Triazolo-linked cinchona alkaloid carbamate anion exchange-type chiral stationary phases: Synthesis by click chemistry and evaluation

Immobilization strategy based on Huisgen 1,3-dipolar cycloaddition (click chemistry) of 10,11-didehydrocinchona tert-butylcarbamates to azido-grafted silica gels has been evaluated for preparation of novel chiral stationary phases (CSP 1-3). The resultant 1,2,3-triazole-linked CSPs were tested under various mobile phase conditions (polar organic and reversed phase mode) with a representative set of structurally diverse racemic acids including N-protected aminoacids, aromatic and aryloxycarboxylic acids as well as binaphthol phosphate. The chiral recognition performance of the C3-triazole-linked CSPs was found to mirror largely that of the known C3-thioether-linked CSP in terms of elution order, enantioselectivity and retention behavior. In an effort to assess the non-specific binding expressed as retention increment of these triazole-linked CSPs, the parent azidopropyl- and triazole-modified silica materials (thus not containing the chiral head ligand) were studied independently. Compared with the corresponding CSPs, the analyte retention on the azidopropyl control column was very low, and practically negligible on the corresponding triazole-modified reference column. Only minor losses in analyte retention behavior (<5%) were observed with triazole-linked CSPs after two month of continuous use with polar-organic and reversed-phase-type mobile phases, highlighting the excellent stability of the 1,2,3-triazole linker.     

Resolution of phthalans obtained by ortho-litiathion of aryloxiranes by enantioselective high-performance liquid chromatography: Performances of various chiral stationary phases

The HPLC separation of the enantiomers of six phthalans (1,3-dihydrobenzo[c]furans) synthesized as racemic mixtures from ortho-lithiated aryloxiranes was accomplished in the normal-phase mode using seven polysaccharide-derived and Pirkle-type chiral stationary phases (CSPs) and n-hexane/2-propanol mixtures as mobile phases. Separation and resolution factors up to 1.6 and 4.2, respectively, were obtained. The performances of various CSPs with regard to the same compound were, however, quite different not only between the two types of CSPs but also within the same type (polysaccharide-derived or Pirkle-type). Also diastereomeric pairs of phthalans show different enantioseparation using the same CSP.     

Super/Subcritical Fluid Chromatography Separations with Four Synthetic Polymeric Chiral Stationary Phases

New synthetic polymeric chiral selectors were developed recently as chiral stationary phases. They were tested with supercritical fluid mobile phases made of CO₂ plus an alcohol modifier and 0.2% v/v trifluoroacetic acid. The polymeric N,N′-(1S,2S)-1,2-cyclohexanediyl-bis-2-propenamide (P-CAP), the polymeric N,N′-[(1R,2R)]-1,2-diphenyl-1,2-ethanediyl] bis-2-propenamide (P-CAP-DP), the polymeric trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide (DEABV) and the polymeric N,N′-[(1R,2R)-1,2-diphenyl-1,2-ethanediyl] bis-4-vinylbenzamide (DPEVB) were bonded to 5 μm silica particles and used to prepare four columns that were tested with a set of 88 chiral compounds with a wide variety of chemical functionalities. All 88 test compounds were separated on one or more of these “related” polymeric CSPs. Forty-three enantiomeric pairs were separated in SFC conditions by only one of the CSPs. Twenty pairs were separated by two CSPs and 18 and 7 enantiomeric pairs were separated by 3 and all 4 CSPs, respectively. The three P-CAP, P-CAP-DP and DEABV CSPs have equivalent success being able to separate 49 enantiomeric pairs of the studied set with respectively 12, 14 and 20 at baseline (R _s  > 1.5). The DPEVB CSP was significantly less efficient separating only 18 chiral compounds with only one at baseline. The great advantage of the SFC mobile phases is the rapid separation, witch most achieved in less than 5 min.     

Transition from enantioselective high performance to ultra-high performance liquid chromatography: A case study of a brush-type chiral stationary phase based on sub-5-micron to sub-2-micron silica particles

Three brush-type chiral stationary phases (CSPs) differing in the particle size of the starting silica particles have been prepared by covalent grafting of the π-acidic bis-(3,5-dinitrobenzoyl)-derivative of trans-1,2-diaminocyclohexane (DACH-DNB). Starting silica particles of 4.3, 2.6 and 1.9 micron were used to generate the final CSPs using an improved, highly reproducible synthetic methodology, that allowed to assemble and surface-graft the whole chiral selector in only two steps. The different CSPs have been packed in columns of various length and diameters, and fully characterized in terms of flow permeability, kinetic performances and enantioselectivity using a set of test solutes. Very high speed and high resolution applications together with stereodynamic HPLC examples are demonstrated on the columns with reduced particle diameters, on which separations of several enantiomeric pairs are routinely obtained with analysis times in the 15–40 s range.     

Enantioseparation of 1-Phenyl-1,2,3,4-tetrahydroisoquinoline on Polysaccharide-Based Chiral Stationary Phases

The enantiomers of 1-phenyl-1,2,3,4-tetrahydroisoquinoline have been directly separated on polysaccharide-based chiral stationary phases (CSPs). The normal phase separation of (S)- and (R)-1-phenyl-1,2,3,4-tetrahydroisoquinoline was accomplished by screening of the immobilized Chiralpak IC column with different eluents. The effect of mobile phase type on retention, selectivity and resolution was studied. 2-Propanol or ethanol/n-hexane/ethanolamine mixtures were applied as mobile phases by screening of following polysaccharide-based immobilized (Chiralpak IA, Chiralpak IC) and coated (Lux Cellulose-1, Lux Cellulose-2, Lux Amylose-2) CSPs. Polar organic and reversed-phase conditions were also tested for direct enantioseparation of 1-phenyl-1,2,3,4-tetrahydroisoquinoline.     

Stationary phase-related investigations of quinine-based zwitterionic chiral stationary phases operated in anion-, cation-, and zwitterion-exchange modes

The concept of recently introduced Cinchona alkaloid-type zwitterionic chiral stationary phases (CSPs) is based on fusing key cation- and anion-exchange (CX, AX) moieties in one single low-molecular mass chiral selector (SO) with the resulting CSPs allowing enantiomer separations of a wide range of chiral ionizable analytes comprising acids, bases, and zwitterionic compounds. Herein, we report principal, systematic investigations of the ion-exchange-type retention mechanisms available with the novel zwitterionic CSPs in nonaqueous polar organic mode. Typical CX and AX processes, corresponding to the parent single ion exchangers, are confirmed also for zwitterionic CSPs. Also the mechanism leading to recognition and retention of zwitterions was found to be ion exchange mediated in a zwitterion-exchange (ZX) mode. In both AX and CX modes the additional ionizable group within the SO besides the site responsible for the respective ion-exchange process could be characterized as an intramolecular counterion (IMCI) that effectively participates in the ion-exchange equilibria and thus, contributes to solute elution. In the ZX mode both oppositely charged groups of the zwitterionic SO were found not only to be the sites for simultaneous ion pairing with the analyte but also functioned as IMCIs at the same time. The main practical consequences of the IMCI feature were significant reduction of the amounts and even elimination of acidic and basic additives required in the eluent systems to afford analyte elution while still providing faster analysis than the parent single ion-exchanger-type CSPs. The set of ten structurally different zwitterionic CSPs employed in this study facilitated the establishment of correlations between chromatographic behavior of the CSPs with particular SO elements, thereby supporting the understanding of the working principles of these novel packing materials on a molecular level.     

Preparation of chiral stationary phase via activated carbamate intermediate for liquid chromatographic optical resolution

Summary Chiral stationary phases (CSPs) for liquid chromatography were prepared by the way of an activated carbamate intermediate. The amino group of aminopropylsilyl silica gel was first activated by carbamylation with disuccinimido carbonate (DSC). The obtained activated carbamate silica gel (ACsil) proved useful as an intermediate for the preparation of urea-type CSPs. The reaction of ACsil with (S)- of (R)-1-(α-naphthyl)-ethylamine gave naphthylethylurea type CSPs. These CSPs were also obtained directly from aminopropylsilyl silica gel by its reaction with optically active (S)- or (R)-succinimido 1-(α-naphthyl)ethyl carbamate (SINEC). Several phenylthiohydantoin amino acid enantiomers and p-bromophenylcarbamyl amino acid enantiomers were resolved on the CSPs by elution with aqueous mobile phase.     

Liquid chromatographic resolution of beta-amino acids on CSPs based on optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6

Two chiral stationary phases (CSPs) based on optically active (3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 covalently bonded to silica gel were utilized for the first time for the resolution of racemic β-amino acids using high performance liquid chromatography. All of the 10 β-amino acids tested were resolved on the CSP containing residual silanol-protecting n-octyl groups, while only five β-amino acids were resolved on the CSP containing residual silanol groups. The superiority of the CSP containing residual silanol-protecting n-octyl groups and the characteristic retention behaviors of the two enantiomers on the CSP were rationalized to stem from the removal of the residual silanol groups, which can otherwise induce the non-enantioselective retention of the analytes, and the improved lipophilicity of the CSP. The elution orders of the two enantiomers of β-amino acids were identical on the two CSPs and, consequently, it was concluded that the two CSPs were concluded to utilize identical chiral recognition mechanisms. The different elution orders of the analytes were proposed to be attributed to the presence or absence of π-π interactions between the CSP and analytes.     

Synthesis and chiral recognition of novel amylose derivatives containing regioselectively benzoate and phenylcarbamate groups

A new class of regioselectively substituted amylose derivatives bearing three different substituents at 2-, 3- and 6-positions, and two different substituents at 2-position and 3-, 6-positions were synthesized by a sequential process based on the esterification of 2-position of a glucose unit. Their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). Each derivative had its own characteristic recognition ability depending on the arrangement of side chains at the three positions. Among the derivatives, amylose 2-(4-t-butylbenzoate) and amylose 2-(4-chlorobenzoate) series exhibited high chiral recognition. Some racemates can be efficiently separated on these derivatives as well as on the amylose tris-3,5-dimethylphenylcarbamate, which is commercially available as Chiralpak AD and one of the most powerful CSPs. The structures of the amylose derivatives were also investigated by circular dichroism spectroscopy.     

Chiral phenethylamine synergistic tricarboxylic acid modified β-cyclodextrin immobilized on porous silica for enantioseparation

In this work, a series of chiral phenethylamine synergistic tricarboxylic acid modified β-cyclodextrin bonded stationary phase for high performance liquid chromatography (HPLC) were synthesized via a simple one-pot synthesis approach. Various racemates (aryl alcohols, flavanones, triazoles, benzoin, etc.) were well separated on the tricarboxylic acid modified chiral stationary phases in both normal and reversed modes with good reproducibility and stability, and the influence of mobile phase composition on resolution (R_s) were deeply investigated. The RSD values of R_s for repeatability and column-to-column were below 1.28% and 3.05%, respectively. Hence, the fabrication of tricarboxylic acid modified chiral stationary phase (CSPs) is a new efficient strategy to improve the application of β-cyclodextrin as CSPs in the field of chromatography.     

Effect of the Orientation of Amide Linkage Groups on the Enantioselectivity of Two Related Synthetic Polymeric Chiral Stationary Phases

Two new synthetic polymeric chiral stationary phases (CSPs) based on trans-(1S,2S)-cyclohexanedicarboxylic acid bis-4-vinylphenylamide (I) and trans-N,N′-(1R,2R)-cyclohexanediyl-bis-4-ethenylbenzamide (II) monomers were prepared and evaluated by normal phase high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). A variety of chiral compounds were separated on these two new CSPs. The different orientation of the amide groups in the two CSPs resulted in a striking difference in the enantioselectivity properties of these two CSPs. Their differences in enantioselectivity with HPLC and SFC were compared.     

Cationic cyclodextrins chemically-bonded chiral stationary phases for high-performance liquid chromatography

Two covalently bonded cationic β-CD chiral stationary phases (CSPs) prepared by graft polymerization of 6^A-(3-vinylimidazolium)-6-deoxyperphenylcarbamate-β-cyclodextrin chloride or 6^A-(N,N-allylmethylammonium)-6-deoxyperphenylcarbamoyl-β-cyclodextrin chloride onto silica gel were successfully applied in high-performance liquid chromatography (HPLC). Their enantioseparation capability was examined with 12 racemic pharmaceuticals and 6 carboxylic acids. The results indicated that imidazolium-containing β-CD CSP afforded more favorable enantioseparations than that containing ammonium moiety under normal-phase HPLC. The cationic moiety on β-CD CSPs could form strong hydrogen bonding with analytes in normal-phase liquid chromatography (NPLC) to enhance the analytes’ retention and enantioseparations. In reversed-phase liquid chromatography (RPLC), the analytes exhibited their maximum retention when the pH of mobile phase was close to their pK_a value. Inclusion complexation with CD cavity and columbic/ionic interactions with cationic substituent on the CD rim would afford accentuated retention and enantioseparations of the analytes.     

Recent trends in chiral separations on immobilized polysaccharides CSPs

Polysaccharide CSPs are recognized widely in chiral chromatography but the introduction of immobilized phases (Chiralpak IA, Chiralpak IB and Chiralpak IC columns) is a remarkable achievement. The immobilized CSPs can be used with organic, normal and reversed phase modes; even with prohibited solvents too (tetrahydrofuran, chlorofom, dichloromethane, acetone, 1,4-dioxane, ethylacetate, and certain other ethers). Their susceptibilities to work with a wide range of solvents have increased the range of applications including chiral recognition mechanisms. Besides, these are also useful for monitoring the progress of stereo-specific reactions; normally need prohibited solvents. The present review describes the various aspects of commercial available immobilized chiral columns. Attempts have been made to discuss immobilized polysaccharides CSPs, immobilized vs coated CSPs, comparison of immobilized CSPs, method development, optimization, chiral recognition mechanism and applications. The chiral recognition capabilities of commercial columns were in the order of Chiralpak IA > Chiralpak IB > Chiralpak IC columns; but complimentary to each other. Of course, these CSPs are not fully developed and need more advancements and applications. Definitely, the future of immobilized CSPs is quite better. Hopefully, in the coming years they will be the choice of the chromatographers for chiral separations in liquid chromatography.     

Chromatographic Enantioresolution of Six Purine Derivatives Endowed with Anti-Human Breast Cancer Activity

Three (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purine derivatives (1–3) and three (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-2-ylmethyl)-9H-purine derivatives (4–6) showed relevant activity against human breast cancer cell line MCF-7, when assayed as racemates. The relevant structural analogy among these compounds stimulated us to evaluate and compare their chromatographic behaviour with five polysaccharide-based chiral stationary phases (CSPs). For a column screening purpose, four cellulose-based (Chiralcel OD-H, Chiralpak IB, Lux Cellulose-2, and Sepapak-4; CSPs 1–4, respectively) and an amylose-based (Lux Amylose-2; CSP 5) CSPs were initially assayed by employing the same “standard” eluent mixture. With CSP 2, the performance from “non standard” eluent systems was also evaluated. The different type and position of the substituents onto the carbamate moiety, the coated or immobilized nature of the modified polymer chain, and the different type of winding as well as the eluent composition were found to deeply affect the enantiorecognition mechanism. While the different winding of the polymer and the derived different morphology of the binding cleft in the 2,5-disubstituted chloromethyl phenyl carbamate amylose-based CSP 5 was found to be unsuited to get profitable enantio-discriminations of all the analyzed compounds, CSP 3 and CSP 4 produced the highest α and R _S values in the enantioselective analysis of four out of six racemates (that is 1–3 and 6). In CSP 2, the conformational change of the polysaccharide chain upon immobilization produced a profound influence on the chromatographic behaviour of compounds 1–6. A relevant improvement of the enantioresolution quality of CSP 2 was obtained when used in combination with “non standard” solvents as constituents of the mobile phase mixture.     

Comparative Enantioseparation of Seven Amino Alcohols on Teicoplanin‐Based Chiral Stationary Phases

Abstract

The macrocyclic antibiotics represent a relatively new class of chiral selectors in separation science and teicoplanin‐based chiral stationary phases (CSP) have been used successfully in a number of applications in high‐performance liquid chromatography. In the present studies, we self‐prepared two bonded CSPs–teicoplanin (TE) and teicoplanin phenyl isocyanate (TE‐Phe). Seven amino alcohols, propranolol, bisoprolol fumarate, atenolol, salbutamol, isoproterenol, metoprolol, and labetalol were enantioseparated on both self‐made CSPs using methanol as mobile phase and acetic acid (HOAc) and triethylamine (TEA) as mobile phase additives. On both CSPs, the different enantioseparation behavior of analytes with different structure was compared. The influence of the concentration of mobile phase additives (HOAc and TEA) on the enantioseparation was investigated. In all conditions, the retention factors (k′) of seven analytes on TE‐Phe CSP were larger than that on TE CSP. However, the separation factors (α) and resolutions (Rs) on TE‐Phe CSP were smaller than that on TE CSP. The results indicated that the derivatized TE‐Phe CSP is not efficient as original teicoplanin CSP. Our observations also suggested that, for teicoplanin‐based CSPs, π‐π interactions and dipole‐dipole between solutes and CSPs mainly contribute to the retention of solutes on CSPs while hydrogen bonding and steric interactions play important roles in the chiral recognition for teicoplanin‐based CSPs.     

Novel cyclodextrin chiral stationary phases for high performance liquid chromatography enantioseparation: Effect of cyclodextrin type

Three novel chiral stationary phases (CSPs) were prepared by regioselective chemical immobilization of mono(6^A-N-allylamino-6^A-deoxy)perphenylcarbamoylated (PICD) α-, β-, and γ-cyclodextrins (CDs) onto silica support via hydrosilylation. Their enantioseparation properties in high performance liquid chromatography (HPLC) were evaluated with a large spectrum of racemates including flavanone compounds, β-adrenergic blockers, amines and non-protolytic compounds. The effect of CD's cavity size on enantioseparation abilities was studied and discussed. The results indicated that CD's surface loading at silica support played an important role in the enantioseparation on these CSPs under normal-phase conditions while inclusion phenomena contributed the major driving force under reverse-phase conditions. As expected, α-PICD demonstrated the best resolutions towards flavonone and most aromatic alcohols under normal-phase conditions with the highest surface loading; while Fujimura's competitive inclusion model can be applied to explain the better enantioseparations towards β-adrenergic blockers, amines and non-protolytic compounds with α- and β-PICD CSPs. γ-PICD CSP showed superior enantioseparation ability for sterically encumbered analytes like flavanone compounds under both normal-phase and reversed phase conditions.     

Rational optimization of the Whelk-O1 chiral stationary phase using molecular dynamics simulations

Rational in silico optimization of the Whelk-O1 chiral stationary phase (CSP) has been carried out based on the chiral recognition mechanism extracted from previous molecular dynamics simulations [C.F. Zhao, N.M. Cann, Anal. Chem. 80 (2008) 2426] of this CSP. Three modified CSPs have been examined. The first two are designed to increase selectivity by reducing the docking probability of the less retained analyte. The third modified selector reverses the amide bridge to introduce a structural motif found in the popular carbamate-derivatized polysaccharide CSPs [Y. Okamoto, M. Kawashima, K. Hatada, J. Am. Chem. Soc. 106 (1984) 5357]. For each modified selector, an atomistic model has been obtained through extensive ab initio calculations. The effect of selector modification is then evaluated via simulations of the modified interface in the presence of target analytes. Simulation results show that the separation factors are increased for the modified CSPs but in some cases elution orders are reversed. The Whelk-O1 CSP was originally designed to separate naproxen [W.H. Pirkle, C.J. Welch, B. LAmm, J. Org. Chem. 57 (1992) 3854]. With this in mind, molecular dynamics simulations of naproxen are compared for the original, and the modified, selectors.     

Investigations of mobile phase contributions to enantioselective anion- and zwitterion-exchange modes on quinine-based zwitterionic chiral stationary phases

Novel chiral stationary phases (CSPs) based on zwitterionic Cinchona alkaloid-type low-molecular mass chiral selectors (SOs), as they have been reported recently, were investigated in HPLC towards effects on their chromatographic behavior by mobile phase composition. Mobile phase characteristics like acid-to-base ratio and type of acidic and basic additives as well as effect of type of bulk solvents in nonaqueous polar organic and aqueous reversed-phase (RP) eluent systems were varied in order to illustrate the variability and applicability of zwitterionic CSPs with regard to mobile phase aspects. Chiral SOs of the five zwitterionic CSPs investigated herein contained weak and strong cation-exchange (WCX, SCX) sites at C9- and C6′-positions of the Cinchona alkaloid scaffold which itself accommodated the weak anion-exchange (WAX) site. The study focused on zwitterion-exchange (ZX) operational mode and chiral amino acids as target analytes. Besides, also the anion-exchange (AX) mode for chiral N-blocked amino acid analytes was considered, because of the intramolecular counterion (IMCI) property available in AX mode. Overall, most general and successful conditions in ZX mode were found to be weakly acidic methanolic mobile phases. In aqueous eluents RP contributions to retention came into play but only at low organic modifier content because of the highly polar character of zwitterionic analytes. At higher acetonitrile content, HILIC-related retention phenomena were observed. When using weakly basic eluent system in AX mode remarkably fast enantiomer separations involving exclusion phenomena were possible with one enantiomer eluting before and the other after void volume.     

The Resolution of Enantiomeric Ibuprofen on Chiral Stationary Phases Containing N-Arylcarbamoyl Derivatives of Phenylglycine

A series of fourteen anilide derivatives of ibuprofen were resolved on six chiral stationary phases (CSPs) derived from N-arylcarbamoyl derivatives of (S)-phenylglycine. Excellent chiral resolutions were achieved on these CSPs. The ionic-type CSPs showed better chiral recognition abilities than the corresponding covalent-type CSPs, and the CSP bearing two chiral centers has better performance than the CSPs bearing only one chiral center. The highest separation factor was achieved using the ionic-type CSP bearing two chiral centers for the resolution of the 3,5-dinitroanilide derivative of ibuprofen. This result is better than those reported in literature for the resolution of ibuprofen on the CSPs derived from amino acids, According to the chromatographic behaviors, the hydrogen bonding interaction, the π-π interactions provided by the phenyl groups in CSPs bearing one chiral center, and the phenylethylcarbamoyl moiety in CSPs bearing two chiral centers dominate the chiral recognition.