Separation Of Enantiomers Research Articles

Multistate Aggregation-Induced Chiroptical Properties of Enantiopure Disulfide-Mediated Bispyrene Macrocycles.

A chiral bispyrene macrocycle designed for exclusive intermolecular excimer fluorescence upon aggregation was synthesized by a double hydrothiolation of a bis-enol ether macrocycle followed by intramolecular oxidation of free thiols. Unusually high stereoselectivity was achieved for the thiol-ene additions under templated conditions and Et3 B/O2 radical initiation. After enantiomer separation (chiral stationary phase HPLC), aqueous conditions provoked aggregation. Detailed structural evolution was afforded by ECD/CPL monitoring. Three regimes can be observed and characterized by strong modifications in chiroptical patterns under, at, or above a 70 % H2 O : THF threshold. In luminescence, high glum dissymmetry factors values were obtained, up to 0.022, as well as a double sign inversion of CPL signals during the aggregation, a behavior rationalized by time-dependent density functional theory (TDDFT) calculations. Langmuir layers of enantiopure disulfide macrocycles were formed at the air-water interface and transferred onto solid substrates to afford Langmuir-Blodgett films, which were then studied by AFM and UV/ECD/fluorescence/CPL.

Spatial Separation of Enantiomers by Field-Modulated Surface Scattering

A new optomechanical chiral resolution scheme for molecules possessing a permanent dipole moment is proposed. The approach involves the following molecular manipulation sequence: (i) a molecular alignment electromagnetic pulse, (ii) a time-delayed dipole orienting static electric field, and (iii) scattering from a frictional surface. The method offers various control parameters including pulse duration and intensity, static field strength, delay time, surface dynamic friction coefficient, and incident molecular velocity that can be tuned to optimize spatial enantioseparation. The scheme is demonstrated for the prototypical HSOH chiral molecule, predicting an enantiomeric excess as high as 50%.

Separation and quantification of the urinary enantiomers of 2-hydroxyglutaric acid by capillary electrophoresis with capacitively coupled contactless conductivity detection: Application to the diagnosis of D- and L-2-hydroxyglutaric aciduria.

2-hydroxyglutaric aciduria is an inherited neurometabolic disorder with two major types: D-2-hydroxyglutaric aciduria and L-2-hydroxyglutaric aciduria. An easy and fast capillary electrophoresis system combined with a capacitively coupled contactless conductivity detection method was developed for the enantioseparation and determination of D- and L-2-hydroxyglutaric acid in urine. D- and L-2-hydroxyglutaric acids were separated using vancomycin as the chiral selector. The optimal separation conditions for enantiomers were achieved by the use of a buffer containing 50mM 4-(N-morpholino) butane sulfonic acid solution (pH 6.5), an electroosmotic flow modifier (0.001% [w/v] polybrene), and 30mM vancomycin as chiral selector. The analysis time was 6min under optimal conditions. The optimized and validated method was successfully implemented for quantifying D- and L-2-hydroxyglutaric aciduria in patients' urine, without any pretreatment step. The linearity of the method was determined to be in the range of 2-100mg/L for D- and L-2-hydroxyglutaric acid in urine. The precision (relative standard deviation%) was obtained at about 7%. For D- and L-2-hydroxyglutaric acids, the limits of detection were 0.567 and 0.497mg/L, respectively.

Continuous enantiomeric separation using water-oil-water segmented flow system

The multiphase segmented flow, which consisted of the material, extraction, and recovery phases, was applied to the chiral separation. The use of simultaneous extraction and back-extraction with multiple phases aims to prevent the accumulation of target material in the extraction phase by efficiently removing it in the recovery phase, thereby enabling extraction performance beyond the equilibrium. This study demonstrated the chiral separation of an aqueous racemic amino acid derivative (3,5-dinitrobenzoyl-(R,S)-leucine) with a cinchona alkaloid chiral host. A droplet manipulation system comprising PFA manifolds and valves was utilized to alternately form an acidic material phase slug and a basic recovery phase slug in a 1-octanol oil phase containing the chiral host. Two aqueous phases were collected separately using valves controlled by an optical sensor signal. The manipulation system achieved stable operation across various conditions, including flow rate, residence time, and slug lengths. The enantiomeric excess, which represents the purity of the chirality, could be increased to 50 % through 200 s extraction in a one-step operation.

Spin‐Controlled Helical Quantum Sieve Chiral Spectrometer (Adv. Mater. 24/2023)

Identification of Chiral Enantiomers In article number 2209125, Hossam Haick and co-workers report a molecular-spin-sensitive-antenna (MSSA) made up of organically functionalized graphene layers on a fibrous helical cellulose network. The MSSA structure combines relativistic spin–orbit interaction coupled with organic spin-coherence and offers spatiotemporal chiral molecule separation, recognition, and selective identification of enantiomers. Coupled with neuromorphic artificial intelligence, it enables rapid classification of chiral molecules, resulting in portable spin-controlled spectrometry.

Peptide-derived coordination frameworks for biomimetic and selective separation.

Peptide-derived metal-organic frameworks (PMOFs) have emerged as a class of biomimetic materials with attractive performances in analytical and bioanalytical chemistry. The incorporation of biomolecule peptides gives the frameworks conformational flexibility, guest adaptability, built-in chirality, and molecular recognition ability, which greatly accelerate the applications of PMOFs in enantiomeric separation, affinity separation, and the enrichment of bioactive species from complicated samples. This review focuses on the recent advances in the engineering and applications of PMOFs in selective separation. The unique biomimetic size-, enantio-, and affinity-selective performances for separation are discussed along with the chemical structures and functions of MOFs and peptides. Updates of the applications of PMOFs in adaptive separation of small molecules, chiral separation of drug molecules, and affinity isolation of bioactive species are summarized. Finally, the promising future and remaining challenges of PMOFs for selective separation of complex biosamples are discussed.

In situ growth preparation of a new chiral covalent triazine framework core-shell microspheres used for HPLC enantioseparation.

Themanufacturing of chiral covalent triazine framework core-shell microspheres CC-MP CCTF@SiO2 composite is reportedas stationary phase for HPLC enantioseparation. The CC-MP CCTF@SiO2 core-shell microspheres were prepared by immobilizing chiral COF CC-MP CCTF constructed using cyanuric chloride and (S)-2-methylpiperazine on the surface of activated SiO2 through an in-situ growth approach. Various racemates as analytes were separated on the CC-MP CCTF@SiO2-packed column. The experimental results indicate that 19 pairs of enantiomers were well separated on the CC-MP CCTF@SiO2-packed column, including alcohols, phenols, amines, ketones, and organic acids. Among them, there are 17 pairs of enantiomers that can achieve baseline separation with good peak shapes. Their resolution values on this chiral column are between 0.4 and 5.61. The influences of analyte mass, column temperature, and composition of the mobile phase on the resolution of enantiomers were studied. In addition, the chiral resolution ability of CC-MP CCTF@SiO2-packed column was compared with the commercial chiral chromatographic columns (Chiralpak AD-H and Chiralcel OD-H columns) and some CCOF@SiO2 chiral columns (β-CD-COF@SiO2, CTpBD@SiO2, and MDI-β-CD-modified COF@SiO2). The CC-MP CCTF@SiO2-packed column exhibited some unique advantages and can complement these chiral columns in chiral separations. The research results show that the CC-MP CCTF@SiO2 chiral column offered high column efficiency (e.g., 17680 plates m-1 for ethyl mandelate), low column backpressure (5-9 bar), high enantioselectivity, and excellent chiral resolution ability for HPLC enantioseparation with good stability and reproducibility. The relative standard deviations (RSD) (n = 5) of the retention time, and peak areas by repeated separation of ethyl mandelate are 0.23% and 0.67%, respectively. It demonstrates that the CC-MP CCTF@SiO2 core-shell microsphere composite has great potential in enantiomeric separation by HPLC.

Enantiomeric Separation and Degradation of Benoxacor Enantiomers in Horticultural Soil by Normal-Phase and Reversed-Phase High Performance Liquid Chromatography.

The separation of benoxacor enantiomers on six commercial chiral columns was investigated by high-performance liquid chromatography (HPLC) under normal-phase and reversed-phase conditions. The mobile phases included hexane/ethanol, hexane/isopropanol, acetonitrile/water, and methanol/water. The effects of the chiral stationary phases (CSPs), temperature, and mobile phase composition and ratio on the separation of benoxacor enantiomers were examined. Under normal-phase conditions, the two benoxacor enantiomers were completely separated on Chiralpak AD, Chiralpak IC, Lux Cellulose-1, and Lux Cellulose-3 columns and partially separated on a Lux Cellulose-2 column. Under reversed-phase conditions, benoxacor enantiomers were completely separated on a Lux Cellulose-3 column and partially separated on Chiralpak IC and Lux Cellulose-1 columns. Normal-phase HPLC performed better than reversed-phase HPLC for the separation of benoxacor enantiomers. As the column temperature increased from 10 °C to 4 °C, the enthalpy (ΔH) and entropy (ΔS) results indicated that the resolution was strongly affected by the temperature and that the lowest temperature did not always produce the best resolution. An optimized separation method on the Lux Cellulose-3 column was used to investigate the stability of benoxacor enantiomers in solvents and the degradation of benoxacor enantiomers in three types of horticultural soil. Benoxacor enantiomers were stable, and degradation or racemization were not observed in methanol, ethanol, isopropanol, acetonitrile, hexane, or water (pH = 4.0, 7.0, and 9.0). In three horticultural soils, the degradation rate of S-benoxacor was faster than that of R-benoxacor, resulting in soil enrichment with R-benoxacor. The results of this study will help to improve the risk assessment of enantiomer levels of benoxacor in the environment.

Unraveling complexation and separation of novel asymmetric uranyl-5-methoxy-2-(4-methoxy-6’-(quinazolin-2-yl)-[2,2’-bipyridin]-6-yl) quinazoline to chiral fungicides R/S-metalaxyls and R/S-benalaxyls

ABSTRACT Chiral fungicides R/S-metalaxyls(R/S-MTLs) and R/S-benalaxyls(R/S-BNLs) have different biotoxicity. It is very important to unravel molecular recognition and enantiomeric separation of R/S-MTLs or R/S-BNLs. In this paper, we designed a novel asymmetric ligand: 5-methoxy-2-(4-methoxy-6”-(quinazolin-2-yl)-[2,2”−bipyridin]-6-yl)quinazoline(MMQBQ), further constructed a new asymmetric receptor Uranyl-MMQBQ by coordination of MMQBQ with uranyl. The complexation and separation of receptor Uranyl-MMQBQ with guests R/S-MTLs or R/S-BNLs were systematically studied using density functional theory (DFT) method. The respects of ESP, MBO, RFC, LOL, EDDM, EST-NOCV, QTAIM, FMOs, IGMH, and Gibbs free energy changes were calculated and analyzed. The results indicated that the MMQBQ could form a stable receptor Uranyl-MMQBQ with uranyl, which could selectively recognize and separate chiral R/S-MTLs or R/S-BNLs by receptor’s U coordinating to carbonyl oxygens of guests. For R/S-MTLs, separation factors(SF S/R) of Uranyl-MMQBQ were more than 16 in water, chlorobenzene, toluene, carbon tetrachloride and cyclohexane, enantioselectivity coefficients(ESCs) ranged of 99.92%-94.24% in the above four solvents. While for R/S-BNLs, ESCs of Uranyl-MMQBQ were over 99% and SF S/R values were a range of 533.97–2839.36 in the above organic solvents. The findings provide valuable information for design of novel uranyl ligand and its complexes, meanwhile supply useful guidance for experiments on separation of other chiral fungicides in environmental protection.

Determination of amphetamine enantiomers in urine by conductive vial electromembrane extraction and ultra-high performance supercritical fluid chromatography tandem mass spectrometry.

Separation and quantification of amphetamine enantiomers are commonly used to distinguish between consumption of prescription amphetamine (mostly S-amphetamine) and illicit forms of the drug (racemate). In this study, electromembrane extraction with prototype conductive vials was combined with ultra-high performance supercritical fluid chromatography (UHPSFC-MS/MS) to quantify R- and S-amphetamine in urine. Amphetamine was extracted from 100 μL urine, diluted with 25 μL internal standard solution and 175 μL 130 mM formic acid, across a supported liquid membrane (SLM) consisting of 9μL of a 1:1(w/w) mixture of 2-nitrophenyloctyl ether (NPOE) and bis(2-ethylhexyl)phosphite (DEHPi) into an acceptor phase containing 300 μL 130 mM formic acid. The extraction was facilitated by the application of 30 V for 15 min. Enantiomeric separation was achieved using UHPSFC-MS/MS with a chiral stationary phase. The calibration range was 50-10,000 ng/mL for each enantiomer. The between-assay CV was ≤5%, within-assay CV ≤ 1.5%, and bias within ±2%. Recoveries were 83%-90% (CV ≤ 6%), and internal standard corrected matrix effects were 99-105 (CV ≤ 2%). The matrix effects ranged from 96% to 98% (CV ≤ 8%) when not corrected by the internal standard. The EME method was compared with a chiral routine method that employed liquid-liquid extraction (LLE) for sample preparation. Assay results were in agreement with the routine method, and the mean deviation between methods was 3%, ranging from -21% to 31%. Finally, sample preparation greenness was assessed using the AGREEprep tool, which resulted in a greenness score of 0.54 for conductive vial EME, opposed to 0.47 for semi-automated 96-well LLE.

Chiral gold nanoparticles/graphene oxide membranes with ultrahigh and stable permeance for sieving and enantioseparation

In accurately balancing membrane permeability and separation coefficient, effectively preventing membrane collapse in separation process is the core to improve the efficiency of membrane separation for two-dimensional materials. Here, we used L-cysteine (L-Cys) functionalized gold nanoparticles as scaffolds and spacers and fabricated a multi-effect separation membrane with GO nanosheets. The as-prepared membrane performed the following as a separation membrane: (1) As a robust spacer, the gold nanoparticles, prevented the collapse of the GO sheet and thus the membrane showed stability; (2) The existence of amphiphilic L-cysteine enabled the possibility of regulating the GO sheets layer spacing between 16.9 and 17.8 Å by pH, which achieves controllable ultrahigh permeance for size sieving. The maximum permeation rate of the membrane can reach 216.6 L m−2 h−1 bar−1, which is much greater than that of pure GO membrane; (3) Further, we demonstrate the following properties of the as-prepared membranes embedded with amphiphilic chiral gold nanoparticles for the enantiomer separation. The membrane has high enantioselectivity for Pen racemates, and its chiral separation factor and permeation rate can reach 1.83 and 21.7 L m−2 h−1 bar−1, respectively.

Chirale Kontrolle von Molekülen in der Gasphase mittels Mikrowellenpulsen

AbstractMikrowellen Drei‐Wellen Mischen ist ein bemerkenswerter, neuartiger Ansatz für tief gehende Untersuchungen an chiralen Molekülen. Diese nicht‐lineare und kohärente Technik, die auf resonanten Mikrowellenpulsen basiert, dient als robuste Methode zur Unterscheidung von Enantiomeren chiraler Moleküle und zur Bestimmung des Enantiomerenüberschusses, sogar für komplexe Mischungen chiraler Moleküle. Neben solchen analytischen Anwendungen erlaubt uns die Verwendung maßgeschneiderter Mikrowellenpulse die Kontrolle und Manipulation von Chiralität auf der Molekülebene. Der vorliegende Artikel liefert einen Überblick zu jüngsten Entwicklungen im Bereich des Mikrowellen Drei‐Wellen Mischens und seine Erweiterung zu enantiomerenspezifischem Populationstransfer. Letzteres ist ein entscheidender Schritt in Richtung Enantiomerentrennung – energetisch und schließlich auch räumlich. Im letzten Abschnitt präsentieren wir neue experimentelle Ergebnisse zur Verbesserung des enantiomerenspezifischen Populationstransfers, bei denen wir einen Enantiomerenüberschuss von 40 % in dem Rotationsenergieniveau von Interesse allein mit Hilfe von Mikrowellenpulsen erreichen konnten.

Engineering UiO-68-Typed Homochiral Metal–Organic Frameworks for the Enantiomeric Separation of Fmoc-AAs and Mechanism Study

Homochiral metal-organic frameworks (HMOFs) have been widely investigated in the application of enantiomeric separation. Nonetheless, it remains a significant challenge to explore the effect of multiple weak interactions between HMOF adsorbents and chiral adsorbates on enantiomeric separation performance still. In this work, robust chiral amine-alcohol-functionalized UiO-68-typed Zr-HMOFs 1-3 with the same hydrogen-bonding sites but slightly different π-binding sites were prepared for the enantioseparation of amino acid derivatives (Fmoc-AAs) with large π-binding groups. As a consequence of multiple host-guest interactions, these Zr-HMOFs exhibit speedy adsorption and high adsorption capacity for Fmoc-L/D-AAs and dissimilar enantioselectivity for the adsorption of their enantiomers. Materials 1 and 2 exhibit excellent enantioselective separation performance for Fmoc-valine with a single terminal π-binding group, while material 3 displays excellent enantioselective separation performance for Fmoc-phenylalanine and Fmoc-tryptophan with π-binding groups at both ends. As evidently demonstrated by our experimental and density functional theory (DFT) computational results, when the number of π-binding groups preset in the confined chiral space of adsorbents matches the number of π-binding groups of chiral adsorbates, the synergism of π-π or σ-π interactions will increase enantioselectivity; otherwise, the competition interactions from redundant identical binding sites will weaken enantioselectivity. Our case not only provides a tremendously typical system for investigating the collaborative discrimination of multiple weak interactions and exploring the impact of relatively excessive binding sites of HMOF adsorbents or chiral adsorbates on the enantioselective separation performance but also provides guidance for targeted functional modifications of high-performance chiral porous materials.

Length-Dependent Enantioselectivity of Carbon Nanotubes by Gel Chromatography

High-purity enantiomer separation of chiral single-wall carbon nanotubes (SWCNTs) remains a challenge compared with electrical type and chirality separations due to the limited selectivities for both chirality and handedness, which is important for an exploration of their properties and practical applications. Here, we performed length fractionation for enantiomer-purified SWCNTs and found a phenomenon in which the enantioselectivities were higher for longer nanotubes than for shorter nanotubes due to length-dependent interactions with the gel medium, which provided an effective strategy of controlling nanotube length for high-purity enantiomer separation. Furthermore, we employed a gentler pulsed ultrasonication instead of traditional vigorous ultrasonication for preparation of a low-defect long SWCNT dispersion and achieved the enantiomer separation of single-chirality (6,5) SWCNTs with an ultrahigh enantiomeric purity of up to 98%, which was determined by using the linear relationship between the normalized circular dichroism intensity and the enantiomeric purity. Compared with all results reported previously, the present enantiomeric purity was significantly higher and reached the highest level reported to date. Due to the ultrahigh selectivity in both chirality and handedness, the two obtained enantiomers exhibited perfect symmetry in their circular dichroism spectra, which offers standardization for characterizations and evaluations of SWCNT enantiomers.

Enantioseparation/Recognition based on nano techniques/materials.

Enantiomers show different behaviors in interaction with the chiral environment. Due to their identical chemical structure and their wide application in various industries, such as agriculture, medicine, pesticide, food, and so forth, their separation is of great importance. Today, the term "nano" is frequently encountered in all fields. Technology and measuring devices are moving towards miniaturization, and the usage of nanomaterials in all sectors is expanding substantially. Given that scientists have recently attempted to apply miniaturized techniques known as nano-liquid chromatography/capillary-liquid chromatography, which were originally accomplished in 1988, as well as the widespread usage of nanomaterials for chiral resolution (back in 1989), this comprehensive study was developed. Searching the terms "nano" and "enantiomer separation" on scientific websites such as Scopus, Google Scholar, and Web of Science yields articles that either use miniaturized instruments or apply nanomaterials as chiral selectors with a variety of chemical and electrochemical detection techniques, which are discussed in this article.

Preparation of Chiral Porous Organic Cage Clicked Chiral Stationary Phase for HPLC Enantioseparation.

Porous organic cages (POCs) are a new subclass of porous materials, which are constructed from discrete cage molecules with permanent cavities via weak intermolecular forces. In this study, a novel chiral stationary phase (CSP) has been prepared by chemically binding a [4 + 6]-type chiral POC (C120H96N12O4) with thiol-functionalized silica gel using a thiol-ene click reaction and applied to HPLC separations. The column packed with this CSP presented good separation capability for chiral compounds and positional isomers. Thirteen racemates have been enantioseparated on this column, including alcohols, diols, ketones, amines, epoxides, and organic acids. Upon comparison with a previously reported chiral POC NC1-R-based column, commercial Chiralpak AD-H, and Chiralcel OD-H columns, this column is complementary to these three columns in terms of its enantiomeric separation; and can also separate some racemic compounds that cannot be separated by the three columns. In addition, eight positional isomers (iodoaniline, bromoaniline, chloroaniline, dibromobenzene, dichlorobenzene, toluidine, nitrobromobenzene, and nitroaniline) have also been separated. The influences of the injection weight and column temperature on separation have been explored. After the column has undergone multiple injections, the relative standard deviations (RSDs) for the retention time and selectivity were below 1.0 and 1.5%, respectively, indicating the good reproducibility and stability of the column for separation. This work demonstrates that POCs are promising materials for HPLC separation.

Development of an enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of methorphan and its O-demethylated metabolite in human blood and its application to post-mortem samples

In the present work an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the separation and quantitative determination of dextro - and levo -methorphan and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. The separation of enantiomers of methorphan and metabolites was performed on the polysaccharide-based chiral column Lux AMP in combination with acetonitrile and 5 mM aqueous ammonium bicarbonate pH 11 in the ratio 50:50 (%, v/v) as mobile phase with the flow rate 1 mL/min. The mass spectrometer was operated in scheduled multiple reaction monitoring (MRM) mode, with four transitions for each dextromethorpan, levomethorphan, dextrorphan and dextromethorphan-d3 and two transitions for each levorphanol, levorphanol-d3 and dextrorphan-d3. Application of this method to human post-mortem blood samples confirmed cases of severe overdosing with dextromethorphan, levomethorphan, and less commonly with both.

Synthesis of Cyclodextrin-based MOFs incorporating amino acid chiral ligands for chiral separation of naproxen enantiomers

The design of adsorbent materials capable of efficiently separating chiral molecules were a promising way to achieve selective recognition and separation of racemic enantiomers. Here, a γ-Cyclodextrin (γ-CD) based metal–organic framework (MOF) with D-Histidine (D-His) as a chiral ligand was synthesized for the first time to construct a functional chiral adsorption separation material (D-His@γ-CD-MOF), which can be used as an adsorbent for Naproxen enantiomer (S-/R-Npx). D-His@γ-CD-MOF provided a large number of chiral recognition sites for the selective enrichment of S-Npx based on the realization of the spatial advantage of the MOF structure, and the e.e. % value of a single separation of Npx racemates could reach 20.0 %. Meanwhile, the intermolecular binding patterns were predicted by computer simulations, and the chiral adsorption was attributed to the difference in affinity due to the difference in π-lone pair miscellaneous and hydrogen bonding force capacities of D-His@γ-CD-MOF and S-/R-Npx. Therefore, considering the stereospecific structure of D-His@γ-CD-MOF as well as the lone pair electron and hydrogen bonding coordination ability, it has great prospects for application as a chiral resolution agent.

Enantioseparation and mechanism study on baclofen by capillary electrophoresis and molecular modeling

Enantioselective analysis of chiral drugs plays a significant role in chemistry, biology and pharmacology. Baclofen, an antispasmodic chiral drug, has been widely studied due to the obvious differences in toxicity and medical activity between enantiomers. Herein, a simple and efficient method for separation of baclofen enantiomers by capillary electrophoresis was established without complicated sample derivatization and expensive instruments. Then, the molecular modeling and density functional theory were used to simulate and investigate the chiral resolution mechanism of electrophoresis, the calculated intermolecular forces were directly presented by visualization softwares. Moreover, the theoretical and experimental electronic circular dichroism (ECD) spectra of ionized baclofen were compared, and the configuration of dominant enantiomer in the nonracemic mixture can be determined by ECD signal intensity, which was proportional to the electrophoresis peak area difference of the corresponding enantiomer excess experiments. In this way, the peak order identification and configuration quantification of baclofen enantiomers in electrophoretic separation were successfully achieved without relying on a single standard.

Preparation and chromatographic performance of chiral peptide-based stationary phases for enantiomeric separation.

This study presents the development of three new chiral stationary phases. They are based on silica modified with peptides containing phenylalanine and proline. Successful analyses and characterizations were conducted using Fourier transform infrared spectra, elemental analysis, and thermogravimetric analysis. After this, the enantioselective performance of the three chiral peptide-based columns was evaluated. The evaluation used 11 racemic compounds under normal-phase high performance liquid chromatography mode. Optimized enantiomeric separation conditions were established. Under these conditions, the enantiomers of flurbiprofen and naproxen were successfully separated on CSP-1 column: the separation factor of these was 1.27 and 1.21, respectively. In addition, the reproducibility of the CSP-1 column was also investigated. The results of the investigation illustrated that the stationary phases have good reproducibility (RSD = 0.73%, n = 5).