Enantioseparation of Amino Acids on a Chiral Stationary Phase Derived from L‐α‐Norleucinyl‐ and Pyrrolidinyl‐disubstituted Cyanuric Chloride by High‐Performance Liquid Chromatography

A silica‐based chiral stationary phase (CSP) derived from L‐α‐norleucinyl‐ and pyrrolidinyl‐disubstituted cyanuric chloride was prepared for the enantioseparation of methyl esters of N‐(3,5‐dinitrobenzoyl) amino acids by high‐performance liquid chromatography. The chromatographic results show that effective enantioseparation of methyl esters of N‐(3,5‐dinitrobenzoyl)amino acids, except for proline, was achieved on this chiral stationary phase. The chromatographic resolution of racemic n‐propyl ester of N‐(3,5‐dinitrobenzoyl)valine on CSP‐B is better than that of racemic methyl ester of N‐(3,5‐dinitrobenzoyl)valine on CSP‐B or CSP‐A reported previously (J. Chromatogr. A, 676 (1994) 303). The comparison of the chromatographic results obtained in this study with those on CSP‐A reported previously reveals that steric effect, instead of hydrophobic interaction, between the n‐butyl group attached to the chiral center of the chiral selector and the alkyl group attached to the chiral center of the chiral selectand plays a significant role in chiral discrimination. The increase in the selectivity factor of methyl esters of N‐(3,5‐dinitrobenzoyl)amino acids with bulky alkyl groups was examined on CSP‐B.     

Nonlinear effects in enantioselective chromatography: prediction of unusual elution profiles of enantiomers in non-racemic mixtures on an achiral stationary phase doped with small amounts of a chiral selector

Nonlinear effects caused by molecular association of enantiomers in non-racemic mixtures can cause unexpected effects in chiroptics, NMR spectroscopy, homogeneous catalysis, and chromatography. Herein we present a theoretical model to simulate and verify unusual elution orders of enantiomers on an achiral stationary phase doped with a small amount of a chiral selector or achiral columns coupled with columns doped with a chiral selector. Scenarios with strong, medium, and weak associations of enantiomers, different separation efficiencies typical for flash chromatography and liquid chromatography, and the influence of the enantioselectivity of the chiral selector on the complex equilibria have been investigated. The findings presented here are of importance for the validation of the determination of enantiomeric ratios in not fully separated elution zones as well as for the preparative separation of non-racemic enantiomeric mixtures on chiral stationary phases bonded to achiral matrices.     

Last ten years (2008–2018) of chiral ligand‐exchange chromatography in HPLC: An updated review

Chiral ligand‐exchange chromatography is one of the elective strategies for the direct enantioresolution of small chelating compounds: amino acids, diamines, amino alcohols, diols, small peptides, etc. Unlike other methods, the interaction between chiral selector and analyte enantiomers is mediated by a cation, thus producing diastereomeric ternary complexes. Two main approaches are conventionally applied in chiral ligand‐exchange chromatography. The first relies upon chiral stationary phases where the chiral selector is either covalently immobilized or physically adsorbed onto suitable packing materials (coated phases). In the second approach, chiral molecules are added to the eluent, thus generating chiral eluent systems. Among the advantages of chiral ligand‐exchange chromatography, the generation of UV/vis‐active metal complexes, and the use of commercially available or easy‐to‐synthesize chiral selectors, in combination to rather inexpensive achiral columns for coated phases and chiral eluents, are noteworthy. Besides amino acids and amino alcohols, other species have proven suitable for chiral ligand‐exchange chromatography applications. Recently, the use of either chiral ionic liquids or micellar liquid chromatography systems as well as the successful off‐column formation of diastereomeric complexes have expanded the selectivity profiles and application fields. All of these issues are touched in the review, shedding light to the contributions appeared in the last decade.     

Monolithic silica columns functionalized with substituted polyproline‐derived chiral selectors as chiral stationary phases for high‐performance liquid chromatography

In this study, two polyproline‐derived chiral selectors are bonded to monolithic silica gel columns. In spite of high chiral selector coverage, the derivatization was found to have only a slight effect on the hydrodynamics of the mobile phase through the column. The enantioseparation ability of the resulting chiral monolithic columns was evaluated with a series of structurally diverse racemic test compounds. When compared to analogous bead‐based chiral stationary phases, higher enantioseparation and broader application domain were observed for monolithic columns. Moreover, the increase in flow rate produces a minor reduction of resolution, which permits to shorten analysis time. Additionally, increased loadability defines chiral polyproline derived monoliths as adequate for preparative chromatography.     

Enantiomeric separation of alcohols and amines on a proline chiral stationary phase by gas chromatography

A new chiral stationary phase for gas chromatography was prepared by covalently attaching a diproline chiral selector that has proven to be effective in liquid chromatography to a methylhydrosiloxane-dimethylsiloxane copolymer. With this new chiral stationary phase for GC, racemic aromatic alcohols could be resolved without derivatization. Racemic aromatic and aliphatic amines could also be resolved after derivatization of the amino groups with trifluoroacetic anhydride or isopropyl isocyanate. On this stationary phase, the isopropyl isocyanate derivatives of amines showed higher enantioselectivity than the trifluoroacetic anhydride derivatives. In both the enantiomeric separations of alcohols and derivatized amines, the aromatic racemic analytes showed higher enantioselectivities than their aliphatic analogs. Some of the alpha-amino and alpha-hydroxy aromatic acids could also be separated after derivatization to N-trifluoroacetyl methyl esters for amino acids or O-trifluoroacetyl methyl esters for hydroxyl acids.     

Structure‐enantioselectivity relationships for the study of chiral recognition in peptide enantiomer separation on cinchona alkaloid‐based chiral stationary phases by HPLC: Influence of the N‐terminal protecting group

Eleven different N‐terminal protecting groups (acetyl, benzoyl, FMOC, etc.) were employed for the HPLC separation of oligoalanine peptide enantiomers containing up to six amino acids. Isocratic HPLC separations were performed using a hydro‐organic buffered mobile phase and 4 mm ID columns containing three different chiral anion exchange stationary phases based on cinchona alkaloid‐derived chiral selectors. For most peptides successful separations could be obtained with all protecting groups, although those comprising aromatic moieties were found to yield higher enantioselectivities than those with aliphatic residues, since they are capable of undergoing favourable π‐π interactions with the selector. Systematic investigations concerning the presence or absence of structural features of related protecting groups showed that the use of protecting groups that are optimally adjusted to the binding pocket of the chiral selector effects a significant gain in enantioselectivity. At the same time these studies provided new insights into the chiral recognition mechanism.     

Liquid chromatographic enantioseparation of limonene‐based carbocyclic β‐amino acids on zwitterionic Cinchona alkaloid‐based chiral stationary phases

The eight stereoisomers of limonene‐based carbocyclic β‐amino acids containing three chiral centers have been directly separated on chiral stationary phases containing Cinchona alkaloid‐based zwitterionic selectors. The effects of bulk solvent composition of the mobile phase, the nature of base additives, counterion concentration, and the structure of selector on the enantiorecognition were studied. Experiments were performed at constant mobile phase composition in the temperature range 5–40°C to study the effect of temperature. Thermodynamic parameters were calculated on the basis of the plots of ln α versus 1/T curves. The enthalpically or entropically driven enantioseparations were found to depend strongly on the structures of analyte and selector. The eight stereoisomers of limonene‐based carbocyclic β‐amino acids could be differentiated as well‐separated peaks in a traditional 1D chromatographic system in two runs by applying the two complementary ZWIX(+)™ and ZWIX(–)™ columns.     

Chiral HPLC and physical characterisation of orthoconic antiferroelectric liquid crystals

New orthoconic antiferroelectric liquid crystalline materials were synthesised and characterised in their racemic forms and as (S) enantiomers. The materials possess oligo-methylene spacers of different lengths in semi-fluorinated achiral chains and lateral substitution by fluorine at two different positions of the molecular core. For comparison purposes, analogical materials without fluorine lateral substitutions were also prepared. Polysaccharide chiral stationary phases based on two different chiral selectors were used for the separation of the enantiomers of the individual racemic mixtures by high-performance liquid chromatography. A baseline separation of (S) and (R) enantiomers was obtained for four of the six studied liquid crystalline materials. Two of the materials were partially separated under the optimised separation conditions. The elution order of the individual enantiomers in the racemic mixtures was successfully assigned, as pure (S) enantiomers of all the studied materials were available. Both the position of the fluorine atom within the molecular core and the size of the achiral moiety had significant effects on the separation of the individual enantiomers of the studied compounds. Moreover, it was also found that the structure of the chiral stationary phase selector significantly influenced the enantiomeric resolution.     

Green high‐performance liquid chromatography enantioseparation of lansoprazole using a cellulose‐based chiral stationary phase under ethanol/water mode

A simple and environmentally friendly reversed‐phase high‐performance liquid chromatography method for the separation of the enantiomers of lansoprazole has been developed. The chromatographic resolution was carried out on the cellulose‐based Chiralpak IC‐3 chiral stationary phase using a green and low‐toxicity ethanol‐aqueous mode. The effects of water content in the mobile phase and column temperature on the retention of the enantiomers of lansoprazole and its chiral and achiral related substances have been carefully investigated. A mixed‐mode hydrophilic interaction liquid chromatography and reversed‐phase retention mechanism operating on the IC‐3 chiral stationary phase allowed us to achieve simultaneous enantioselective and chemoselective separations in water‐rich conditions. The enantiomers of lansoprazole were baseline resolved with a mobile phase consisting of ethanol/water 50:50 without any interference coming from chiral and achiral impurities within 10 min.     

High‐performance liquid chromatography enantioseparation of polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases under multimodal elution

An investigation on the high‐performance liquid chromatography enantioseparation of 12 polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases is described. The overall study was directed toward the generation of efficient separations in order to obtain pure atropisomers that will serve as ligands for building homochiral metal organic frameworks. Four coated columns—namely, Lux Cellulose‐1, Lux Cellulose‐2, Lux Cellulose‐4, and Lux Amylose‐2—and two immobilized columns—namely, Chiralpak IC and IA—were used under normal, polar organic, and reversed‐phase elution modes. Moreover, Chiralcel OJ was considered under normal‐phase and polar organic conditions. The effect of the chiral selector and mobile phase composition on the enantioseparation, the enantiomer elution order and the beneficial effect of nonstandard solvents were studied. The effect of water in the mobile phase on the enantioselectivity and retention was investigated and retention profiles typical of hydrophilic interaction liquid chromatography were observed. Interesting phenomena of solvent‐induced enantiomer elution order reversal occurred under normal‐phase mode. All the considered 4,4′‐bipyridines were enantioseparated at the multimilligram level.     

Liquid‐Phase Enantioselective Chromatographic Resolution Using Interpenetrated,Homochiral Framework Materials

Effective separation of mixtures of enantiomers is of continuing interest in analytical and preparative chromatography, with new materials frequently designed and tested. We report two new enantiomerically pure 2D→3D interpenetrated materials used as stationary liquid chromatographic (LC) phases that are shown to resolve selected racemic mixtures with enantiomeric and chemical selectivity. Dicarboxylate ligands derived from amino acids on naphthalene and perylene cores form 2D frameworks that interpenetrate to give 3D structures. Selectivity is initially tested by uptake from solution; subsequent LC methods show that the materials exhibit resolution of racemic analytes in ‘micro‐columns’ and that the two closely related materials show markedly different selectivity for different analytes with much greater activity than the ligands alone. Comparison with a close‐packed analogue suggests that the separation activity is largely due to surface effects.     

Enantiomeric separation of unusual secondary aromatic amino acids

Summary High-performance liquid chromatographic and gas chromatographic methods were developed for the separation of unusual secondary aromatic amino acids. Amino acids containing 1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydronorharmane-1-carboxylic acid and 1,2,3,4-tetrahydro-3-carboxy-2-carboline moieties were synthetized in racemic or chiral forms. The high-performance liquid chromatography was carried out either on a teicoplanin-containing chiral stationary phase or on an achiral C₁₈ column. In the latter case the diastereomers of the amino acids formed by precolumn derivatization with the chiral reagents 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate or 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide were separated. The gas chromatographic analyses were based on separation on a Chirasil-L-Val column. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997     

Enantioseparation of 2‐(substituted phenyl)propanoic acids by high‐speed countercurrent chromatography and investigation of the influence of substituents in enantiorecognition

This paper concentrates on the enantioseparation of racemic 2‐(substituted phenyl)propanoic acids by high‐speed countercurrent chromatography with substituted β‐cyclodextrin as the chiral selector, and an investigation of the influence of the substituent on the benzene ring in enantiorecognition between the chiral selector and enantiomer of each racemate is presented. This is an extension research of our previous work on the enantioseparation of 2‐phenyl propanoic acid derivatives, to investigate the relationship between the value of enantioseparation factor and the different substituent on the benzene ring. In total, ten racemic 2‐(substituted phenyl)propanoic acids were investigated, of which four including 2‐(4‐nitrophenyl)propanoic acid, 2‐(4‐methylphenyl)propanoic acid, 2‐(4‐hydroxyphenyl)propanoic acid, and 2‐(4‐chlorophenyl)propanoic acid, were studied by countercurrent chromatography for the first time, and two racemates were successfully enantioseparated. The distribution ratio and enantioseparation factor for all the ten racemates were determined by enantioselective liquid–liquid extraction. The results showed that an electron‐donating group on the benzene ring presents a higher enantiorecognition induced by chiral selector than that of racemates with an electron‐withdrawing group on the benzene ring.     

Two-component chiral phase transfer catalysts: enantioselective esterification of an N-acylated amino acid

[see reaction]. The first example of a two-component chiral phase transfer catalyst is described which, operating in a biphasic solvent system, preferentially esterifies one enantiomer of a racemic N-acylated amino acid. The two-component catalyst is comprised of an achiral quaternary ammonium ion and a proline-derived chiral selector initially developed for the liquid chromatographic separation of enantiomers.     

Preparation and evaluation of a chiral HPLC stationary phase based on cone calix[4]arene functionalized at the upper rim with l‐alanine units

Here we report a new chiral stationary phase (CSP) immobilized on silica gel based on cone calix[4]arene functionalized at the upper rim with two l ‐alanine units as new chiral selector that has been used in high‐performance liquid chromatography. The CSP was prepared by covalently bonding the allyl groups at the lower rim of calix[4]arene to silica gel by thiol‐ene click chemistry reaction. Elemental analysis of the CSP showed that 120 μmol of chiral selector bonded per gram of silica gel. 1‐Hexene was used for end‐capping of unreacted mercapto groups on silica gel. Since the CSP is chemically bonded to the silica, it can be used in the normal‐phase and reversed‐phase mode and with halogenated solvents as mobile phases, if desired. The chromatographic performance of the CSP was evaluated in the enantioseparation of the 3,5‐dinitrobenzoyl derivatives of some amino acids, diclofop‐methyl and dl ‐mandelic acid.     

A novel pillar[3]trianglimine macrocycle with a deep cavity used as a chiral selector to prepare a chiral stationary phase by thiol-ene click reaction for enantioseparation in high-performance liquid chromatography

A chiral pillar[3]trianglimine (C₆₀H₇₂N₆O₆) with a deep cavity has been developed as a chiral selector and bonded to thiolated silica by thiol-ene click reaction to fabricate a novel chiral stationary phase for enantioseparation in high-performance liquid chromatography. The enantioseparation performance of the fabricated chiral stationary phase has been evaluated by separating various racemic compounds, including alcohols, esters, amines, ketones, amino acids, and epoxides, in both normal-phase and reversed-phase elution modes. In total, 14 and 17 racemates have been effectively separated in these two separation modes, respectively. In comparison with two widely used chiral columns (Chiralcel OD-H and Chiralpak AD-H), our novel chiral stationary phase offered good chiral separation complementarity, separating some of the tested racemates that could not be separated or were only partially separated on these two commercial columns. The influences of analyte mass, mobile phase composition, and column temperature on chiral separation have been investigated. Good repeatability, stability, and column-to-column reproducibility of the chiral stationary phase for enantioseparation have been observed. After the fabricated column had been eluted up to 400 times, the relative standard deviations (n = 5) of resolution (Rs) and retention time of the separated analytes were < 0.39% and < 0.20%, respectively. The relative standard deviations (n = 3) of Rs and retention time for column-to-column reproducibility were < 4.6% and < 5.2%, respectively. This study demonstrated that the new chiral stationary phase has great prospects for chiral separation in high-performance liquid chromatography.     

One‐Dimensionally Disordered Chiral Sorting by Racemic Tiling in a Surface‐Confined Supramolecular Assembly of Achiral Tectons

The aggregation of (pro)chiral/achiral molecules into crystalline structures at interfaces forms conglomerates, racemates, and solid solutions, comparable to known bulk phases. Scanning tunneling microscopy and Monte Carlo simulations were employed to uncover a distinct racemic phase, expressing 1D disordered chiral sorting through random tiling in surface‐confined supramolecularly assembled achiral 4,4′′‐diethynyl‐1,1′:4′,1′′‐terphenyl molecules. The configurational entropy of the 1D disordered racemic tiling phase was verified by analytical modeling, and found to lie between that of a perfectly ordered 2D racemate and a racemic solid solution.     

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector,N‐methacryloyl‐l‐histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d ,l ‐phenylalanine, d ,l ‐tyrosine, and d ,l ‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l ‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l ‐enantiomers of aromatic amino acids migrated before d ‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.     

Chemically L-prolinamide-modified monolithic silica column for enantiomeric separation of dansyl amino acids and hydroxy acids by capillary electrochromatography and mu-high performance liquid chromatography

A silica-based chiral monolithic column prepared by sol-gel process and chemical modification of chiral selector was used for enantioseparation of dansyl amino acids and hydroxy acids by capillary electrochromatography (CEC) and mu-high-performance liquid chromatography (mu-HPLC). L-Prolinamide was modified as a chiral selector. The chiral stationary phase (CSP), the chiral complex of Cu(II) with L-prolinamide, provides an anodic electroosmotic flow (EOF) in CEC. The EOF was found to be dependent on applied electric field strength, the pH, and the composition of mobile phases. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pore. D-Enantiomers migrated before L-enantiomers except for dansyl-(Dns)-DL-Ser. The separation efficiencies of up to 17600 (D) and 13,200 plates m(-1) (L) were achieved for the separation of DL-indole-3-lactic acid.     

Permethylated β-cyclodextrin in liquid poly(oxyethylene) as a stationary phase for capillary gas chromatography

This paper reports the study of poly(oxyethylene) as a solvent for heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (PM-β-CD) and the potential of these mixtures for constructing enantioselective gas chromatography columns. Enantioseparations of volatile racemic mixtures using capillaries coated with 10–50% PM-β-CD diluted in Carbowax 20M were evaluated. The influence of the polymer type on retention, separation and reproducibility over a period of time is critically discussed. The poly(oxyethylene)-based columns were also compared to columns prepared by dilution of PM-β-CD in two polysiloxanes, SE-30 and SE-54, at several temperatures. Finally, we evaluated a new stationary phase prepared by crosslinking the chiral selector to the poly(oxyethylene) matrix.