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.     

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.     

Homochiral metal–organic framework used as a stationary phase for high‐performance liquid chromatography

Metal–organic frameworks are promising porous materials. Chiral metal–organic frameworks have attracted considerable attention in controlling enantioselectivity. In this study, a homochiral metal–organic framework [Co₂(D‐cam)₂(TMDPy)] (D‐cam = d ‐camphorates, TMDPy = 4,4′‐trimethylenedipyridine) with a non‐interpenetrating primitive cubic net has been used as a chiral stationary phase in high‐performance liquid chromatography. It has allowed the successful separation of six positional isomers and six chiral compounds. The good selectivity and baseline separation, or at least 60% valley separation, confirmed its excellent molecular recognition characteristics. The relative standard deviations for the retention time of run‐to‐run and column‐to‐column were less than 1.8 and 3.1%, respectively. These results demonstrate that [Co₂(D‐cam)₂(TMDPy)] may represent a promising chiral stationary phase for use in high‐performance liquid chromatography.     

Synthesis of novel chiral imidazolium stationary phases and their enantioseparation evaluation by high-performance liquid chromatography

Two novel chiral stationary phases (CSPs) were prepared by bonding chiral imidazoliums on the surface of silica gel. The chiral imidazoles were derivatized from chiral amines, 1-phenylethylamine and 1-(1-naphthyl)ethylamine. The obtained CSPs were characterized by Fourier Transform Infrared (FT-IR) spectroscopy and elemental analysis (EA), demonstrating the bonding densities of CSP 1 and CSP 2 were 0.43 mmol g⁻¹ and 0.40 mmol g⁻¹, respectively. These two CSPs could be used to availably separate 8 pharmaceuticals, 7 mandelic acid/its derivatives, 2 1-phenylethylamine derivatives, 1 1,1′-bi-2-naphthol, and 1 camphorsulfonic acid in high-performance liquid chromatography (HPLC). It is found that CSP 1 could effectively enantioseparate most chiral analytes, especially the acidic components, while CSP 2 could enantiorecognize all chiral analytes, although a number of components did not achieve baseline separation. Additionally, the effects of mobile phase composition, mobile phase pH and salt content, chiral selector structures, and analyte structures on the enantiorecognitions of the two CSPs were investigated. It is found that high acetonitrile content in mobile phases was conducive to enantiorecognition. Mobile phase pH and salt content could alter the retention behaviors of different enantiomers of the same chiral compound, resulting in better enantioresolution. Moreover, both chiral selector structures and substituted groups of analytes played a significant role in the separation of chiral solutes.     

Synthesis of novel chiral stationary phases for high-performance liquid chromatography

Three novel chiral selectors 4a-c were synthesized from(S)-amino acids and(R)-1-phenyl-2-(4-methylphenyl)ethylamine.4a-cwere connected to 3-aminopropylsilanized silica gel to be used as the chiral stationary phase for HPLC.Five amino acid derivativesand two pyrethroid insecticides were fairly resolved on these three new chiral stationary phases under normal phase condition.     

Chiral nitrogen-containing calix[4]crown—an excellent receptor for chiral recognition of mandelic acid

A chiral nitrogen-containing calix[4]crown 2 bearing optically pure 1,2-diphenyl-1,2-oxyamino residue at lower rim showed excellent chiral recognition between enantiomers of mandelic acid. Using competitive ¹H NMR titration the ratio of association constants of (S)- and (R)-mandelic acid with the chiral calix[4]crown was determined to be 102, that is 98% de, which is the best result obtained from artificial receptors for the chiral recognition of mandelic acid up to now.     

Enantioseparation of 3‐phenyllactic acid by chiral ligand exchange countercurrent chromatography

3‐Phenyllactic acid is an antimicrobial compound with broad‐spectrum activity against various bacteria and fungus. The observed difference in pharmacological activity between optical isomeric 3‐phenyllactic acid necessitates a method for enantioseparation. Chiral ligand exchange countercurrent chromatography was investigated for the enantioseparation of 3‐phenyllactic acid with a synthesized chiral ligand. A two‐phase solvent system was composed of n‐butanol/hexane/water (0.4:0.6:1, v/v/v) to which N‐n‐dodecyl‐l ‐hydroxyproline was added to the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transitional metal ion. The influence factors were optimized by enantioselective liquid–liquid extraction. Baseline enantioseparation of racemic 3‐phenyllactic acid by analytical high‐speed countercurrent chromatography was achieved. The optical purities of enantiomeric 3‐phenyllactic acid reached 99.0%, as determined by chiral high‐performance liquid chromatography.     

A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 num diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 num ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.     

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl--amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (ln ) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparaton for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Δ_R,SΔH° and Δ_R,SΔS° afforded by Van’t Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP, remarkable increases in enanselectivity were observed for all the compounds, as the result of a “synergistic” effect.     

Preparation of chiral oxazolinyl‐functionalized β‐cyclodextrin‐bonded stationary phases and their enantioseparation performance in high‐performance liquid chromatography

A simple procedure for the synthesis of three new oxazolinyl‐substituted β‐cyclodextrins (6‐deoxy‐6‐R‐(–)‐4‐phenyl‐4,5‐dihydrooxazolinyl‐β‐cyclodextrin, 6‐deoxy‐6‐S‐(–)‐4‐phenyl‐4,5‐dihydrooxazolinyl‐β‐cyclodextrin, and 6‐deoxy‐6‐S‐(–)‐(4‐pyridin‐1‐ium‐4‐methyl‐benzenesulphonate)‐4,5‐dihy‐drooxazolinyl‐β‐cyclodextrin) and their covalent bonding to silica are reported. The ability of these chiral stationary phase columns for separating compounds is also presented and discussed. Twenty‐eight compounds were examined in the polar‐organic mobile phase mode, and 11 β‐nitroethanols were tested in the reversed‐phase mode. Excellent enantioseparations were achieved for most of the analytes, even for several challenging compounds. The rigid and flexible structures of mono‐substituted chiral groups and the fragments around the rim of the β‐cyclodextrin cavity played an important role in the separation process. Factors such as π–π stacking, dipole–dipole interactions, ion‐pairing, and steric hindrance effects were found to affect the chromatographic performance. Moreover, the buffer composition, and percentages of organic modifiers in the mobile phase, were investigated and compared. The mechanisms involved in the separation were postulated based on the chromatographic data.     

High‐performance liquid chromatographic enantioseparation of isoxazoline‐fused 2‐aminocyclopentanecarboxylic acids on a chiral ligand‐exchange stationary phase

The application of a chiral ligand‐exchange column for the direct high‐performance liquid chromatographic enantioseparation of unusual β‐amino acids with a sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate‐Cu(II) complex as chiral selector is reported. The investigated amino acids were isoxazoline‐fused 2‐aminocyclopentanecarboxylic acid analogs. The chromatographic conditions were varied to achieve optimal separation. The effects of temperature were studied at constant mobile phase compositions in the temperature range 5–45°C, and thermodynamic parameters were calculated from plots of lnk or lnα versus 1/T. Δ(ΔH°) ranged from –2.3 to 2.2 kJ/mol, Δ(ΔS°) from –3.0 to 7.8 J mol^?1 K^?1 and –Δ(ΔG°) from 0.1 to 1.7 kJ/mol, and both enthalpy‐ and entropy‐controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. The sequence of elution of the enantiomers was determined in all cases.     

Retention prediction of o-phthalaldehyde amino acid derivatives in reversed-phase liquid chromatography

Summary Retention prediction of o-phthalaldehyde amino acid derivatives in reversed-phase liquid chromatography has been investigated. The retention of all derivatives could be predicted within about 10% relative error under the appropriate separation conditions in both isocratic and gradient-elution modes.     

Evaluation of differences between Chiralpak IA and Chiralpak AD‐RH amylose‐based chiral stationary phases in reversed‐phase high‐performance liquid chromatography

Polysaccharide‐based chiral stationary phases can be used for the enantioselective separation of a wide range of structurally different compounds. These phases are available with chiral selectors coated or immobilized on silica gel support. The means of attachment of the chiral selector to the carrier can influence the separation performance of these stationary phases. This paper deals with evaluation of differences in the separation abilities of coated Chiralpak AD‐RH versus immobilized Chiralpak IA amylose‐based stationary phases in the reversed–phase mode of high–performance liquid chromatography. A set of chiral analytes was separated under acidic and basic conditions. Differences were observed in the enantioseparation potential of the tested phases. The linear‐free energy relationship and additional evaluation of ionic interactions were used to ascertain whether the interactions that participate in retention and enantioseparation are affected by the means of preparation of these phases. All the interactions covered by the linear‐free energy relationship were significant for the studied phases and their absolute values were almost always higher for the coated phase. Ionic interactions were found to be more important on the immobilized stationary phase but did not contribute to any improvement in the enantioselective separation performance.     

Preparation of a new chiral stationary phase for HPLC based on the (R)- 1-phenyl-2-(4-methylphenyl)ethylamine amide derivative of (S)-valine and 2-chloro-3,5-dinitrobenzoic acid: enantioseparation of amino acid derivatives and pyrethroid insecticides

A novel chiral stationary phase (CSP) for HPLC was prepared by bonding (R)-1-phenyl-2-(4-methylphenyl)ethylamine amide derivative of (S)-valine to aminopropyl silica gel through a 2-amino-3,5-dinitro-1-carboxamido-benzene unit. The CSP was used for the separation of some amino acid derivatives and pyrethroid insecticides by chiral HPLC. Satisfactory baseline separation required optimization of the variables of mobile phase composition. Use of dichloromethane as modifier in the mobile phase gave baseline separations of amino acid derivatives. The two enantiomers of fenpropathrin and four stereoisomers of fenvalerate were baseline separated using hexane-dichloromethane-ethanol as mobile phase. The results show that the enantioselectivity of the new CSP is better than Pirkle type 1-A column for these compounds. Only partial separations were observed for the stereoisomers of cypermethrin and cyfluthrin, which gave even and eight peaks, respectively.     

Synthesis and enantioseparation behaviors of novel immobilized 3,5‐dimethylphenylcarbamoylated polysaccharide chiral stationary phases

Two new polysaccharide‐derived chiral selectors, namely, 6‐azido‐6‐deoxy‐3,5‐dimethylphenylcarbamoylated amylose and 6‐azido‐6‐deoxy‐3,5‐dimethylphenyl carbamoylated cellulose, were synthesized under homogeneous conditions and immobilized onto aminized silica gel by the Staudinger reaction, resulting in two new immobilized polysaccharide chiral stationary phases (CSPs). Their enantioseparation performances were investigated under normal‐phase mode by HPLC. Among 17 analytes, baseline separations of 12 pairs of enantiomers are achieved on the immobilized cellulose CSP, which demonstrates that this new cellulose material exhibits almost the same enantioseparation performance as the coated cellulose CSP. In addition, the amylose‐derived CSP presents limited enantiorecognition ability but certain complementarity with the immobilized and coated cellulose‐based materials. Neither metolachlor nor paclitaxel side chain acids are separated on two cellulose‐derived CSPs, but effective separations are obtained on the immobilized amylose column.     

Ionic liquid as a mobile phase additive in high‐performance liquid chromatography for the simultaneous determination of eleven fluorescent whitening agents in paper materials

In the present study, 11 4,4′‐diaminostilbene‐2,2′‐disulfonic acid based fluorescent whitening agents with different numbers of sulfonic acid groups were separated by using an ionic liquid as a mobile phase additive in high‐performance liquid chromatography with fluorescence detection. The effects of ionic liquid concentration, pH of mobile phase B, and composition of mobile phase A on the separation of fluorescent whitening agents were systematically investigated. The ionic liquid tetrabutylammonium tetrafluoroborate is superior to tetrabutylammomnium bromide for the separation of the fluorescent whitening agents. The optimal separation conditions were an ionic liquid concentration at 8 mM and the pH of mobile phase B at 8.5 with methanol as mobile phase A. The established method exhibited low limits of detection (0.04–0.07 ng/mL) and wide linearity ranges (0.30–20 ng/mL) with high linear correlation coefficients from 0.9994 to 0.9998. The optimized procedure was applied to analyze target analytes in paper samples with satisfactory results. Eleven target analytes were quantified, and the recoveries of spiked paper samples were in the range of 85–105% with the relative standard deviations from 2.1 to 5.1%. The obtained results indicated that the method was efficient for detection of 11 fluorescent whitening agents.     

Enantioseparation of aldols by high‐performance liquid chromatography on polysaccharide‐based chiral stationary phases that bear chlorinated substituents

Two families of aldols, obtained from the condensation of aromatic aldehydes with cyclohexanone or acetone (ten examples in each group), were analyzed by high‐performance liquid chromatography in normal phase elution mode on three polysaccharide‐based chiral stationary phases of the Lux series, namely, Lux Cellulose‐2, Lux Cellulose‐4 and Lux Amylose‐2, which share the common feature of chlorinated substituents in the chiral selectors. Following simple optimization steps, the enantioseparation of all aldols derived from cyclohexanone was achieved and the highest values of separation factor (α, 1.32 < α < 2.20) and resolution (R_s, 4.5 < R_s <17.2) were observed on Lux Cellulose‐2, with the only exception of the 4‐nitro‐substituted derivative that was better resolved on Lux Cellulose‐4. On the contrary, Lux Amylose‐2 was the best choice for aldols derived from acetone and only specific analytes in this group were resolved on the cellulose‐based supports. A variable‐temperature study of selected compounds allowed us to determine thermodynamic parameters of the enantioseparation process, which was enthalpy‐controlled in all the cases except one.     

HPLC enantioseparation of amino acids and their protected derivatives used in peptide synthesis with pre-column chiral derivatization

Summary Indirect chiral separation methods based on enantiomeric derivatizations were developed in order to monitor optical purity of uncoded amino acids and a new series of amino acid derivatives. Marfey's reagent was used for derivatization of amino groups: whilst boxyl groups were derivatised with (1R, 2R)-or (1S, 2S)-2-amino-1(4-nitrophenyl)-1,3-propanediol reagents were used, respectively. The separations of diastereomeric derivatives formed via derivatization were optimized in RP-HPLC and NP-HPLC systems. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

高效液相色谱手性流动相法拆分酮基布洛芬对映体

以Lichrospher C18为分析柱, 将β-环糊精、2,6-二甲基-β-环糊精、2,3,6-三甲基-β-环糊精分别作为手性流动相添加剂, 系统地研究了R,S-酮基布络芬对映体在HPLC系统中的拆分. 建立了以2,3,6-三甲基-β-环糊精为手性流动相添加剂分离R,S-酮基布络芬对映体方法.