Comparison of core-shell versus fully porous particle packings for chiral liquid chromatography productivity

A loading and productivity study was done using three racemates on vancomycin and teicoplanin-bonded chiral stationary phases of different particle formats. Two columns were packed with 2.7 μm superficially porous particles and two columns were packed with identically bonded 5 μm fully porous particles. The last two columns were packed with specially synthesized 4.5 μm vancomycin and teicoplanin superficially porous particles. The loading of different chiral compounds showed that the columns filled with 2.7-μm chiral stationary phases were inappropriate for preparative separations due to their very low permeability which precluded high flow rates. However, columns containing 4.5 μm superficially porous (core-shell) particles were as effective for small-scale preparative chiral separations as columns filled with classical 5 μm fully porous particles. Comparing the 4.5 μm superficially porous particles and 5 μm fully porous particles teicoplanin columns, the observed respective productivities of 270 and 265 mg/g chiral phase/h for 5-methyl-5-phenyl hydantoin enantiomers were obtained. Particular attention was given to the peculiar case of the mianserin enantiomeric separation on vancomycin columns that gave observed productivities of 200 and 205 mg/g chiral phase/h on the 4.5 μm superficially porous particles and 5 μm fully porous particles, respectively.     

Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

Enantioselective Liquid Chromatographic Separations Using Macrocyclic Glycopeptide-Based Chiral Selectors

Numerous chemical compounds of high practical importance, such as drugs, fertilizers, and food additives are being commercialized as racemic mixtures, although in most cases only one of the isomers possesses the desirable properties. As our understanding of the biological actions of chiral compounds has improved, the investigation of the pharmacological and toxicological properties has become more and more important. Chirality has become a major issue in the pharmaceutical industry; therefore, there is a continuous demand to extend the available analytical methods for enantiomeric separations and enhance their efficiency. Direct liquid chromatography methods based on the application of chiral stationary phases have become a very sophisticated field of enantiomeric separations by now. Hundreds of chiral stationary phases have been commercialized so far. Among these, macrocyclic glycopeptide-based chiral selectors have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance. This review focuses on direct liquid chromatography-based enantiomer separations, applying macrocyclic glycopeptide-based chiral selectors. Special attention is paid to the characterization of the physico-chemical properties of these macrocyclic glycopeptide antibiotics providing detailed information on their applications published recently.     

High efficiency,narrow particle size distribution,sub-2 μm based macrocyclic glycopeptide chiral stationary phases in HPLC and SFC

State of the art chiral chromatography still employs 3–5 μm bonded or immobilized chiral selectors in 10–25 cm columns. With the availability of 1.9 μm narrow particle size distribution (NPSD) silica, it is now possible to make ever shorter, high efficiency columns practical for sub-minute chiral separations. Three macrocyclic glycopeptides (teicoplanin, teicoplanin aglycone, and vancomycin) were bonded onto 1.9 μm NPSD particles. Such packed columns had ∼80% lower backpressure as compared to polydisperse (PD) 1.7 μm silica materials when using the same mobile phase. The decreased backpressure allowed for diminution of frictional heating and allowed for the use of the 1.9 μm NPSD particle based columns at high flow rates. The 1.9 μm NPSD particle based columns showed up to 190,000 plates m⁻¹ for chiral molecules and 210,000 plates m⁻¹ for achiral probes. Representative enantiomeric separations are shown for wide classes of compounds, including different types of amino acids, β-blockers, and pharmaceutically important heterocyclic compounds such as oxazolidinones. Applications in three liquid chromatography modes, namely, reversed phase, polar organic mode and normal phase chiral separations were shown with resolution values ranging from 1.5 to 5.7. Additionally, the same columns were used with supercritical fluid chromatography (SFC) for ultrafast separations.     

Enantiomeric impurities in chiral catalysts,auxiliaries, and synthons used in enantioselective syntheses. Part 4

The enantiomeric purity of chiral reagents used in asymmetric syntheses directly affects the apparent reaction selectivity and the product’s enantiomeric excess. Herein, 46 recently available chiral compounds were evaluated in order to determine their actual enantiomeric compositions. They have not been assayed previously and/or have been introduced after 2006, when the last comprehensive evaluation of commercially available chiral compounds was reported. These compounds are widely used in asymmetric syntheses as chiral synthons, catalysts, and auxiliaries. The enantioselective analysis methods include HPLC approaches using Chirobiotic, Cyclobond and LARIHC series chiral stationary phases, and GC approaches using Chiraldex chiral stationary phases. Accurate, efficient assays for selected compounds are given. All enantiomeric test results were categorized within five impurity levels (i.e., <0.01%, 0.01–0.1%, 0.1–1%, 1–10% and >10%). Different batches of the same reagent from the same company can have different levels of enantiomeric impurities. Many of the reagents tested were found to have less than 0.1% enantiomeric impurities. Only one of the chiral compounds was found to have an enantiomeric impurity exceeding 10%.     

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro‐liquid chromatography system. Fully porous, core–shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub‐2 μm fully porous as well as the 2.7 μm core–shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56–2.69) before correction for extra‐column contribution compared to normal‐bore columns. Moreover, the influence of extra‐column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub‐2 μm fully porous particle packed column for ultra‐fast liquid chromatography.     

Evaluation of ethoxynonafluorobutane as a safe and environmentally friendly solvent for chiral normal-phase LC-atmospheric pressure chemical ionization/electrospray ionization-mass spectrometry

Coupling normal-phase LC separation methods to atmospheric pressure ionization (API)-mass spectrometry (MS) for detection can be problematic because of the possible detonation hazard and because nonpolar solvents do not support ionization of the analyte. Unlike achiral separations, enantiomeric separations can be very sensitive to small changes in the separation environment. Thus, completely substituting the main mobile phase component of a normal-phase LC solvent for an environmentally friendly, nonflammable fluorocarbon-ether as a safe and effective solvent must be thoroughly evaluated before it can be recommended for enantioselective separations with API-MS detection. Ethoxynonafluorobutane (ENFB) was used as a normal-phase solvent for the enantioselective separation of 15 compounds on two macrocyclic glycopeptide chiral stationary phases (CSPs) and a new polymeric chiral stationary phase. The chromatographic figures of merit were compared between results obtained with the ENFB mobile phases and traditional heptane-based mobile phases. In addition, the limits of detection (LOD) using the API-MS compatible ENFB were examined, as well as flow rate sensitivities and compatibilities with common polar organic modifier. ENFB is a safe and effective solvent for enantioselective normal-phase/API-MS analyses.     

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.     

Thin-layer chromatography enantioseparations on chiral stationary phases: a review

The current state of chiral separations by thin-layer chromatography using chiral stationary phases is reviewed. Both stationary phases essentially constituted by the chiral selector and those obtained by the impregnation of achiral plates with appropriate chiral selectors are described. Particular attention is paid to commercial and non-commercial cellulose and cellulose-derivative plates, as well as commercially available Chiralplate?, which are currently the most widely used. Some of the most important results obtained to date are reported and discussed; the examples provided illustrate the very wide range of structurally different solutes that can be readily resolved into their enantiomers by planar chromatographic methods. Special attention is paid to the discussion of the retention and resolution factors that influence chiral discrimination. The quantitative analysis of enantiomers is also discussed, especially from the point of view of determination of enantiomeric purity.     

Evaluation and Comparison of a 3,5‐Dimethylphenyl Isocyanate Teicoplanin with Phenyl Isocyanate Teicoplanin Chiral Stationary Phase Using RP‐HPLC

HPLC enantiomeric separations of 8 α‐amino acids were achieved using two self‐made chiral stationary phases (CSP)–phenyl isocyanate teicoplanin (Phe‐TE) and 3,5‐dimethylphenyl isocyanate teicoplanin (DMP‐TE), using reversed phase mobile phases. The Phe‐TE or the DMP‐TE CSP was prepared from the TE using derivative agents, phenyl isocyanate or 3,5‐dimethylphenyl isocyanate, respectively. The chromatographic results were given as the retention, selectivity, resolution factor and the enantioselective free energy difference corresponding to the separation of the two enantiomers. The effect of pH, organic modifier type and amount were discussed, and the stereoselectivities for two TE‐based CSPs were compared. The chiral selectivity factor for six α‐amino acids on DMP‐TE is somewhat bigger than that on Phe‐TE CSP under reversed phase (RP) mode. Comparison of the enantiomeric separations using self‐made Phe‐TE and DMP‐TE was conducted to gain a better understanding of the chiral recognition mechanism of the macrocyclic glycopeptide CSP.     

Enantiomeric separation of racemic sulphoxides on chiral stationary phases by gas and liquid chromatography

Summary The enantiomeric resolution of seven racemic sulphoxides on chiral stationary phases has been investigated by gas and liquid chromatography. In gas chromatography the separations were performed on octakis-(2,6-di-O-pentyl-3-O-butyryl)-γ-cyclodextrin (FS Lipodex-E) and heptakis-(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin (DMP-β-CD). Both stationary phases were suitable for separation of the enantiomers of the sulphoxides. With one exception for each series all racemetes could be resolved on both stationary phases; FS Lipodex-E was more enantioselective than DMP-β-CD, whereas the latter seemed more generally applicable. Liquid chromatographic separations with Chiralcel-OB as stationary phase were significantly improved by optimization of mobile phase composition and temperature. Resolution factors up to R_s=6 were achieved indicating that the improved separations could now be easily used for preparative purposes.     

Recent accomplishments in the field of enantiomer separation by CEC

The present review intends to summarize recent developments in the field of enantioselective separations and analysis by CEC. It covers studies published in English language in common peer-reviewed journals within the period between 2003 and 2006. Both, methods making use of chiral mobile phase additives as well as chiral stationary phases for electrochromatographic enantiomer separations, are reviewed. Achievements that have been made on the various column technologies, such as open-tubular, particle-packed, inorganic, organic and particle-fixed (hybrid-type) monolithic as well as molecularly imprinted polymer phases, are discussed.     

硅胶基质高效液相色谱填料研究进展

高效液相色谱(HPLC)不仅是一种有效的分析分离手段,也是一种重要的高效制备分离技术。色谱柱是HPLC系统的核心,不同性能的填料是HPLC广泛应用的基础。硅胶是开发最早、研究最为深入、应用最为广泛的HPLC固定相基质,其制备方法主要有喷雾干燥法、溶胶-凝胶法、聚合诱导胶体凝聚法及模板法等。近年来,亚2μm小粒径硅胶、核-壳型硅胶、双孔径硅胶、介孔性硅胶、有机杂化硅胶等新型硅胶应用于HPLC并取得了色谱分离技术的飞速发展,例如基于亚2μm填料的超高压液相色谱技术、基于核-壳型填料的快速分离技术、基于杂化硅胶填料的高温液相色谱技术等。硅胶经表面化学键合、聚合物包覆等有机改性可制得先进的大分子限进填料、温敏性填料、手性填料等,大大扩展了HPLC的应用范围。本文对液相色谱用硅胶的制备方法、改性与修饰方法以及硅胶基质固定相的评价方法加以系统综述,概述了新型硅胶在HPLC中的应用进展,并对硅胶基质填料的发展方向与应用前景进行了展望。     

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.     

HPLC semi‐preparative separation of diclazuril enantiomers and racemization in solution

Diclazuril has been widely used in poultry feed for prevention and treatment of coccidiosis, and its chiral separation is rarely reported. Herein, semi‐preparative separation method of diclazuril enantiomers has been developed through normal‐phase high‐performance liquid chromatography. Effects of chiral stationary phases, alcoholic modifiers, and column temperature on separation of diclazuril were discussed in detail. Both the single‐urea‐bound 4‐chlorophenylcarbamoylated β‐cyclodextrin and amylose tris(3,5‐dimethylphenylcarbamate)‐coated chiral stationary phases showed strong ability in separation of diclazuril by using n‐hexane–trifluoroacetic acid–ethanol. Then, semi‐preparative separation of diclazuril was carried out through stacked injection, and the "enantiomeric excess" purities of two fractions were over 98%. Next, the electronic circular dichroism profiles of these two fractions in ethanol solution displayed the mirror image of each other in the range 360–200 nm. Moreover, effects of acidic/basic additive, time, and temperature on racemization of diclazuril enantiomers in ethanol solution have been studied in detail through normal‐phase high‐performance liquid chromatography. Racemization of diclazuril enantiomers was remarkably accelerated through adding triethylamine at high temperature. We envision that this systematic investigation of diclazuril at an enantiomeric level would provide valuable information in future studies involving enantioselective bioactive, metabolic, and toxicological activities.     

Gas Chromatographic Separation of Enantiomers on Cyclodextrin Derivatives

In investigations concerned with the phenomenon of molecular chirality, the use of gas chromatography for the enantiomeric analysis of stable, volatile compounds is a technique of steadily growing importance. ^[1] In the last three years an important breakthrough in gas-chro-matographic separation of enantiomers has been achieved by using alkylated cyclodextrins (α, β, and γ) as chiral stationary phases in high-resolution capillary columns. In academic and commercial practice two different and complementary strategies have been adopted up to now. In the first, alkylated cyclodextrins are diluted with polysiloxanes and coated on glass or fused silica capillary columns. In the second, lipophilic per-n-pentylcyclodextrins and hydrophilic di-n-pentyl- and hydroxyalkylpermethylcyclodextrins are coated directly in the form of liquid phases onto suitably pretreated glass or fused silica surfaces. These techniques permit enantiomer separations not only for polar diols and alcohols, derivatized hydroxycarboxylic acids, amino acids, sugars, and alkyl halides, but also for nonpolar alkenes, cyclic saturated hydrocarbons, and metal π complexes. An important aspect for practical applications is that in many cases the enantiomers can be separated without previous derivatization. Whereas the resolution of racemates of unfunctionalized hydrocarbons is attributed to an enantioselective host–guest inclusion complex, some observations indicate that for polar guest molecules additional enantioselective interactions are also involved. The new chiral stationary phases can be used over a wide range of temperatures (25 to 250°C). The technique described is likely to become widely adopted as a simple, accurate and highly sensitive method for the enantiomeric analysis of chiral compounds that can be vaporized without decomposition. It will also stimulate future research aimed at finding universal cyclodextrin phases and elucidating the mechanisms of enantioselectivity.     

Enantiomeric Resolution of a Chiral Sulfoxide Series by LC on Synthetic Polymeric Columns with Multimodal Elution

The liquid chromatography enantiomeric separation of a series of 17 chiral sulfoxides was systematically investigated using multimodal elution with the new synthetic polymeric stationary phases P-CAP, P-CAP DP and DEAVB. The sulfoxide series was composed of aryl alkyl sulfoxides, benzoimidazole sulfoxides and the drugs modafinil, albendazole sulfoxide, omeprazole, lansoprazole, pantoprazole and rabeprazole. This work examines the effectiveness of the polymeric chiral stationary phases for the separation of chiral sulfoxides and describes the superiority of DEABV for these separations in three different elution modes. The first ever reversed phase enantiomeric separations on these columns is demonstrated.     

Chiral silica-based monoliths in chromatography and capillary electrochromatography

Chiral-modified silica-based monoliths have become well-established stationary phases for both high performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The silica-based monoliths were fabricated either in situ in the capillaries for nano-HPLC and CEC or in a mould for “conventional” HPLC. The present review summarizes the chiral modification of silica monoliths and the recent development in the field of enantioselective separations by nano-HPLC and CEC.     

Efficient preparative separation of 6‐(4‐aminophenyl)‐5‐methyl‐4, 5‐dihydro‐3(2H)‐pyridazinone enantiomers on polysaccharide‐based stationary phases in polar organic solvent chromatography and supercritical fluid chromatography

6‐(4‐Aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone is a key synthetic intermediate for cardiotonic agent levosimendan. Very few studies address the use of chiral stationary phases in chromatography for the enantioseparation of this intermediate. This study presents two efficient preparative methods for the isolation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone in polar organic solvent chromatography and supercritical fluid chromatography using polysaccharide‐based chiral stationary phases and volatile organic mobile phases without additives in isocratic mode. Under optimum conditions, Chiralcel OJ column showed the best performance (α = 1.71, Rs = 5.47) in polar organic solvent chromatography, while Chiralpak AS column exhibited remarkable separations (α = 1.81 and Rs = 6.51) in supercritical fluid chromatography with an opposite enantiomer elution order. Considering the sample solubility, runtime and solvent cost, the preparations were carried out on Chiralcel OJ column and Chiralpak AS column (250 × 20 mm i.d.; 10 µm) in polar organic mode and supercritical fluid chromatography mode with methanol and CO₂/methanol as mobile phases, respectively. By utilizing the advantages of chromatographic techniques and polysaccharide‐based chiral stationary phases, this work provides two methods for the fast and economic preparation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone, which are suitable for the pharmaceutical industry.     

Separation of the enantiomers of 2-phenylcyclopropanecarboxylate esters by capillary gas chromatography on derivatized cyclodextrin stationary phases

Summary Separation of the enantiomers of 2-phenylcyclopropanecarboxylate esters has been investigated on derivatized cyclodextrin chiral stationary phases (CD CSPs) to enable direct determination of the enantiomeric purity of the products of enantioselective cyclopropanation. Four stereoisomers of these chiral compounds could be resolved to baseline on permethylated β-cyclodextrin CSP. Some unusual phenomena, iso-enthalpy retention behavior and entropically driven chiral separation, were observed for the enantioseparation of 2-phenylcyclo-propanecarboxylates on the CD CSPs. Thermodynamic parameters were evaluated and an enthalpy-entropy compensation effect was observed forn-alkyl esters of 2-phenylcyclopropanecarboxylate separated on CD CSPs.