The type of organic solvent for the mobile phase appeared to have a dramatic influence on the quality of the separation. Based on the results, a screening strategy was proposed. Enantioseparation was observed in more than 85% of the tested compounds and analysis times of last eluted peak were usually below 10 min.     

Cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia for chiral separation in high performance liquid chromatography.

Porous zirconia particles are very robust material and have received considerable attention as a stationary phase support for HPLC. We prepared cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia (CDMPCCZ) as a chiral stationary phase (CSP) for separation of enantiomers of a set of 14 racemic compounds in normal phase (NP) and reversed-phase (RP) liquid chromatography. Retention and enantioselectivity on CDMPCCZ were compared to those on CDMPC-coated zirconia (CDMPCZ) to see how the change in immobilization method of the chiral selector affects the retention and chiral selectivity. In NPLC, retention was longer and the number of resolved racemates was smaller on CDMPCCZ than on CDMPCZ. However, chiral selectivity factors for some resolved racemates were better on CDMPCCZ than on CDMPCZ. The longer retention on CDMPCCZ is likely due to strong, non-chiral discriminating interactions with the carbon layer on CDMPCZ. In RPLC only two racemates were resolved on CDMPCCZ, but retention times were shorter than, and resolutions were comparable to, those in NPLC, indicating a potential for improving chromatographic performance of the CDMPCCZ column in RPLC with optimized column preparation and separation conditions.     

Efficient preparative separation of β‐cypermethrin stereoisomers by supercritical fluid chromatography with a two‐step combined strategy

An efficient two‐step method has been developed for the separation of β‐cypermethrin stereoisomers by supercritical fluid chromatography with polysaccharide chiral stationary phases. With respect to retention, selectivity, and resolution of β‐cypermethrin, the effects of chiral stationary phases, cosolvents, mobile phases, and column temperature have been studied in detail. Through a two‐step separation, β‐cypermethrin was firstly separated by using a cellulose‐derived chiral stationary phase to obtain two stereoisomeric pairs, and further resolved on an amylose‐based chiral stationary phase to produce four enantiopure stereoisomers. The electronic circular dichroism patterns of the first‐ and the third‐eluted isomers in methanol solution showed the mirror image of each other in the wavelength range 200∼300 nm, indicating that they were a pair of enantiomers. Moreover, the second‐ and the fourth‐eluted isomers were also enantiomers. This proposed two‐step strategy showed low solvent consumption, fast separation speed, and high‐purity, which may provide an effective approach for preparative separation of compounds with multiple chiral centers and difficult‐to‐separate multicomponent samples.     

高效液相色谱法测定手性四面体金属簇合物对映体过量值

在自制的直链淀粉-三(苯基氨基甲酸酯)(ATPC)高效液相色谱手性固定相(HPLC—CSP)上，优化了手性四面体金属簇合物的手性分离条件，测定了不同合成条件下得到的手性四面体金属族合物CoMo(C0)5C5H4C(O)CH3(μη^2-HC≡CCH2OH)的对映体过量值(e.e)。结果表明：高效液相色谱手性固定相法是拆分这类化合物的一种理想方法。     

Enantioseparation tuned by solvent polarity on a β‐cyclodextrin clicked chiral stationary phase

The efficient enantioseparation of 26 racemates has been achieved with the perphenylcarbamoylated cyclodextrin clicked chiral stationary phase by screening the optimum composition of mobile phase in high‐performance liquid chromatography. The chromatographic results indicate that both the retention and chiral resolution of racemates are closely related to the polarity of the mobile phases and the structures of analytes. The addition of alcohols can significantly tune the enantioseparation in normal‐phase high‐performance liquid chromatography. The addition of methanol and the ratio of ethanol/methanol or isopropanol/methanol played a key role on the resolution of flavonoids in ternary eluent systems. The chiral separation of flavonoids with pure organic solvent as mobile phase indicates the preferential order for chiral resolution is methanol>ethanol>isopropanol>n‐propanol>acetonitrile.     

亲水作用色谱填料的研究进展

亲水作用色谱是一种新型的色谱分离模式.此类色谱模式集反相色谱的经济廉价与正相色谱的优点于一体,有效补充了反相色谱的不足.简单介绍实验室中合成的新型亲水色谱固定相.     

Definition and system implementation of strategies for method development of chiral separations in normal- or reversed-phase liquid chromatography using polysaccharide-based stationary phases

This paper proposes strategies in normal- and reversed-phase liquid chromatography (NP-HPLC or NPLC and RP-HPLC or RPLC), which were developed using three polysaccharide-based stationary phases. Those strategies are implemented in a knowledge-based system for the chiral separation of drug enantiomers. Each strategy includes a screening and an optimisation stage. The screening stage allows a fast evaluation of separation possibilities and enantioselectivity for many drugs in a short period of time, while the optimisation stage gives the opportunity to enhance, if needed, the initially obtained separation. Different examples demonstrate the effectiveness of the strategies for fast method development.     

Recent development trends for chiral stationary phases based on chitosan derivatives,cyclofructan derivatives and chiral porous materials in high performance liquid chromatography

The separation of enantiomers by chromatographic methods, such as gas chromatography, high‐performance liquid chromatography and capillary electrochromatography, has become an increasingly significant challenge over the past few decades due to the demand of pharmaceutical, agrochemical, and food analysis. Among these chromatographic resolution methods, high‐performance liquid chromatography based on chiral stationary phases has become the most popular and effective method used for the analytical and preparative separation of optically active compounds. This review mainly focuses on the recent development trends for novel chiral stationary phases based on chitosan derivatives, cyclofructan derivatives, and chiral porous materials that include metal‐organic frameworks and covalent organic frameworks in high‐performance liquid chromatography. The enantioseparation performance and chiral recognition mechanisms of these newly developed chiral selectors toward enantiomers are discussed in detail.     

Recent advances in gas chromatography for solid and liquid stationary phases containing metal ions

This review is devoted to the application of metal complexes as column packings and liquid stationary phases in gas chromatography. Particular attention is paid to the stationary phases with nitrogen-containing functional groups (e.g., amine and ketoimine) and β-diketonates on the modified silica surface. The review also concerns the results of the research on metallomesogenes and chiral stationary phases. The factors influencing the retention mechanism in complexation gas chromatography are discussed. Practical application of the metal chelate-containing chromatographic packings for analytical separation of organic substances is considered.     

Study on the Retention Behavior of Aromatic Carboxylic and Sulfonic acid on a New Anion Exchange Column

Ion chromatography (IC) has gradually developed into a preferred method for the determination of inorganic anions. And in recent years some low molecular aliphatic acid can be also separated in the ion exchange column with the development of stationary phase. But for the determination of aromatic ionic compounds there are some problems. The aromatic anions show enhanced retention due to interaction with the π electrons of the aromatic backbone. Although the addition of an organic modifier can alleviate the difficulty, it is not the ultimate solution. IonPac AS20 column was developed using a unique polymer bonding technology and its substrate coating is aliphatic backbone. The polymer is completely free of any π electron‐containing substituents in the AS20 column. In this paper, the retention behavior of aromatic carboxylic and sulfonic acid on two hydroxide‐selective columns, IonPac AS11‐HC, AS16, and the new column AS20 was also studied. The result showed that the retentions of ten compounds on three columns were different with each other because of their different column characteristics. Among them 4‐chlorobenzene sulfonic acid, 3,5‐dihydric benzoic acid and salicylic acid obviously exhibited the weakest retention on the IonPac AS20. It was showed that π‐π bond function between anion and stationary phases was weakened in AS20 column because its polymer was completely free of any π electron‐containing substituents. So in this paper the AS20 was selected as an analytical column to separate ten aromatic ionic compounds, fumaric acid with conjugate bond included. The retention behavior, separation of the ten compounds and effect of temperature on their retention in the anion‐exchange column AS20 (2 mm) were studied. The result showed that those compounds could be separated with each other when running in gradient program and the organic modifier was unnecessary during the separation. So it is showed that AS20 column can be used as a separating column because its polymer is completely free of any π electron‐containing substituents. Finally, the effect of temperature on the retention behavior in AS20 column was studied and it was showed that the retention of nine compounds exhibited endothermic behavior.     

An insight into the use of dimethylphenyl carbamate cyclofructan 7 chiral stationary phase in supercritical fluid chromatography: The basic comparison with HPLC

Cyclofructan‐based chiral stationary phases were previously shown as a promising possibility for separation of chiral compounds in high performance liquid chromatography. In this work retention and enantiodiscrimination properties of the 3,5‐dimethylphenyl carbamate cyclofructan 7 chiral stationary phase are described in supercritical fluid chromatography. The results obtained in both of the separation methods were compared. A set of compounds with axial or central chirality was used as analytes. The effect of mobile phase composition, that is, addition of different alcohol modifiers and/or trifluoroacetic acid to carbon dioxide, was examined in the supercritical system. Similarly, mobile phases composed of hexane modified with propan‐2‐ol and/or trifluoracetic acid were used in liquid chromatography. A linear free energy relationship model was utilized for characterization of interactions that are decisive for retention and separation in both techniques. Dispersion interactions showed similar negative values using both methods. The main contribution of hydrogen bond acidity was also comparable for both methods. The propensity to interact with n‐ and/or π‐electron pairs of solutes was significant only in the supercritical system.     

Enantioseparation of chiral sulfonates by liquid chromatography and subcritical fluid chromatography

Tert‐butylcarbamoyl‐quinine and ‐quinidine weak anion‐exchange chiral stationary phases (Chiralpak® QN‐AX and QD‐AX) have been applied for the separation of sodium β‐ketosulfonates, such as sodium chalconesulfonates and derivatives thereof. The influence of type and amount of co‐ and counterions on retention and enantioresolution was investigated using polar organic mobile phases. Both columns exhibited remarkable enantiodiscrimination properties for the investigated test solutes, in which the quinidine‐based column showed better enantioselectivity and slightly stronger retention for all analytes compared to the quinine‐derived chiral stationary phase. With an optimized mobile phase (MeOH, 50 mM HOAc, 25 mM NH₃), 12 of 13 chiral sulfonates could be baseline separated within 8 min using the quinidine‐derivatized column. Furthermore, subcritical fluid chromatography (SubFC) mode with a CO₂‐based mobile phase using a buffered methanolic modifier was compared to HPLC. Generally, SubFC exhibited slightly inferior enantioselectivities and lower elution power but also provided unique baseline resolution for one compound.     

N-苄氧甲酰基-α-氨基膦酸二苯酯有机磷化合物高效液相 色谱保留和手性识别 机理的研究

在正相条件下,首次对一系列N-苄氧甲酰基-α-氨基膦酸二苯酯化合物在环糊精类固定相CYCLOBONDISN和Pirkle型固定相SumichiralOA4700上实现了高效液相色谱手性折分,探索运用定量结构-对映异构体选择性保留关系的方法,将对映异构体的色谱保留和溶质分子描述参数相关性联系建立定量方程,结比研究了这二种不同类型的手性固定相对该系列有机磷化合物的色谱保留和手性识别机理,结果表明：对该系列化合物而言,Pirkle型手性因定相SumichiralOA4700的色谱手性折分能力明显优于在β-环糊精上衍生引入了额外的与前者类同的Pirkle型不对称中心的环糊精类固定相CYCLOBONDISN;环糊精类固定相CYCLOBONDISN在正相色谱条件下,包结机理不起主要作用,其作用方式更接近"Pirkle"型手性固定相;虽然二者具有类同的Pirkle型不对称中心,但是,手性识别机理差异显著,在CYCLOBONDISN手性固定相上,对N-苄氧甲酰基-α-氨基膦酸二苯酯化合物色谱折分贡献较大的是其logP和Angle参数相应的相互作用,环糊精提供的不对称性环境对手性识别有重要影响;而SumichiralOA4700对该系列化合物的手性识别与locD和TE相应的作用力相关。     

亲水作用色谱固定相及其在中药分离中的应用

亲水作用色谱(HILIC)作为一种分离极性化合物的液相色谱模式,近年来越来越受到关注和重视。一方面是因为强极性化合物的分离问题引起了各个研究领域的重视,如药物分析、代谢组学、蛋白质组学等研究领域都不同程度地涉及强极性化合物的分离问题;另一方面是由于HILIC具有流动相组成简单、分离效率较高、与质谱兼容以及反压较低等优势。固定相是HILIC发展和应用的基础,本文主要从固定相分子结构的角度对HILIC固定相的结构特征、保留特性以及应用概况等进行了综述。对传统正相色谱固定相用于HILIC以及专门设计的HILIC固定相进行了介绍,评述了各自的优缺点和应用概况;对近年来HILIC固定相在中药分离中的应用进行了介绍;并对HILIC固定相的发展进行了展望。     

β-环糊精类液相色谱固定相及其手性拆分应用研究进展

β-环糊精是以1,4-糖苷键首尾相连的环状低聚糖,具有手性识别功能,特别是衍生化的β-环糊精键合固定相,对手性化合物具有良好的拆分能力。本文介绍了β-环糊精的结构和性质,综述了各种衍生化的β-环糊精液相色谱键合固定相及其在手性化合物拆分中的应用。     

Evaluation of differences between Chiralpak IA and Chiralpak AD‐RH amylose‐based chiral stationary phases in reversed‐phase high‐performance liquid chromatography

Polysaccharide‐based chiral stationary phases can be used for the enantioselective separation of a wide range of structurally different compounds. These phases are available with chiral selectors coated or immobilized on silica gel support. The means of attachment of the chiral selector to the carrier can influence the separation performance of these stationary phases. This paper deals with evaluation of differences in the separation abilities of coated Chiralpak AD‐RH versus immobilized Chiralpak IA amylose‐based stationary phases in the reversed–phase mode of high–performance liquid chromatography. A set of chiral analytes was separated under acidic and basic conditions. Differences were observed in the enantioseparation potential of the tested phases. The linear‐free energy relationship and additional evaluation of ionic interactions were used to ascertain whether the interactions that participate in retention and enantioseparation are affected by the means of preparation of these phases. All the interactions covered by the linear‐free energy relationship were significant for the studied phases and their absolute values were almost always higher for the coated phase. Ionic interactions were found to be more important on the immobilized stationary phase but did not contribute to any improvement in the enantioselective separation performance.     

Chiral Separations in Normal‐phase Liquid Chromatography: Updating a Screening Strategy with a Chlorine‐containing Polysaccharide‐based Selector

The screening conditions of an existing column and mobile phase selection strategy for chiral compounds in normal‐phase high performance liquid chromatography (NP‐HPLC) were tested for their applicability on Chiralpak IC, which is a chiral stationary phase (CSP) made by immobilising cellulosic tris (3,5‐dichlorophenyl‐carbamate) on silica gel. In this study, the applicability of the optimization steps of the existing separation strategy was examined using 36 compounds representing the three possible resolution situations that occur after screening. The cumulative number of separated compounds is 27 (75.0 %), and the cumulative number of baseline separated compounds is 19 (52.8 %).