Chromatographic Separation Of Enantiomers Research Articles

A Validated Chiral Chromatography Method for Enantiomeric Separation of Pomalidomide in Human Plasma.

In the present work, new chiral stationary phase high-performance liquid chromatography (CSP-HPLC) method was established and validated for the quantification of pomalidomide (PMD) enantiomers in human plasma. The chromatographic enantiomeric separation was achieved on a Daicel-CSP, Chiralpack IA 4.6 × 250mm, 5μm; because of its advantages of high degree of retention, high resolution capacity, better reproducibility, ability to produce lower back pressure and low degree of tailing. The mobile phase was maintained as methanol: glacial acetic acid (499.50ml:50μL). Ultraviolet wavelength for detection was 220nm. PMD enantiomer-I and enantiomer-II were separated at 8.83 and 15.34min, respectively. Limit of detection and limit of quantification for each enantiomer and the calibration curve of standard PMD was linear in range between 10-5,000ngmL-1. The method was validated according to The International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH(Q2R1)) specific guidelines. We found no interference peak with PMD chromatogram obtained. This is a simple, reliable and specific method for detection and quantification of enantiomer of PMD in human plasma sample.

Chiroptical Generation, Switching, and Long‐Term Memory in Supramolecular Azobenzene‐Pendant Polymer: Regulation by Cellulose Peralkyl Esters, D‐/L‐Glucose Permethyl Esters, Solvents, UV Light Irradiation, and Thermal Annealing Process†

Comprehensive SummaryVarious optically active polymers are known to afford sophisticated chirality‐related functionalities, i.e., asymmetric catalysis, chiroptical switching and memory in UV‐vis‐NIR region, chromatographic separation of enantiomers, and sensors for molecular chirality. Recently, material researchers have paid much attention to the design of chiral supramolecular architectures from achiral polymers upon intermolecular interactions with help of greener biosources. The present article reports an instantaneous generation of ambidextrous supramolecules revealing light‐driven chiroptical switching/memory in UV‐vis region when achiral azobenzene‐containing vinylpolymers are non‐covalently interacted with alkyl ester derivatives of natural cellulose and D‐/L‐glucose. It was recognized that the semi‐synthetic biomaterials efficiently work as chirality‐inducing scaffoldings to several achiral and optically inactive molecules, oligomers, and polymers. Our successful results shed light on a new approach of how inexpensive poly‐/mono‐saccharide derivatives can afford supramolecular chiroptical systems with the azobenzene pendant polymer as aggregates in suspension and liquid‐crystalline films with minimal energy, time, and cost.

Direct determination of tyrosine and tryptophane enantiomers in pumpkin (Cucurbita moschata) by HPLC-UV/Vis: Effect of cooking treatment on enantiomers profile

An analytical method to determine the content of tryptophan (Trp) and tyrosine (Tyr) enantiomers in pumpkin (Cucurbita moschata) is developed and validated. The method consists of a sample preparation procedure with a direct liquid chromatographic separation of enantiomers on a Whelk-O2 chiral stationary phase. The chromatographic separation was performed under gradient reversed-phase conditions in which the effect of pH and organic modifier were studied. A multivariate design of experiments was applied in the optimization of the extraction procedure. After optimization the complete analytical process was validated. Great accuracy was achieved with recovery between 92–109% with a relative standard deviation of less than 10%. Limits of detection obtained ranged between 0.04 and 0.07 mg L-1. The resulting analytical method is applied with success to dose the content of Trp and Tyr and their enantiomers in several pumpkin cultivars. The impact of several traditional cooking methods on the content of these amino acids was also studied. The obtained data showed that studied pumpkin is a good source of free Trp and Tyr, mainly L-Trp. The results confirm the natural presence of D-Trp in this vegetable. Finally, the results demonstrate that the levels of all analytes are modified during traditional cooking treatments.

Development of a liquid chromatographic method for enantioseparation of Eflornithine using (S)-α-ethyl benzylamine as a chiral derivatizing agent

ABSTRACT Eflornithine (2-fluoromethyl-DL-ornithine) is a potent and irreversible inhibitor that selectively targets ornithine decarboxylase, a key enzyme in the polyamine biosynthesis pathway. Due to the lack of chromophoric moiety in the eflornithine structure, its detection via UV detector is difficult. Thus, direct high-performance liquid chromatographic (HPLC) separation of enantiomers of Eflornithine is not feasible, and pre-column derivatization is required for its determination through chiral HPLC columns. We developed an indirect HPLC method using (S)-α-ethyl benzylamine as a chiral derivatizing agent for the enantioseparation of Eflornithine. The diastereomers thus produced were then separated via LiChrospher C18 column (5 μm particle size, L × I. D. 25 cm × 4.6 mm). The mobile phase used was a mixture of acetonitrile and 0.1% aq. TFA and varied in linear gradients of 30–70% of acetonitrile for 30 min run at a flow rate of 1.0 mL min−1 and UV detection at 320 nm. The separation parameters were optimized by altering the mobile phase composition and flow rate. The findings revealed that the chromatographic separation was accomplished within 15 min, with resolution values greater than 4.5 for Eflornithine enantiomers. The detection and quantitation limits were 9.26 ng mL− 1 and 18.52 ng mL− 1 for Eflornithine enantiomers.

Development and validation of a novel chiral chromatographic method for separation of lenalidomide enantiomers in human plasma.

Lenalidomide (LND) is an analogue of thalidomide that is second generation immunomodulatory drugs (IMiDs). LND contains asymmetric carbon atom and exist R and S enantiomer. S (-) form of enantiomer are considered to be more potent and biologically active in tumor cell. It is available in racemic form for clinical use. The study aims to develop and validate enantiomer separation of LND in human plasma. The chromatographic enantiomeric separation was achieved on a Daicel-CSP, Chiralpack IA 4.6 × 250 mm_5 μm. The mobile phase was constituted in combination of methanol:glacial acetic acid at a concentration of 499.50 ml: 50 μl. UV wavelength detection was 220 nm. The RSD% for all validation parameters was found to be within the acceptable limit. The chiral chromatographic (chiral stationary phase-high-performance liquid chromatography [CSP-HPLC]) method developed and validated for the quantitative estimation of LND enantiomers S (-) and R (+) in human plasma sample is accurate, precise, robust, stable and selective.

Preparation and evaluation of a chiral porous organic cage based chiral stationary phase for enantioseparation in high performance liquid chromatography

Porous organic cages (POCs) are a new kind of porous molecular materials, which have gained widespread interest in many fields due to their intriguing properties, including excellent molecular solubility, inherent molecular cavity and rich host-guest chemistry. To date, many chiral POCs have been explored as chiral stationary phases (CSPs) for gas chromatographic (GC) separation of enantiomers. However, the applications of chiral POCs for high performance liquid chromatography (HPLC) enantiomeric separation is extremely rare. In this study, we report the construction of thiol-ene click reaction for the preparation of CSP for HPLC by using a [4+8]-type chiral POC NC4-R as chiral selector. The fabricated CSP showed good chiral resolution performance not only in normal-phase HPLC (NP-HPLC) but also in reversed-phase HPLC (RP-HPLC). Seventeen and ten racemates were well resolved in the two separation modes, respectively, including ketones, esters, alcohols, phenols, amines, ethers, organic acids, and amino acids. Moreover, the fabricated column also shows good chiral recognition complementarity to two popular chiral HPLC columns (Chiralpak AD-H and Chiralcel OD-H columns) and previously reported chiral POC NC1-R-based HPLC column, which can resolve some racemates that unable to be resolved by the two commercially available chiral HPLC columns and NC1-R-based column. The relative standard deviation (RSD) values (n = 4) of retention time and resolution (Rs) of analytes separated on the column were less than 0.3 % and 0.5 % after it was subjected to different injections, showing the good reproducibility and stability of the NC4-R-based column. This work demonstrated high potentials of chiral POCs for HPLC enantioseparation and the applicability of chiral POC-based HPLC columns can be broadened by developing more chiral POCs with diverse structures as chiral selector for HPLC.

'Ab Ovo' Chiral Phases and Chiral Reagents for Liquid Chromatographic Separation and Isolation of Enantiomers.

The de-novo approach of mixing chirally pure reagents or Cu(II)-L-amino acid complexes in the slurry of silica gel for preparing TLC plates was reported from author's laboratory and was successful for separation and isolation of enantiomers. Using high molar absorptivity molecules, e. g., 1,5-difluoro-2,4-dinitrobenzene and cyanuric chloride, more than 38 new chiral derivatizing reagents were synthesized in our laboratory by straightforward nucleophilic substitution with simple chiral auxiliaries. Besides, (S)-naproxen, (S)-ketoprofen, and (S)-levofloxacin were used as chiral platforms. A conceptual approach using both achiral phases in chromatography for enantioseparation was also adopted. 1 H NMR and DFT based software were used to explain structures of non-covalent and covalent diastereomeric pairs and determination of configuration and separation mechanism. The methods can be easily used to determine and control enantiomeric purity with advantages over a variety of commercial chiral phases.

Racemate Resolution of Alanine and Leucine on Homochiral Quartz, and Its Alteration by Strong Radiation Damage.

Homochiral proteins orchestrate biological functions throughout all domains of life, but the origin of the uniform l-stereochemistry of amino acids remains unknown. Here, we describe enantioselective adsorption experiments of racemic alanine and leucine onto homochiral d- and l-quartz as a possible mechanism for the abiotic emergence of biological homochirality. Substantial racemate resolution with enantiomeric excesses of up to 55% are demonstrated to potentially occur in interstitial pores, along grain boundaries or small fractures in local quartz-bearing environments. Our previous hypothesis on the enhanced enantioselectivity due to uranium-induced fission tracks could not be validated. Such capillary tubes in the near-surface structure of quartz have been proposed to increase the overall chromatographic separation of enantiomers, but no systematic positive correlation of accumulated radiation damage and enantioselective adsorption was observed in this study. In general, the natural l-quartz showed stronger enantioselective adsorption affinities than synthetic d-quartz without any significant trend in amino acid selectivity. Moreover, the l-enantiomer of both investigated amino acids alanine and leucine was preferably adsorbed regardless of the handedness of the enantiomorphic quartz sand. This lack of mirror symmetry breaking is probably due to the different crystal habitus of the synthetic z-bar of d-quartz and the natural mountain crystals of l-quartz used in our experiments.

Controllable organosilane monolayer density of surface bonding using silatranes for thiol functionalization of silica particles for liquid chromatography and validation of microanalytical method for elemental composition determination

The present work systematically investigates a new strategy for the functionalization of silica gel using alkyl silatrane chemistry instead of alkylsilanes for synthesis of chromatographic stationary phases. In this work, silica was chemically modified for further functionalization by a thiol-ene click reaction. Thus, 3-mercaptopropylsilatrane (MPS) was used which is capable to form self-assembled monolayers (SAM) on top of silanol surfaces in a controlled manner as previously shown for silicon wafers. The utility of this chemistry for stationary phase synthesis in liquid chromatography was not evaluated yet. Hence, silica surface modifications using MPS were studied in comparison to established 3-mercaptopropyltrimethoxysilane (MPTMS) chemistry. First, the employed elemental analysis method was validated and it showed excellent intra-day and inter-day precisions (typically less than 5% RSD). It could be shown that the reaction kinetics of MPS was roughly 35-times faster than with MPTMS. After 30 min reaction time with MPS, the thiol content reached 74% of the maximal coverage. Due to controlled chemistry with MPS, which does not lead to oligomeric siloxane network at the silica surface, the ligand coverage was lower. However, multiple silanization cycles with MPS led to a dense surface coverage (around 4 µmol m−2). 29Si cross polarization/magic angle spinning (CP/MAS) solid-state NMR revealed distinct T1/T2/T3 ratios for MPS and MPTMS materials with up to 80% T3 (indicative for trifunctional siloxane linkage) for MPS and around 20% T3 for MPTMS. This indicates a more homogeneous, thinner monolayer film of MPS on the silica surface, as compared to an irregular thick oligomeric siloxane network with MPTMS. Bonding of quinine carbamate as chiral selector afforded an efficient chiral stationary phase (CSP) for chromatographic enantiomer separation. Separation factors were comparable to MPTMS-bonded CSP, however, chromatographic efficiency was much better for the MPS-bonded CSP. H/u curves indicated a reduced mass transfer resistance by roughly factor 3 for MPS- compared to MPTMS-bonded CSP. This confirms better chromatographic performance of surfaces with homogeneous monolayer compared to network structures on the silica surface which suffer from poor stationary phase mass transfer.

A β-cyclodextrin covalent organic framework used as a chiral stationary phase for chiral separation in gas chromatography

Chiral covalent organic frameworks (CCOFs) featuring chirality, stability, and good porosity have attracted a considerable amount of attention due to their important applications, such as asymmetric catalysis, chiral separation, and chiral recognition. In this study, a β-cyclodextrin (β-CD) covalent organic framework (β-CD-COF) diluted with polysiloxane OV-1701 was explored as a novel chiral stationary phase (CSP) for gas chromatography (GC) separation of racemates. The β-CD-COF coated capillary column had excellent selectivity, not only for the separation of linear alkanes, linear alcohols, fatty acid methyl esters mixture, the Grob mixture and positional isomers, but also for the resolution of chiral compounds, including chiral alcohols, aldehydes, ethers, and amino acid derivatives. In addition, the β-CD-COF-coated capillary column presented good repeatability and reproducibility. This work indicated the great potential of the CCOFs coated capillary column for the chromatographic separation of enantiomers.

Chiral Recognition for Chromatography and Membrane-Based Separations: Recent Developments and Future Prospects.

With the rapid development of global industry and increasingly frequent product circulation, the separation and detection of chiral drugs/pesticides are becoming increasingly important. The chiral nature of substances can result in harm to the human body, and the selective endocrine-disrupting effect of drug enantiomers is caused by differential enantiospecific binding to receptors. This review is devoted to the specific recognition and resolution of chiral molecules by chromatography and membrane-based enantioseparation techniques. Chromatographic enantiomer separations with chiral stationary phase (CSP)-based columns and membrane-based enantiomer filtration are detailed. In addition, the unique properties of these chiral resolution methods have been summarized for practical applications in the chemistry, environment, biology, medicine, and food industries. We further discussed the recognition mechanism in analytical enantioseparations and analyzed recent developments and future prospects of chromatographic and membrane-based enantioseparations.

Chromatographic Enantiomer Separation of Chiral Amines as Fluorene-2-carboxaldimine Derivatives Using Polysaccharide-derived Chiral Stationary Phases

Chromatographic Enantiomer Separation of Chiral Amines as Fluorene-2-carboxaldimine Derivatives Using Polysaccharide-derived Chiral Stationary Phases

Liquid Chromatographic Separation and Thermodynamic Investigation of Ethyl Nipecotate Enantiomers on Immobilized Amylose-Based Chiral Stationary Phase.

Ethyl nipecotate enantiomers are widely used as chiral building blocks in the synthesis of drug substances. An efficient and economic chiral high-performance liquid chromatographic method for determination of enantiomeric purity of ethyl nipecotate is developed and validated. Chiral separation was achieved on immobilized amylose-based stationary phase using a mixture of n-hexane: ethanol: diethylamine (80:20:0.1, v/v/v) as mobile phase. Resolution between R-(-)-ethyl nipecotate (REN) and S-(+)-ethyl nipecotate (SEN) peaks was found to be 3.59. The detector response of SEN exhibited an excellent linearity over the concentration range of 4.5-120μgmL-1. The limit of detection and limit of quantification for SEN were 0.016 and 0.045μg and REN were 0.015 and 0.043μg, respectively. The influence of column oven temperatures on chiral retention and separation was studied in the range of 25°C to 50°C; the thermodynamic parameters ΔH°, ΔS° and ΔG° were evaluated from van't Hoff plots and used to explain the strength of interaction between analyte and stationary phase.

Separating Enantiomers of Haloalkanes and Alcohols on a Stationary Phase Based on the Supramolecular Structure of Melamine with Induced Chirality

A new chiral stationary phase is proposed that is based on the supramolecular structure of melamine self-assembled in the mode of the induction of chirality. Induction is achieved via mechanical stirring in accordance with the Kondepudi effect in order to form a layer of a supramolecular structure with predominantly one type of chiral supramolecular domains on the surface of the sorbent. The resulting inert support with 1% melamine is used for the gas chromatographic separation of enantiomers of 2-butanol, 1‑methoxy-2-propanol, 2-bromobutane, 2-chlorobutane, and 2-bromopentane. The coefficients of selectivity and the separation of enantiomers are calculated. No separation of enantiomers is observed on a column conditioned at 100°C. After conditioning the column at 200°C, a series of successful separations of racemates is obtained in the 60–110°C range of temperatures. The highest coefficient of selectivity for haloalkanes is obtained at 70–75°C, so 2-bromobutane has α = 1.62 at 70°C, 2-chlorobutane has α = 1.23 at 75°C, and 2‑bromopentane has α = 1.68 at 70°C. The coefficients of selectivity for 2-butanol and 1-methoxy-2-propanol are highest at 80 and 100°C (α = 1.45 and 2.24), respectively. It is assumed that the ability of the suprastructure of melamine to exhibit enantioselectivity is associated with the formation of cavities capable of chiral recognition.

Enantiodiscrimination of Chiral Amines as Naphthaldimine Derivatives on Polysaccharide-derived Chiral Stationary Phases by Normal Phase HPLC

Normal phase high-performance liquid chromatographic enantiomeric separation of chiral amines as naphthaldimine derivatives was performed on amylose...

Determination of Chemical and Enantiomeric Purity of α‐Amino Acids and their Methyl Esters as N‐Fluorenylmethoxycarbonyl Derivatives Using Amylose‐derived Chiral Stationary Phases

Liquid chromatographic enantiomer separation and simultaneous determination of chemical and enantiomeric purity of α‐amino acids and their methyl esters as N‐fluorenylmethoxycarbonyl (FMOC) derivatives was performed on three covalently bonded type chiral stationary phases (CSPs) derived from amylose derivatives. The enantiomer separation of α‐amino acid esters as N‐FMOC derivatives was better than that of the corresponding acids, especially for CSP 1 and 2. Chemical impurities as the corresponding racemic acids present in several commercially available racemic amino acid methyl esters were observed to be 0.49–17.50%. Enantiomeric impurities of several commercially available L‐amino acid methyl esters were found to be 0.03–0.58%, whereas chemical impurities as the corresponding racemic acids present in the same analytes were found to be 0.13–13.62%. This developed analytical method will be useful for the determination of chemical and enantiomeric purity of α‐amino acids and/or esters as N‐FMOC derivatives using amylose‐derived CSPs.

Atropisomeric Separation of PCB-95 by HPLC

2,2’,3,5’,6-Pentachlorobiphenyl (PCB-95) is an environmentally significant chiral PCB, of which enantioselective toxicity, biodegradation and chiral stability studies have been limited to date, as no commercially available enantiomers exist for PCB-95 and due to the lack of an efficient preparatory chiral separation method. A selective, sensitive, and rapid high-performance liquid chromatography with UV detection (HPLC-UV) method has been developed and validated for the chromatographic separation and quantitation of PCB-95 enantiomers. In this study, we resolved enantiomers of PCB-95 using a cellulose tris (4-methylbenzoate) Chiralcel OJ- H column. After evaluating mobile phase compositions and temperatures, optimum separation and detection were obtained with isocratic 100% n-hexane as the mobile phase, a column temperature of 20°C, a flow rate of 1 mL/min, and a detection wavelength of 280 nm. The total run time was 8 minutes. Enantiomer purity was confirmed using enantioselective gas capillary chromatography-electron capture detection. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to limit of detection, limit of quantification, precision, linearity, robustness and ruggedness.

Liquid Chromatographic Enantiomeric Separation of Chiral Aliphatic Amines Using 2-Hydroxynaphthaldehyde as a Derivatizing Agent on Polysaccharide-Derived Chiral Stationary Phases

A new liquid chromatographic enantiomer separation of chiral amines as 2-hydroxynaphthaldimine derivatives on several coated and immobilized chiral stationary phases (CSPs) derived from polysaccharides is described. 2-Hydroxynaphthaldehyde was introduced as a derivatizing agent for the enantiomeric separation of chiral aliphatic amines under the normal phase conditions, and was found to both enhance detection sensitivity and provide suitable interaction sites for enantiodiscrimination. Cellulose-derived CSPs, in general, exhibited better enantiomeric separation than amylose-derived CSPs. Amongst the examined six covalently bonded and four coated-type CSPs, covalently bonded Chiralpak IC, which contains a cellulose-based chiral selector, showed the best enantioseparation. The method was applied to the determination of the enantiomeric purity of commercially available (R)- and (S)-leucinol. The enantiomeric impurities of the investigated analytes procured from two suppliers were found to be 0.06–1.20%. The developed analytical method was also validated in accordance with ICH guidelines and proved to be enantioselective, sensitive and effective for the enantiomeric separation of chiral aliphatic amines as 2-hydroxynaphthaldimine derivatives under ultraviolet detection.

Liquid chromatographic separation of the enantiomers of N-fluorenylmethoxycarbonyl α-amino acids on a covalently bonded type polysaccharide-derived chiral stationary phase under fluorescence detection

The liquid chromatographic enantiomer separation of N-fluorenylmethoxycarbonyl (FMOC) α-amino acids was performed using a covalently immobilized CSP (Chiralpak IB) with cellulose...

Effect of Secondary Equilibria on the Adsorption of Ibuprofen Enantiomers on a Chiral Stationary Phase with a Grafted Antibiotic Eremomycin

The chromatographic separation of ibuprofen enantiomers on a Nautilus-E chiral stationary phase with a grafted eremomycin antibiotic at high column loading is accompanied by distortion of the shape of chromatographic peaks. A model is proposed to explain this phenomenon. A number of factors are considered in the model: the ionization of ibuprofen in the mobile phase, the pH change in the mass transfer zone caused by ionization, and competitive adsorption involving buffer components. Simulations performed using this model within the theory of nonequilibrium chromatography allow the shape of chromatograms for large amounts of S- and R-ibuprofen samples to be predicted. The adsorption mechanism is found to be mainly ion-exchange. The contribution from the molecular adsorption of ibuprofen to the total retention is shown to be several percent.