Analytical and semi-preparative HPLC enantioseparation of novel pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety using immobilized polysaccharide chiral stationary phases

The direct HPLC enantioseparation of a novel series of chiral pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety was performed on two immobilized polysaccharide chiral stationary phases (Chiralpak IA, Chiralpak IC) using n-hexane (n-Hex)/dichloromethane (DCM) mobile phase with 5% alcohol additive. Good baseline separation of the enantiomers was achieved using amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA) on analytical scale. The analytical method was further scaled up to semi-preparative loading to obtain small amounts of both the enantiomers of pyridazin-3(2H)-one derivative. The semi-preparative resolution of all compounds was successfully achieved with n-hexane/dichloromethane/ethanol (EtOH) as mobile phase using a semi-preparative Chiralpak IA column. The first fractions were isolated with purities of >99.9% (enantiomeric excess (e.e.), and the second fractions were obtained with purities of >98.2% (enantiomeric excess). The assignment of the absolute configuration was established for the F1 fraction of compound a-2 by single-crystal X-ray diffraction method.     

Study of the enantiomeric separation of the anticholinergic drugs on two immobilized polysaccharide‐based chiral stationary phases by HPLC and the possible chiral recognition mechanisms

In this work, the enantiomeric separation of ten anticholinergic drugs was first examined on two derivative polysaccharide chiral stationary phases (CSPs), i.e., Chiralpak ID and Chiralpak IA in the normal phase mode. Except for scopolamine hydrobromide, the remaining nine analytes could be completely separated with good resolutions using both columns under the optimized mobile phase conditions. And the enantiomeric discrimination ability of the studied CSPs towards nine analytes was in the order of Chiralpak ID > Chiralpak IA. The influences of organic modifier types, alcohol content, and base/acid additives on the enantiomeric separation were evaluated and optimized. According to the experimental results, the effect of the structures of analytes on enantiomeric separation was discussed. Additionally, the chiral recognition mechanisms were proposed based on the thermodynamic analysis of the experimental data.     

Comparative studies of immobilized polysaccharide derivatives chiral stationary phases for enantioseparation of furanocoumarins and dihydroflavones and discussion on chiral recognition mechanism

Enantiomeric separation of furanocoumarins and dihydroflavones compounds were systematically studied in the normal-phase mode using four different polysaccharide-type chiral stationary phases, namely, Chiralpak IA, Chiralpak IC, Chiralpak IG, and Chiralpak IK-3 by high-performance liquid chromatography. The effect of alcohol modifiers and alcohol content on enantiomeric separation was evaluated for the separation of furanocoumarins and dihydroflavones. All the eight compounds have achieved baseline separation with the resolutions ranging between 1.52 and 23.11. For a better insight into the enantiorecognition mechanisms, thermodynamic analysis was carried out. The mechanisms of chiral recognition have been discussed. Among four chiral columns, Chiralpak IG exhibited the most universal and the best enantioseparation ability toward furanocoumarins and dihydroflavones when used n-hexane-isopropanol and n-hexane-ethanol as mobile phase, respectively. The steric hindrance, hydrogen bonding, and π-π interaction played major roles in chiral recognition on Chiralpak IG. By comparing four chiral columns, this work systematically analyzed the separation methods of furanocoumarins and dihydroflavones for the first time and reported some active chiral ingredients of traditional Chinese medicine that have never been separated, which provided a further insight into the enantioseparation of furanocoumarins and dihydroflavones on chiral stationary phases.     

新型含苯并噻唑α-氨基膦酸酯类化合物在淀粉类手性固定相上的液相色谱分离

本文运用涂敷型(Chiralpak AD-H)和键合型(Chiralpak IA)两种淀粉类手性固定相高效液相色谱法,进行了新型含苯并噻唑α-氨基膦酸酯类化合物的手性分离。从色谱分离的保留因子(k)、分离系数(α)和分离度(Rs)三个方面考察了两种类型色谱柱的分离性能,上述化合物在Chiralpak IA柱上能够得到较好的基线分离。同时,讨论了温度、流动相极性和目标分析物的结构等因素对Chiralpak IA柱分离性能的影响。由于键合型固定相较稳定的性能,使某些非常规的溶剂(如THF)成功地应用于手性α-氨基膦酸酯类化合物的分离。     

Immobilized versus coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases for the enantioselective separation of cyclopropane derivatives by liquid chromatography

The solvent versatility of Chiralpak IA, a new chiral stationary phase (CSP) containing amylose tris(3,5-dimethylphenylcarabamate) immobilized onto silica gel, is investigated for the enantioselective separation of a set of cyclopropane derivatives using ethyl acetate or dichloromethane (DCM) as non-standard mobile phase eluent and diluent, respectively in high-performance liquid chromatography (HPLC). A comparison of the separation of cyclopropanes on both immobilized and coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) in HPLC using a mixture of n-hexane/2-propanol (90/10 and 99/1, v/v) as mobile phase with a flow rate of 0.5 ml/min and UV detection at 254 nm, is demonstrated. The optimized method of separation is used for an online HPLC monitoring for the Rh(II)-catalyzed asymmetric intermolecular cyclopropanations in dichloromethane. Direct analysis techniques without further purification, workup or removal of dichloromethane were summarized. The method provides an easy and direct determination of the enantiomeric excess of the cyclopropanes and selectivity of the catalyst used without any further work up.     

Comparative studies of immobilized chiral stationary phases based on polysaccharide derivatives for enantiomeric separation of 15 azole compounds

Immobilized polysaccharide‐based columns showed excellent enantioselectivity in normal phase separation mode. In this work, enantioseparation abilities of four immobilized polysaccharide‐derived chiral stationary phases (Chiralpak IA, Chiralpak IB, Chiralpak IC, and Chiralpak ID) toward 15 azole compounds were evaluated. Separation was carried out using n‐hexane as mobile phase with ethanol, 1‐propanol, 1‐butanol, and 2‐propanol as modifiers. And twelve compounds have achieved baseline separation with the resolutions ranging between 2.05 and 21.73. The enantioseparation on the four polysaccharide‐based chiral columns using different alcohol modifiers was compared. In general, the best separation performance was identified as Chiralpak IC, which was able to resolve 11 compounds to baseline and two partially under the screening conditions. Separation on Chiralpak IB was not satisfactory, because only four compounds were baseline separated.     

Application and comparison of immobilized and coated amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases for the enantioselective separation of beta-blockers enantiomers by liquid chromatography

A direct liquid chromatographic enantioselective separation of a set of β-blocker enantiomers on the new immobilized and conventional coated amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) was studied using methanol as mobile phase and ethanolamine as an organic modifier (100:0.1, v/v). The separation, retention and elution order of the enantiomers on both columns under the same conditions were compared. The effect of the immobilization of the amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA) on the chiral recognition ability was noted when compared to the coated phase (Chiralpak AD) which possesses a higher resolving power than the immobilized one (Chiralpak IA). A few racemates, which were not or poorly resolved on the immobilized Chiralpak IA were most efficiently resolved on the coated Chiralpak AD. However, the immobilized phase withstand solvents like dichloromethane when used as an eluent or as a dissolving agent for the analyte. The versatility of the immobilized Chiralpak IA in monitoring reactions performed in dichloromethane using direct analysis techniques without further purification, workup or removal of dichloromethane was studied on a representative example consisting of the lipase-catalyzed irreversible transesterification of a β-blocker using either vinylacetate or isopropenyl acetate as acyl donor in dichloromethane as organic solvent.     

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.     

多糖类手性固定相超临界流体色谱法分离手性化合物

采用超临界流体色谱法(SFC),在多糖固定相Chiralpak IA、IB、IC、ID、IE和IF上成功拆分了11种手性化合物。分离结果表明,这6支手性色谱柱对这些手性化合物具有良好的手性识别互补性,均可以在10 min之内得到良好的分离结果,具有较好的实用性。改性剂甲醇、乙醇和异丙醇对手性化合物的保留时间以及手性选择性均具有良好的调节作用,需要根据不同手性物质在手性柱上的分离情况加以区别,选择使用,并调节改性剂至合适的比例。针对键合型固定相溶剂通用性的特征,特殊改性剂的应用也有助于优化手性分离。     

在多糖衍生物类色谱柱上手性拆分β-氨基醇及β-羟基 硫醚类化合物

在以正己烷-异丙醇为移动相的体系中,用ChiralcelOD,ChiralcelOJ及ChiralpakAD作为手性固定相对13种β-氨基醇及β-羟基硫醚类化合物对映体进行HPLC手性拆分,这些化合物至少能在一支柱上得到基线级分离。考察了它们于不同浓度配比的这类洗脱体系中在柱上的色谱行为。实验表明化合物取代基的性质明显影响它们在手性柱上的拆分。手性固定相与外消旋样品上的极性基团之间的氢键作用和π-π作用可能是进行手性识别的主要原因。方法已用于非手性环氧化合物不对称开环反应产物β-氨基醇及β-羟基硫醚类化合物的光学纯度鉴定。     

Characterization of Column Hold-Up Volume with Static and Dynamic Methods on an Immobilized Polysaccharide-Based Chiral Stationary Phase

Polysaccharide-based chiral stationary phases (CSPs) are efficient for enantioseparation of many chiral compounds. Immobilized polysaccharide CSP, as used in the Chiralpak IA column, is a new configuration that was recently introduced for application in chiral separation. As shown in several previous studies, the characteristics of Chiralpak IA columns cannot be simply extrapolated from the coated version. In this study, hold-up volume of a Chiralpak IA column was evaluated by static and dynamic methods. The static pyconometry method gave similar hold-up volumes either as an average value from a range of solvents or a direct measurement from the carbon tetrachloride-isopropanol (IPA) solvent pair. The dynamic method with 1,3,5-tri-tert-butylbenzene (TTBB) was influenced by the ratio of n-hexane and 2-propanol in the mobile phase but not by the dissolving solvent of TTBB. The two methods resulted in the same hold-up volume of ∼3.0 mL. TTBB showed weaker retention on the IA column after correction of isobaric thermal expansion of the mobile phase. During temperature variations in the range of 15–50 °C, the hold-up volume of TTBB was highly reproducible. Results of this study improve our understanding of the chromatographic features of the immobilized polysaccharide IA column.     

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.     

多糖衍生物手性固定相上采用酸性或碱性添加剂的流动相拆分β受体阻滞剂对映体

考察了多糖类手性固定相在含有酸性或碱性添加剂的流动相下高效液相色谱法拆分β受体阻滞剂对映体的效果。色谱条件: 流动相为10%～30%(体积分数,下同)乙醇-正己烷(含0.1%三氟乙酸)和10%～30%乙醇-正己烷(含0.1%三乙胺),流速1.0 mL/min,紫外检测波长254 nm。结果表明,在直链淀粉-三(3,5-二甲基苯基氨基甲酸酯)衍生物手性固定相(Chiralpak AD和Chiralpak IA)上拆分β受体阻滞剂对映体,酸性添加剂的流动相体系与碱性添加剂的流动相体系相比,碱性添加剂的流动相的拆分效果比酸性添加剂的流动相要好。而在纤维素-三(3,5-二甲基苯基氨基甲酸酯)衍生物的手性固定相(Chiralcel OD和Chiralpak IB)上分离β受体阻滞剂,比较酸性添加剂的流动相与碱性添加剂的流动相的拆分效果,发现酸性添加剂的流动相条件下对映体的保留减弱,但对映体的选择性增大,特别是在Chiralcel OD上,酸性添加剂的流动相体系对对映体的选择性非常理想,而且随着流动相中酸性添加剂含量的增加,β受体阻滞剂对映体的分离效果更佳。     

Study of the enantiomeric separation of an acetamide intermediate by using supercritical fluid chromatography and several polysaccharide based chiral stationary phases

Four chiral stationary phases, based on the phenylcarbamate derivatives of amylose or cellulose: Chiralcel OD-H, Chiralpak AD, Lux Cellulose-2 and Lux Amylose-2, were evaluated for the enantiomeric separation of an acetamide chiral intermediate, the (4S-trans)-4-(ethylamino)-4-(N-acetamide)-5,6-dihydro-(6S)-methyl-4H-thieno-[2,3-b]thiopyran-7,7-dioxide, using SFC. The effect of the different modifiers and temperatures, on the separation, was also studied. The chiral separation could not be achieved using the Chiralpak AD column, nevertheless the other columns provided excellent results with analysis times close to 6 min and resolutions higher than 2. The highest enantioresolutions and retentions were obtained with the Lux Cellulose-2 column and 2-propanol as organic modifier. The isoelution temperatures were estimated from the van't Hoff plots, and in all the cases they were above the temperature range studied which means that the enantiomeric separation was enthalpy driven.     

Chiral Separation of Organic Phosphonate Compounds on Polysaccharide-Based Chiral Stationary Phases

Four polysaccharide-based chiral stationary phases have been used to separate the enantiomers of fourteen O,O-dialkyl-1-benzyloxycarbonyl-aminoarylmethyl phosphonates. These polysaccharide-based chiral stationary phases are Chiralpak AD, Chiralpak AS, Chiralcel OG and Chiralcel OJ. The data obtained indicate that the chiral separation ability for these organophosphonate compounds are in the order Chiralpak AD > Chiralcel OG > Chiralcel OJ > Chiralpak AS. With Chiralpak AD, all of the studied compounds could be easily baseline separated. Those two polysaccharides possess different chiral discrimination mechanism due to of the difference of the conformational structures of amylose and cellulose. The chiral discrimination of derivatized amylose chiral stationary phases were based on the stereogenic fit of the analytes in the helical structures of amylose and the transient diastereomeric complex formation between the analyte and the amylose CSP through π–π interaction H-bond interactions and induced dipole interactions exerted by the substituents on the analyte molecules. The chiral discrimination, in case of derivatized cellulose chiral stationary phase is based on the stereogenic fit of the analytes in the grooves of cellulose followed by interactions mentioned above between the analytes and the cellulose CSP.     

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。     

Analytical and semipreparative high performance liquid chromatography enantioseparation of new substituted 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyrazoles on polysaccharide-based chiral stationary phases in normal-phase, polar organic and reversed-phase conditions

The direct HPLC enantioseparation of five pairs of new chiral pyrazole derivatives on coated cellulose- and amylose-based chiral stationary phases (Chiralpak AD, Chiralcel OJ and Chiralcel OJ-RH) and new immobilised amylose-based Chiralpak IA CSP was performed. Very high enantioselectivity factor (alpha) values were achieved in polar organic and reversed-phase conditions by using OJ-RH as CSP. Chiralpak IA exhibited an excellent chiral resolving ability in normal-phase mode and it allowed the enantioseparation of analytes investigated with resolution factors (Rs) >20. Due to its bonded nature, it was successfully employed at analytical and semipreparative scale in combination with normal-phase eluents containing "non-standards" solvents such as acetone.     

高效液相色谱法手性分离二茂铁衍生物

建立了4个单手性和3个双手性(含有手性中心和面手性)的二茂铁衍生物在Chiralpak IC(纤维素-三(3,5-二氯苯基氨基甲酸酯))和Chiralpak IE3(直链淀粉-三(3,5-二氯苯基氨基甲酸酯))手性固定相上的高效液相色谱分离方法。4个单手性二茂铁衍生物中有3个可以在Chiralpak IE3固定相上实现基线分离,另外1个则在Chiralpak IC手性固定相上实现基线分离。3个双手性二茂铁衍生物可在Chiralpak IC手性固定相上实现基线分离。研究表明,这两种手性固定相对二茂铁衍生物具有较好的手性识别作用,并且具有互补作用。这一研究结果可为手性二茂铁化合物的分离提供借鉴和参考。     

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.     

在多糖衍生物类色谱柱上手性拆分乙炔基氮杂环丙烷类化合物

在以正己烷－异丙醇为移动相的体系中，用ＣｈｉｒａｌｐａｋＡＤ和ＣｈｉｒａｌｃｅｌＯＤ作为手性固定相对１５种乙炔基氮杂环丙烷类化合物对映体进行了ＨＰＬＣ手性拆分。这些化合物至少在一支柱上能基线级分离。