Enantioseparation of vesamicol and novel vesamicol analogs by high-performance liquid chromatography on different chiral stationary phases

High-performance liquid chromatography enantioseparation of vesamicol and six novel azaspirovesamicols (amino alcohols) was accomplished on different chiral stationary phases (CSPs) by using an optical rotation based chiral detector for identification of the resolved enantiomers. The Pirkle-type column Reprosil Chiral-NR was found to be most suitable for chiral resolution in normal phase (NP) mode; all compounds could be enantioseparated successfully. Also the cellulose-based column Reprosil Chiral-OM showed appropriate separation properties by using NP conditions. The amylose-type column Reprosil Chiral-AM-RP was most suitable for enantioseparation in reversed phase (RP) mode; five out of seven compounds were resolved. This CSP showed a considerably higher capability for chiral recognition of vesamicol derivatives in RP mode than the corresponding cellulose-based column Reprosil Chiral-OM-RP. Enantioseparation with the teicoplanin aglycone-based column Reprosil Chiral-AA was successful under polar ionic mobile phase conditions.     

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.     

Enantioselective liquid-solid extraction for screening of structurally related chiral stationary phases

Summary An enantioselective liquid-solid batch extraction method is described for the screening of novel chiral stationary phases (CSPs) during optimization studies of chiral selectors derived form a common lead structure. Extraction enantioselectivity (α) values can be calculated from the enantiomeric excess ee-values of the selectand, which are measured in the liquid phase by enantioselective HPLC. Extraction α-values have been correlated with chromatographic α-values. The influence was studied of several experimental parameters of the assay (pH_a, buffer concentration, temperature, selector/selectand and phase ratio) on the enantiomeric excess (ee) values of the selectands and the enantioselectivity of the CSPs, respectively. The derived statistically significant model has then been implemented to predict chromatographic α-values of novel CSPs. For example, an ee of 89.3% for DNB-Leu as selectand could be achieved in batch extraction for a novel synthesized but mechanistically similarly-acting CSP derived form quinine. This corresponds to a calculated extraction α-value of 17.7. Based on this α_extraction a chromatographic α-value of 28.8 was predicted by the linear correlation model; the experimental HPLC α-value of 31.7 was in good agreement and demonstrated the validity of the proposed screening method. The method is particularly helpful in SO optimization studies.     

Analytical and semipreparative chiral separation of cis‐itraconazole on cellulose stationary phases by high‐performance liquid chromatography

cis‐Itraconazole is a chiral antifungal drug administered as a racemate. The knowledge of properties of individual cis‐itraconazole stereoisomers is vital information for medicine and biosciences as different stereoisomers of cis‐itraconazole may possess different affinity to certain biological pathways in the human body. For this purpose, either chiral synthesis of enantiomers or chiral separation of racemate can be used. This paper presents a two‐step high‐performance liquid chromatography approach for the semipreparative isolation of four stereoisomers (two enantiomeric pairs) of itraconazole using polysaccharide stationary phases and volatile organic mobile phases without additives in isocratic mode. The approach used involves the separation of the racemate into three fractions (i.e. two pure stereoisomers and one mixed fraction containing the remaining two stereoisomers) in the first run and consequent separation of the collected mixed fraction in the second one. For this purpose, combination of cellulose tris‐(4‐methylbenzoate) and cellulose tris‐(3,5‐dimehylphenylcarbamate) columns with complementary selectivity for cis‐itraconazole provided full separation of all four stereoisomers (with purity of each isomer > 97%). The stereoisomers were collected, their optical rotation determined and their identity confirmed based on the results of a previously published study. Pure separated stereoisomers are subjected to further biological studies.     

Cationic cyclodextrins chemically-bonded chiral stationary phases for high-performance liquid chromatography

Two covalently bonded cationic β-CD chiral stationary phases (CSPs) prepared by graft polymerization of 6^A-(3-vinylimidazolium)-6-deoxyperphenylcarbamate-β-cyclodextrin chloride or 6^A-(N,N-allylmethylammonium)-6-deoxyperphenylcarbamoyl-β-cyclodextrin chloride onto silica gel were successfully applied in high-performance liquid chromatography (HPLC). Their enantioseparation capability was examined with 12 racemic pharmaceuticals and 6 carboxylic acids. The results indicated that imidazolium-containing β-CD CSP afforded more favorable enantioseparations than that containing ammonium moiety under normal-phase HPLC. The cationic moiety on β-CD CSPs could form strong hydrogen bonding with analytes in normal-phase liquid chromatography (NPLC) to enhance the analytes’ retention and enantioseparations. In reversed-phase liquid chromatography (RPLC), the analytes exhibited their maximum retention when the pH of mobile phase was close to their pK_a value. Inclusion complexation with CD cavity and columbic/ionic interactions with cationic substituent on the CD rim would afford accentuated retention and enantioseparations of the analytes.     

Analytical and semipreparative resolution of ranolazine enantiomers by liquid chromatography using polysaccharide chiral stationary phases

Novel HPLC methods were developed for the analytical and semipreparative resolution of new antianginal drug ranolazine enantiomers. Good baseline enantioseparation was achieved using cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC) chiral stationary phases (CSPs) under both normal-phase and polar organic modes. The validation of the analytical methods including linearity, LODs, recovery, and precision, and the semipreparative resolution of ranolazine racemate were carried out using methanol as mobile phase without any basic and acidic additives under polar organic mode, using CDMPC CSPs. At analytical scale, the elution times of both enantiomers were less than 7.5 min at 20 degrees C and 1.0 mL/min, with the separation factor (a) 1.88 and the resolution factor (R(s)) 2.95. At semipreparative scale, about 14.3 mg/h enantiomers could be isolated and elution times of both enantiomers were less than 13 min at 2.0 mL/min. To increase the throughput, the technique of overlapping injections was used. The first eluted enantiomer was isolated with a purity of 99.6% enantiomer excess (e.e.) and > 99.0% yield. The second enantiomer was isolated with a purity of 98.8% e.e. and > 99.0% yield. In addition, optical rotation and circular dichroism spectroscopy of both ranolazine enantiomers isolated were also investigated.     

Supercritical fluid chromatography in chiral separations: Evaluation of equivalency of polysaccharide stationary phases

Nine different chiral columns based on covalently immobilized or coated tris(3,5‐dimethylphenylcarbamate) cellulose and amylose have been explored. We evaluated their respective enantioselective potential including the enantioseparation and qualitative characteristics of peaks. The generic screening conditions were using gradient elution from 5 to 40% organic modifier/CO₂ during 3 min with about 40 enantiomer pairs. Primary screening was carried out using ten different mobile phases varying in type of additives while using one representative amylose‐ and one cellulose‐based column. The complete evaluation of all nine columns was then carried out using three best performing organic modifiers: (1) methanol + 0.1% trifluoroacetic acid + 0.1% diethylamine, (2) isopropanol + 0.1% trifluoroacetic acid + 0.1% diethylamine, and (3) methanol + 0.1% ammonium hydroxide. Equivalency of different columns with the same chiral selector was not confirmed. Columns with the same stationary phase but different supports or manufacturing methods displayed differences in enantioselectivity and general performance. The similarity corresponded to 62 and 63% for the three cellulose‐coated columns taking CEL1 as the reference. The similarity was 67% for the pair of amylose‐based coated columns. For immobilized columns, the similarity was 69 and 59% for celluloses and amyloses pairs, respectively. The best performing column based on success rate of enantioseparation was Chiralcel OD‐3 when using methanol + 0.1% trifluoroacetic acid and 0.1% diethylamine combined additive.     

Enantiomer separation by gas chromatography on cyclodextrin chiral stationary phases

Fused silica capillary columns coated with several alkyl or acyl cyclodextrin derivatives, especially those of α- and β-cyclodextrins, are suitable for the enantiomer separation of a wide variety of volatile compounds of different molecular size and functionality. Positional isomers and more than 250 pairs of optical isomers have been resolved, including chiral hydrocarbons, acetals, ethers, epoxides, carbonates, lactones, esters, acids, ketones, aldehydes, alcohols, halocarbons, and also nitrogen-and sulfur-containing compounds. The physical properties of the cyclodextrin derivatives, even those obtained as viscous fluids, could be improved by dissolving them in polysiloxane liquid phases commonly used for GLC.     

新型直链淀粉类手性固定相的制备与手性拆分性能

通过对糖单元2-位进行选择性酯化以及6-位保护与去保护,运用区域选择性方法合成了5种新型直链淀粉类衍生物,分别为直链淀粉-2-苯甲酸酯-3-(4-氯苯基氨基甲酸酯)-6-(3,5-二甲基苯基氨基甲酸酯)、直链淀粉-2-苯甲酸酯-3-(4-氯苯基氨基甲酸酯)-6-(3,5-二氯苯基氨基甲酸酯)、直链淀粉-2-苯甲酸酯-3,6-二(4-氯苯基氨基甲酸酯)、直链淀粉-2-(4-氯苯甲酸酯)-3,6-二(3,5-二氯苯基氨基甲酸酯)和直链淀粉-2-(4-氯苯甲酸酯)-3,6-二(环己基氨基甲酸酯),并将其涂覆在氨丙基硅胶表面制备了HPLC手性固定相。利用核磁共振-氢谱(¹H-NMR)和傅里叶变换红外光谱(FT-IR)技术对所合成衍生物的结构进行了表征和分析,并用HPLC法评价所合成衍生物的手性识别能力。与具有单一取代基直链淀粉类手性固定相的对比分析表明,所合成的新型直链淀粉类手性固定相对于某些对映体具有更为优异的拆分结果。进一步分析表明,2-、3-和6-位取代基的性能和引入位置对直链淀粉衍生物的手性识别能力均有较大的影响。     

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.     

Evaluation of novel amylose and cellulose-based chiral stationary phases for the stereoisomer separation of flavanones by means of nano-liquid chromatography

Three polysaccharide-based chiral stationary phases, Sepapak^® 1, Sepapak^® 2 and Sepapak^® 3 have been evaluated in the present work for the stereoisomer separation of a group of 12 flavonoids including flavanones (flavanone, 4′-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, hesperetin, naringenin) and flavanone glycosides (hesperidin, naringin) by nano-liquid chromatography (nano-LC). The behaviour of these chiral stationary phases (CSPs) towards the selected compounds was studied in capillary columns (100 μm internal diameter (i.d.)) packed with the above mentioned CSPs using polar organic, reversed and normal elution modes. The influence of nature and composition of the mobile phase in terms of concentration and type of organic modifier, buffer type and water content (reversed phase elution mode) on the enantioresolution (R_s), retention factor (k) and enantioselectivity (α) was evaluated. Sepapak^® 3 showed the best chromatographic results in terms of enantioresolution, enantioselectivity and short analysis time, employing a polar organic phase mode. A mixture of methanol/isopropanol (20/80, v/v) as mobile phase enabled the chiral separation of eight flavanones with enantioresolution factor (R_s) in the range 1.15–4.18. The same analytes were also resolved employing reversed and normal phase modes with mixtures of methanol/water and hexane/ethanol at different ratios as mobile phases, respectively. Loss in resolution for some compounds, broaden peaks and longer analysis times were observed with these last two chromatographic elution modes.     

Novel cyclodextrin chiral stationary phases for high performance liquid chromatography enantioseparation: Effect of cyclodextrin type

Three novel chiral stationary phases (CSPs) were prepared by regioselective chemical immobilization of mono(6^A-N-allylamino-6^A-deoxy)perphenylcarbamoylated (PICD) α-, β-, and γ-cyclodextrins (CDs) onto silica support via hydrosilylation. Their enantioseparation properties in high performance liquid chromatography (HPLC) were evaluated with a large spectrum of racemates including flavanone compounds, β-adrenergic blockers, amines and non-protolytic compounds. The effect of CD's cavity size on enantioseparation abilities was studied and discussed. The results indicated that CD's surface loading at silica support played an important role in the enantioseparation on these CSPs under normal-phase conditions while inclusion phenomena contributed the major driving force under reverse-phase conditions. As expected, α-PICD demonstrated the best resolutions towards flavonone and most aromatic alcohols under normal-phase conditions with the highest surface loading; while Fujimura's competitive inclusion model can be applied to explain the better enantioseparations towards β-adrenergic blockers, amines and non-protolytic compounds with α- and β-PICD CSPs. γ-PICD CSP showed superior enantioseparation ability for sterically encumbered analytes like flavanone compounds under both normal-phase and reversed phase conditions.     

High-performance liquid chromatographic enantioseparation of methanobenzazocines

Chiral recognition and resolution of methanobenzazocines was investigated by HPLC using polysaccharide, Pirkle-type, native and derivatized β-cyclodextrin chiral stationary phases. Enantioseparation of phenyl substituted 2,6-methanobenzazocines was achieved with multiple chiral stationary phases throughout the classes described. Chiral resolution of the enantiomers of 1,5-methano-3-methyl-6-oxo-1,2,3,4,5,6-hexahydro-3-benzazocine was produced on both polysaccharide and Pirkle-type phases. In the case of 1,5-methano-3-methyl-6-phenyl-1,2,3,4,5,6-hexahydro-3-benzazocine only a dinitrophenyl substituted β-cyclodextrin produced a separation of enantiomers.     

Synthesis of novel chiral imidazolium stationary phases and their enantioseparation evaluation by high-performance liquid chromatography

Two novel chiral stationary phases (CSPs) were prepared by bonding chiral imidazoliums on the surface of silica gel. The chiral imidazoles were derivatized from chiral amines, 1-phenylethylamine and 1-(1-naphthyl)ethylamine. The obtained CSPs were characterized by Fourier Transform Infrared (FT-IR) spectroscopy and elemental analysis (EA), demonstrating the bonding densities of CSP 1 and CSP 2 were 0.43 mmol g⁻¹ and 0.40 mmol g⁻¹, respectively. These two CSPs could be used to availably separate 8 pharmaceuticals, 7 mandelic acid/its derivatives, 2 1-phenylethylamine derivatives, 1 1,1′-bi-2-naphthol, and 1 camphorsulfonic acid in high-performance liquid chromatography (HPLC). It is found that CSP 1 could effectively enantioseparate most chiral analytes, especially the acidic components, while CSP 2 could enantiorecognize all chiral analytes, although a number of components did not achieve baseline separation. Additionally, the effects of mobile phase composition, mobile phase pH and salt content, chiral selector structures, and analyte structures on the enantiorecognitions of the two CSPs were investigated. It is found that high acetonitrile content in mobile phases was conducive to enantiorecognition. Mobile phase pH and salt content could alter the retention behaviors of different enantiomers of the same chiral compound, resulting in better enantioresolution. Moreover, both chiral selector structures and substituted groups of analytes played a significant role in the separation of chiral solutes.     

Synthesis of novel chiral stationary phases for high-performance liquid chromatography

Three novel chiral selectors 4a-c were synthesized from(S)-amino acids and(R)-1-phenyl-2-(4-methylphenyl)ethylamine.4a-cwere connected to 3-aminopropylsilanized silica gel to be used as the chiral stationary phase for HPLC.Five amino acid derivativesand two pyrethroid insecticides were fairly resolved on these three new chiral stationary phases under normal phase condition.     

Liquid chromatography with mass spectrometry enantioseparation of pomalidomide on cyclodextrin‐bonded chiral stationary phases and the elucidation of the chiral recognition mechanisms by NMR spectroscopy and molecular modeling

A sensitive and validated liquid chromatography with mass spectrometry method was developed for the enantioseparation of the racemic mixture of pomalidomide, a novel, second‐generation immunomodulatory drug, using β‐cyclodextrin‐bonded stationary phases. Four cyclodextrin columns (β‐, hydroxypropyl‐β‐, carboxymethyl‐β‐, and sulfobutyl‐β‐cyclodextrin) were screened and the effects of eluent composition, flow rate, temperature, and organic modifier on enantioseparation were studied. Optimized parameters, offering baseline separation (resolution = 2.70 ± 0.02) were the following: β‐cyclodextrin stationary phase, thermostatted at 15°C, and mobile phase consisting of methanol/0.1% acetic acid 10:90 v/v, delivered with 0.8 mL/min flow rate. For the optimized parameter at multiple reaction monitoring mode 274.1–201.0 transition with 20 eV collision energy and 100 V fragmentor voltage the limit of detection and limit of quantitation were 0.75 and 2.00 ng/mL, respectively. Since enantiopure standards were not available, elution order was determined upon comparison of the circular dichroism signals of the separated pomalidomide enantiomers with that of enantiopure thalidomide. The mechanisms underlying the chiral discrimination between the enantiomers were also investigated. Pomalidomide‐β‐cyclodextrin inclusion complex was characterized using nuclear magnetic resonance spectroscopy and molecular modeling. The thermodynamic aspects of chiral separation were also studied.     

Use of crosslinked polysiloxane stationary phases in open-tubular capillary liquid chromatography

Crosslinked polysiloxane stationary phases were prepared on soda-lime glass capillaries and applied to the separation of polynuclear aromatic hydrocarbons and of phthalates in reversedphase liquid chromatography. Preparation procedures and chromatographic performance of these columns are described.     

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。     

Enantiomeric resolution of new aromatase inhibitors by liquid chromatography on cellulose chiral stationary phases

Analytical HPLC methods using derivatized cellulose chiral stationary phases were developed for the direct enantioseparation of substituted [1-(imidazo-1-yl)-1-phenylmethyl)]-benzothiazolinone and benzoxazolinone derivatives with one chiral center. Those analogues of fadrozole constitute new potent nonsteroidal inhibitors of aromatase (P450 arom). The separations were made using normal phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol, 1-propanol, or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ). The effects of concentration of various aliphatic alcohols in the mobile phase were studied. A better separation was achieved on cellulose carbamate phase compared with the cellulose ester phase. The effects of structural features of the solutes along with the temperature of the column on the discrimination between the enantiomers were examined. Baseline separation (Rs > 1.5) was easily obtained in many cases.