Chiral Stationary Phase Research Articles

Optimized separation of astaxanthin stereoisomers from microbial sources using chiral HPLC.

Astaxanthin (AST) is a high-value antioxidant, and its efficient isolation and utilization are challenging owing to the presence of different stereoisomers from various sources. In the present study, a semi-preparative HPLC method for the efficient separation of AST stereoisomers using a Chiralpak IC chiral column with good loading capacity and chiral recognition ability was successfully developed. The mobile phase was methanol-methyl tert-butyl ether (90 : 10, v/v), with a flow rate of 3.06 mL min-1 and a maximum injection volume of 0.32 mg. The results indicated that the purity of all-trans AST was 97.9% for Haematococcus pluvialis and 97.5% for Phaffia rhodozyma. Additionally, molecular weights and fragmentation patterns analyzed using mass spectrometry were consistent with those of all-trans AST. Linearity validation and reproducibility experiments revealed that all calibration curves had coefficients of determination (R2) greater than 0.999 and a relative standard deviation (RSD) of <3.8%. This is because all-trans AST stereoisomers could undergo specific rotations or spins due to π-π interactions, hydrogen bonding, and inclusion interactions. This process allowed the successful separation of the three all-trans AST optical isomers and provides a theoretical basis for large-scale preparation of all-trans AST stereoisomers from different sources.

Photostability and Chiral Stability Evaluation of &lt;i&gt;d&lt;/i&gt;-Chlorpheniramine Maleate and its Formulations by Chiral HPLC

Photostability and Chiral Stability Evaluation of &lt;i&gt;d&lt;/i&gt;-Chlorpheniramine Maleate and its Formulations by Chiral HPLC

Enantiospecific Synthesis of Planar Chiral Rhodium and Iridium Cyclopentadienyl Complexes: Enabling Streamlined and Computer-Guided Access to Highly Selective Catalysts for Asymmetric C-H Functionalizations.

Chiral cyclopentadienyl (CpX) metal complexes are frequently used in asymmetric catalysis by virtue of their high reactivity and selectivity. Planar-chiral-only rhodium and iridium cyclopentadienyl complexes are particularly promising due to unrestricted chemical space for CpX ligand design while retaining structural simplicity. However, they are currently still niche because of a lack of efficient synthetic strategies that avoid lengthy chiral auxiliary routes or chiral preparatory HPLC resolution of the complexes. To streamline access to such planar-chiral-only CpX-metal complexes, we designed a straightforward, highly enantiospecific, point-to-planar chirality transfer complexation via facially selective concerted-metalation-deprotonation between metal-carboxylate precursor [M(olefin)2OAc]2 and a chiral cyclopentadiene. This entirely avoids the typical stereoablative complexation of an achiral cyclopentadienyl anion that detrimentally yields a racemate. Exploiting the described enantiospecific complexation protocol and a simple divergent synthetic route to suitable chiral cyclopentadienes, we generated a structurally diverse library of new planar chiral Cp-Rh(I), Cp-Ir(I), Cp-Rh(III), and Cp-Ir(III) complexes. Moreover, the enantiospecific complexation step can be concatenated with a preceding Au-catalyzed cyclization in an efficient one-pot process that likely involves an elaborate point-to-axial-to-point-to-planar chirality transfer. Guided by computational selectivity predictions, the structure of a CpX-Rh complex in our library was tuned to optimize reactivity and selectivity in the asymmetric C-H functionalization of a benzamide with various challenging alkenes. With an optimized CpX-Rh complex in hand, we showcased its excellent catalytic performance and high selectivity for refractory alkene substrates that reacted in poor selectivity with previous CpX-Rh catalysts.

Synthesis of enantiomerically enriched β-substituted analogs of (S)-α-alanine containing 1-phenyl-1H-1,2,3-triazole groups

A synthesis of new enantiomerically enriched derivatives of (S)-α-aminopropionic acid, containing in the β-position 1,2,3-triazole groups coupled with a o-, m- and p-substituted phenyl residue, was developed based on Cu(I) catalyzed [3 + 2] cycloaddition of azides with alkynes. As the starting materials was used the square-planar Ni(II)complex of the Schiff base of propargylglycine with the chiral auxiliary BPB (Benzylprolylbenzophenone) and 1,4-substituted phenyl azides. The assignment of the (S)-absolute configuration of the α-carbon atom of the amino acid residue of the main diastereomeric complexes of the cycloaddition products was carried out on the basis of positive Cotton effects in the region of 480–580 nm of the circular dichroism spectra. The target amino acids were isolated from acid hydrolysates of diastereomeric complexes using ion-exchange demineralization and crystallization from aqueous ethanol. Additional confirmation of the absolute configuration and determination of the enantiomeric purity of the target amino acids were carried out by chiral HPLC analysis. As a result, seven new non-proteinogenic (S)-α-amino acids, containing in the β-position a 1,2,3-triazole moiety, were synthesized.

Chiral Differentiation of Chiral Lactides and Chiral Diketones on Native and Phenylcarbamoylated Cyclodextrin Chiral Stationary Phases.

Inclusion complexation, hydrogen bonds, π-π interaction, dipole-dipole interaction, and steric hindrance effect all contribute to the enantioseparation ability of cyclodextrin (CD) or CD derivatives. In this work, one native cationic CD chiral stationary phases (SHCDCSP) and four derivatized CD-CSPs, namely, per(4-trifluoromethyl) phenylcarbamoylated-β-CD CSP (SFPhCDCSP), per(4-chloro) phenylcarbamoylated-β-CD CSP (SCPhCDCSP), per(4-bromo) phenylcarbamoylated-β-CD CSP (SBPhCDCSP), and per(4-methyl) phenylcarbamoylated-β-CD CSP (SMPhCDCSP), were prepared via thioether linkage and applied for the enantioseparation of chiral lactides and chiral diketones in both reverse phase(RP) and normal phase (NP) modes. Most of the studied chiral lactides were found to be well resolved (Rs > 1.5) under RP mode, especially Met-Sty-Ibn (compound 11) is observed to display highest resolutions (Rs = 5.09, Rs = 3.17) among all the analytes on the SFPhCDCSP and SMPhCDCSP. In NP mode, SFPhCDCSP showed excellent chiral recognition ability towards chiral lactides. The comparison study of CD-CSPs reveals the structure of CSPs play a significant role on the enantioselectivities.

Unexpected Formation of a Chiral spiro-system in the Reaction of the Dilithium Derivative of Hexafluorocumyl Alcohol with N-(t-butylsulfenyl)phthalimide.

An unexpected course of the reaction of hexafluorocumyl alcohol dilithium derivative 2 with N-(t-butylsulfenyl)phthalimide (3) has been presented. The process proceeded under mild conditions and resulted in previously undescribed chiral spiro-system- 3',3'-bis(trifluoromethyl)-3H,3'H-1,1'-spirobis(isobenzofuran)-3-one (5) as the only product. A detailed spectral analysis of the product has been provided, and mechanistic aspects have been investigated. Attempts to separate the enantiomers of compound 5 using a semipreparative HPLC method with a chiral stationary phase column have been described. The repeatability of the reaction using analogs of alcohol 4 has also been tested. DFT calculations of absolute configuration assignment have been performed successfully.

Synthesis of Proline-Derived Helical Copolyacetylenes as Chiral Stationary Phases for HPLC Enantioseparation

Synthesis of Proline-Derived Helical Copolyacetylenes as Chiral Stationary Phases for HPLC Enantioseparation

Discovery of new insecticidal cyclic hexapeptides from Fusarium tricinctum HM76 and their biosynthesis

Fungi often produce a variety of natural products that are toxic to insects, making them an important resource for the discovery of new insecticides. In previous screenings, the crude extract from the wheat pathogen Fusarium tricinctum HM76 exhibited notable insecticidal activity. A chemical investigation of the extract led to the isolation of six undescribed cyclic hexapeptides Futrines (1–6). Their structures were determined through elucidation of NMR and HRESIMS data, as well as chiral HPLC analysis. All the isolated compounds were tested for insecticidal activities against Aphis citricola Van der Goo and Tetranychus cinnabarinus Boisduval. Notably, compounds 2 and 6 demonstrated significant insecticidal activities against T. cinnabarinus and A. citricola, with LC50 and LD50 values of 0.212 mg/mL and 2.230 ng/nymph, respectively. Additionally, the biosynthetic gene cluster responsible for the production of futrines was identified through gene knockout, offering insight into their biosynthetic mechanism. These findings highlight the potential of these cyclic hexapeptides as promising candidates for new insecticide development.

Chiral method validation and stereoselective degradation of profoxydim isomers in paddy soil

The herbicide profoxydim, which is chiral in nature, is commonly used for weed control in rice. However, traditional achiral analysis treats profoxydim isomers as a single compound, providing only partial and inaccuracy information assuming identical behavior for the four isomers. To address this issue, a sensitive and straightforward chiral analytical method was developed using QuEChERS and chiral HPLC–MS/MS to determine the residues of all four stereoisomers of profoxydim in soil. The method was validated and found to have good accuracy and precision, with recoveries ranging from 88.27 to 104.93% for dry soil and from 98.25 to 106.53% for soaked soil, with RSD values lower than 11.14%. The limit of detection for the profoxydim isomers was 7.6 µg/kg, and the limit of quantification was 25.0 µg/kg. Furthermore, the proposed method was employed for the first time to investigate the individual degradation of novel isolated stereoisomers of profoxydim in soil and the kinetics and evolution of the generated degradation products (DPs). The degradation of the four stereoisomers followed first-order kinetics and was found to be stereoselective, with isomers 1 and 4 preferentially degrading with half-lives of 14.7 and 15.6 h respectively. The profoxydim enantiomers remained configurationally stable without any interconversion throughout the experiment. The findings of this study provide a robust scientific basis for improving profoxydim risk assessment and highlight the need to characterize chiral agrochemical isomers to better understand their environmental distribution and fate.

DEVELOPMENT AND OPTIMIZATION OF HPLC METHODS FOR THE CHIRAL SEPARATION OF ARYLPYRAZOLE, CHLOROACETANILIDE AND ORGANOCHLORINE PESTICIDES: CASE STUDIES OF BENZOBICYCLON, ACETOCHLOR AND CHLORDANE.

This work is centered on the development and optimization of High-Performance Liquid Chromatographic methods for the separation of chiral pesticides, particularly benzobicyclon, chlordane and acetochlor employing Agilent Technologies Infinity II chiral HPLC system equipped with a UV – 2000 detector. The stereoisomers were successively separated using a standard analytical column of dimensions 250 mm X 4.6 mm. The Cis– stereoisomers of chlordane exhibited partial baseline separation while the Trans – enantiomers were fully separated. Benzobicyclon and acetochlor enantiomers attained full baseline separation under optimized conditions. Chlordane and benzobicyclon were analyzed using normal phase chromatography [NP]c with a mobile phase of 90% Normal hexane and 10% Isopropanol, while acetochlor was analyzed by reverse phase chromatography [RP] c using 70% methanol and 30% water. Benzobicyclon was later spiked into soil sample and subsequently extracted using Environmental Protection Agency (EPA) methods and the extracts were further analyzed with a shorter analytical column of dimensions 75 mm X 4.6 mm. The HPLC separation revealed that the longer column provided more efficient separation for the benzobicyclon stereoisomers than the shorter column as reflected by high separation factors (?). The high recovery rate of the stereoisomers of benzobicyclon and the internal standard from the soil extracts confirmed the effectiveness of the method of extraction and the HPLC system employed.

Stereochemistry of phosphatidylglycerols from thermotolerant bacteria isolated thermal springs

Phosphatidylglycerol (1,2-diacyl-sn-glycero-3-phospho-glycerol) (PG) is one of the most abundant lipids in biological membranes. However, the chirality of the carbon atom in glycerol phosphate differs among the three kingdoms: bacteria, archaea, and eukaryotes. It is commonly assumed that archaea, as well as bacteria and eukaryotes, produce only one isomer of PG. Archaeal membranes consist of phospholipids with glycerol-1-phosphate in the S configuration, while the phospholipids of the other two kingdoms contain glycerol-3-phosphate with (R) stereochemistry. Another chiral atom is found in glycerol with non-esterified hydroxy groups. Considering the high temperatures that accompanied the origin of life on Earth, it becomes obvious that it is necessary to clarify the importance of membrane lipids in early evolutionary times. To reconstruct the effect of high temperatures on membrane lipids, it is ideal to use microorganisms originating from a thermophilic environment analogous to the early Earth, such as the thermal groundwater of the famous spa town of Karlovy Vary. Here, we prepared all four isomers of PG, i.e., (R,S, R,R, S,R), and (S,S), by organic synthesis and analyzed the representation of individual molecular species in seven bacteria isolated from the Karlovy Vary thermal springs using chiral chromatography - mass spectrometry. Our results provide evidence that five of these strains produce all four isomers of PG and that this production is highly dependent on the cultivation temperature. Subsequent analysis by chiral chromatography revealed that the ratio of isomers, enantiomers, and diastereoisomers depends on the cultivation temperature of individual strains.

5]Helicene Based π-Conjugated Macrocycles with Persistent Figure-Eight and Möbius Shapes: Efficient Synthesis, Chiral Resolution and Bright Circularly Polarized Luminescence.

π-Conjugated chiral shape-persistent molecular nanocarbons hold great potential as chiroptical materials, though their synthesis remains a considerable challenge. Here, we present a simple approach using Suzuki coupling of a [5]helicene building block with various aromatic units, enabling the one-pot synthesis of a series of chiral macrocycles with persistent figure-eight and Möbius shapes. Single-crystal structures of 7 compounds were solved, and 22 enantiomers were separated by preparative chiral HPLC. A notable pyrene-bridged figure-eight macrocycle, with its rigid, fully π-conjugated and overcrowded structure, exhibited pure excimer emission and outstanding circularly polarized luminescence (CPL) properties, including a large dissymmetric factor (|glum|=3.8×10-2) and significant CPL brightness (BCPL=710.5 M-1cm-1). This method provides a versatile synthetic platform for producing various chiral D2-symmetric figure-eight macrocycles and singly or triply twisted Möbius macrocycles with C2 and D3 symmetry, offering tunable chiroptical properties for CPL applications.

5]Helicene Based π‐Conjugated Macrocycles with Persistent Figure‐Eight and Möbius Shapes: Efficient Synthesis, Chiral Resolution and Bright Circularly Polarized Luminescence

Abstractπ‐Conjugated chiral shape‐persistent molecular nanocarbons hold great potential as chiroptical materials, though their synthesis remains a considerable challenge. Here, we present a simple approach using Suzuki coupling of a [5]helicene building block with various aromatic units, enabling the one‐pot synthesis of a series of chiral macrocycles with persistent figure‐eight and Möbius shapes. Single‐crystal structures of 7 compounds were solved, and 22 enantiomers were separated by preparative chiral HPLC. A notable pyrene‐bridged figure‐eight macrocycle, with its rigid, fully π‐conjugated and overcrowded structure, exhibited pure excimer emission and outstanding circularly polarized luminescence (CPL) properties, including a large dissymmetric factor (|glum|=3.8×10−2) and significant CPL brightness (BCPL=710.5 M−1cm−1). This method provides a versatile synthetic platform for producing various chiral D2‐symmetric figure‐eight macrocycles and singly or triply twisted Möbius macrocycles with C2 and D3 symmetry, offering tunable chiroptical properties for CPL applications.

Isoquinoline alkaloids with bioactive constituents from the roots and rhizomes of Hylomecon japonica

Isoquinoline alkaloids are important effective chemical components separated from the Hylomecon japonica, which were mostly reported as planar structures. By using chiral HPLC, we have concluded that some natural isoquinoline alkaloids exist as enantiomeric mixtures, providing a novel direction for future research on isoquinoline alkaloids. Finally, three pairs of enantiomeric isoquinoline alkaloids (1–3, including five undescribed compounds-1a/1b, 3a/3b and 2b), together with another eight known isoquinoline alkaloids (4–9) were isolated from the roots and rhizomes of Hylomecon japonica. Their structures were elucidated by NMR, HRESIMS, UV, IR, and their absolute configurations were further defined by quantum chemical calculations of ECD spectra. The cytotoxic activities of these isolated compounds in MCF-7 cells were evaluated by MTT methods. Among them, the alkaloids 1a, 1b, 2b, 3b and other known alkaloids 5a and 5b had good inhibitory effects on MCF-7 cells of breast cancer in vitro, with an IC50 lower than 20 μM.

Synthesis, Chiroptical Properties, and Absolute Configuration Determination of Phenyl‐4‐pyridyl‐2,5‐dipyrimidinylmethane

Phenyl‐4‐pyridyl‐2,5‐dipyrimidinylmethane (1) has been synthesized in five steps from 5‐methylpyrimidine. The structural feature of 1 is the symmetry elements with respect to the 180º rotation of the aryl group along the central C‐C bonds. Due to this structural feature, the compound 1 is expected the good crystallinity despite of the asymmetric molecular structure. The optical resolution of 1 was achieved by high performance liquid chromatography using chiral stationary phase (chiral HPLC). The circular dichroism (CD) spectra of the two fractions showed opposite signs. As expected, the X‐ray crystallographic analysis was successfully performed for both the racemic and enantiomerically pure crystals of compound 1, with the nitrogen atoms being unambiguously assigned in both cases. It was concluded the first‐eluted fraction in chiral HPLC was determined to be S configuration both by the CD calculation for reproducing the experimental CD spectrum and by the anomalous diffraction of X‐ray crystallographic analysis of enantiomerically pure 1.

Possibilities and limitations of computer assisted chiral HPLC method development for ozanimod on polysaccharide based chiral stationary phases.

In this study, a direct HPLC method was developed to determine the enantiomeric purity of the immunomodulatory drug, ozanimod. A systematic method development process was followed, incorporating risk assessment, identification of critical analytical procedure parameters, initial screening of stationary phases, and software-assisted optimization of method parameters. Eight different polysaccharide-based chiral columns were selected to assess chiral separation of enantiomers under polar organic elution mode. The most promising results were obtained using a methanol:2-propanol mixture on the amylose-based Chiralpak AD column. Following this, systematic modeling was conducted using DryLab software to optimize method conditions, including isocratic eluent composition, temperature, and flow rate. Baseline separation was achieved within fifteen minutes using the optimized parameters: Chiralpak AD column thermostated at 10°C, and a mobile phase of methanol:2-propanol: diethylamine, 70:30:0.1 (v/v/v %), delivered at a flow rate of 0.8 mL/min. The developed method was validated according to current guidelines and in silico robustness testing was conducted to determine tolerance limits for critical separation parameters and their impact on enantioresolution. Our findings demonstrate the utility of DryLab, typically employed for reversed-phase achiral separations, in optimizing chiral methods even in polar organic mode. Limitations of the selected approach the development of chiral separation methods are also highlighted.

Simultaneous enantioseparation of citrus flavonoids in chiral microemulsion electrokinetic chromatography using deep eutectic solvents

A novel, deep eutectic solvent (DES)-assisted sodium cholate (SC)-chiral microemulsion electrokinetic chromatography method is presented for separation of five flavonoid glycoside enantiomers, namely hesperidin, naringin, narirutin, eriocitrin, and neoeriocitrin. Herein, we develop a novel, green DES, whose addition considerably enhances the separation performance through multiple synergistic effects. A series of factors notably affecting chiral separation are systematically optimized: type and concentration of cyclodextrin, DES, and SC, oil phase type, and pH. The microemulsion comprises 0.5 % (v/v) n-octanol, 0.6 % (v/v) DES prepared using betaine and citric acid, 20 mM 2-HP-β-CD, 100 mM scdium cholate hydrate, and 15 mM borax buffer under the optimized conditions. Enantioseparations of the five enantiomers are achieved within 7 min, with resolutions ranging from 1.29 to 2.88. The method developed provides an innovative and effective way for chiral separation of enantiomers in complex food matrices.

L-Cysteine-Bonded Polymeric Monolithic Stationary Phase for Enantioseparation of Dansyl Amino Acids in Capillary Liquid Chromatography.

A chiral monolith stationary phase was fabricated by modifying the monolith surface using L-cysteine through a thiol-epoxy click reaction. L-cysteine-bonded polymer monolith was characterized by scanning electron microscopy/energy-dispersive X-ray and attenuated total reflectance Fourier-transformed infrared. The monomer content and modification temperature were carefully optimized to create a polymer monolith with excellent mechanical stability and permeability. Our findings revealed that the column morphology depended significantly on the porogen concentration and modification temperature for its morphology and efficiency. Adequate pores and binding sites were formed with the optimal porogen content, while a higher modification temperature improved the modification yield, enhancing peak shapes and increasing separation efficiency. The column demonstrated its capability for enantioseparation of dansyl glutamic acid, dansyl aspartic acid, dansyl methionine, and dansyl phenylalanine using a 60mM ammonium acetate buffer solution and acetonitrile in a 20:80 v/v ratio. It maintained good mechanical stability and repeatability with no relative standard deviation exceeding 7%. These results indicated that the L-cysteine-bonded polymer monolith has excellent potential as a chiral stationary phase.

Multiwavelength Optical Rotation Detection: An Effective Approach for the Recognition of Analytical and Semi‐Preparative HPLC Enantioseparation of the Chiral Pheromone Olean and Its Stereochemical Characterization

ABSTRACTOlean is the chiral spiroacetal sex pheromone of the female olive fruit fly Bactrocera oleae. Laboratory tests have demonstrated that the (R)‐(−)‐olean enantiomer is active on males, whereas females respond to (S)‐(+)‐olean. Here we present the first HPLC enantioseparation of olean using polysaccharide derivatives as chiral stationary phases and a polarimetric detector equipped with a micro‐flow cell capable of detecting optical rotation at six different wavelengths. Unlike blind UV detection, the polarimetric detector allows the chiral spiroacetal, which lacks a chromophore, to be detected as bisegnate peaks, indicating the opposite sign of the optical rotation. The HPLC enantioseparation was optimized on the coated type amylose‐based Chiralpak AS‐H CSP using a mobile phase consisting of n‐hexane‐2‐propanol 99.99:0.01 (v/v). These conditions were scaled up to a semi‐preparative level and allowed resolution of 2.5 mg of racemic sample in 5 min. Multiwavelength optical rotation detection during HPLC enantioseparation of racemic samples provides a direct readout of the stereochemistry of olean and allows tracking of virtual optical rotation dispersion curves without the need for preliminary collection of enantiomeric samples by semi‐preparative HPLC.

Proportionally controlled dual-chiral covalent organic frameworks via thiol-ene click reaction for efficient enantioseparation

Developing a universally applicable and commercially hopeful chiral material is a significant challenge for the separation and analysis field. In this study, innovative dual-chiral CCOFs ([SH-β-CD-NALC]x-COFs) modified with 6-deoxy-6-mercapto-β-cyclodextrin (SH-β-CD) and N-acetyl-l-cysteine (NALC) are presented. These CCOFs were synthesized using a single, one-step bottom-up approach at room temperature, specifically designed for enantiomer recognition and separation. We investigated the effect of varying ratios of multiple chiral selectors in CCOFs on chiral recognition abilities through adsorption experiments for the first time. The precisely engineered [SH-β-CD-NALC]1/6-COF, with excellent stability, crystallinity, abundant chiral sites, and a greater specific surface area, was well suited as a chiral stationary phase (CSP) in various racemates separation. The results showed satisfactory resolution, column efficiency, stability, and reproducibility. In addition, mechanism studies have revealed that the dual-chiral COF can offer triple selectivity and achieve the effect where 1 + 1 is greater than 2. This work emphasized the advantages of dual-chiral COF in achieving racemic drug separation, providing a new approach for the development of high-performance chiral separation platforms in the future.