Chiral supercritical fluid chromatography on porous graphitic carbon using commercial dimethyl beta-cyclodextrins as mobile phase additive

Using dimethylated-beta-cyclodextrin mixtures (MeCD) as chiral selectors in CO2-polar modifier mobile phase and porous graphitic carbon as solid-phase, chiral supercritical (or subcritical) fluid chromatography was performed. The adsorbed quantity of MeCD onto the porous graphitic carbon (Hypercarb) was measured for various chiral selector concentrations using the breakthrough method with evaporative light scattering detector. The effects of MeCD concentration in the mobile phase, the nature of the polar modifier, the outlet pressure, the column temperature and the nature of the commercial MeCD mixture on the retention and the enantioselectivities were studied. For a given solute, the enantioselectivity is greatly dependent on the commercial MeCD mixture used. The retention mechanism was also studied. From the data, we find that the dominant mechanism for the chiral discrimination is the diastereoisomeric complexation in the mobile phase.     

Temperature and enantioseparation by macrocyclic glycopeptide chiral stationary phases

Seventy-one chiral compounds were separated on four macrocyclic glycopeptide chiral selectors: teicoplanin, its aglycone, ristocetin A and vancomycin, using three possible separation modes: reversed phase with methanol/buffer mobile phases, normal phase with hexane/ethanol mobile phases and polar ionic mode (PIM) with 100% methanol mobile phase with trace amounts of acid and/or base. These 148 separations were studied in a 5-45 degrees C temperature range. Peak efficiencies always increased with temperature, but in only 17% of the separations studied a small increase of the enantioresolution factor was observed. In the majority (83%) of the cases, the enantioresolution decreased or even vanished when temperature increased. All 148 Van't Hoff plots were linear showing that the selector did not change in the temperature range studied. The calculated enthalpy and entropy variations showed that the interaction of the solute with the stationary phase was always enthalpy driven with normal and reversed mobile phases. It could be enthalpy as well as entropy driven with PIM mobile phases strongly dependent on the solute. The plots of delta(deltaH) versus delta(deltaS) were linear in most cases (enthalpy entropy compensation). This observation cannot be used to give clear information on chiral recognition mechanisms, but it allowed identifying specific stationary phase-solute interactions because the points corresponding to the respective thermodynamic parameters were clearly delineated from the general compensation lines.     

Study of tryptophan enantiomer binding to a teicoplanin-based stationary phase using the perturbation technique. Investigation of the role of sodium perchlorate in solute retention and enantioselectivity

The retention of D,L-tryptophan enantiomers on an immobilized teicoplanin column was investigated in relation to the mobile phase sodium perchlorate concentration using the perturbation method to determine the solute distribution isotherms. From the experimental data, it appeared that the bi-Langmuir model was able to describe D- and L-enantiomer retention on the immobilized selector over the salt concentration range. An increase in the apparent enantioselectivity with an increase in sodium perchlorate concentration was observed. The chiral recognition enhancement was governed by (i) an increase in the difference of the adsorption constants for binding to the high-affinity site (aglycone pocket) between the two enantiomers and (ii) enhancement of the number of aglycone chiral regions interacting with D-tryptophan. It is suggested that an ion-pair formation mechanism between perchlorate and solute and/or selector is responsible for this behavior. In addition, this work shows that additional secondary sites on the teicoplanin surface are involved in the apparent enantioselectivity at low sodium perchlorate concentrations.     

Joint use of cyclodextrin additives in chiral discrimination by reversed-phase high-performance liquid chromatography: temperature effects

The temperature dependence of chiral separations was investigated in combined system of reversed-phase (RP) liquid chromatography using two chiral additives: single or β native cyclodextrins and their permethylated derivatives. The model tested compounds of pharmaceutical interest were: methylphenobarbital, mephenytoin, morsuximide and camphor. Taking the localization of a complexation process as a criterion – the combined system with two selectors has been rationalized as occurring in three stages. The influence of temperature (in narrow range of 20°C) on retention and enantioselectivity was studied in; System I (complexation occurs in the mobile phase), in System II (complexation on the stationary phase) and in System III (complexation in both phases together). In System III (as for System I) it has been found that the model compounds could be classified into three groups based on their retention dependence on temperature: retention decrease with temperature decrease, retention increase with temperature decrease or no influence of temperature on retention. For all the compounds investigated, decrease in temperature increases the selectivity. Standard enthalpy (ΔH⁰) and entropy (ΔS⁰) changes of solute transfer between the mobile and the stationary phase and standard enthalpy (ΔH⁰_CD) and entropy (ΔS⁰_CD) changes of complex formation were also calculated. In Systems I and III, if the complexation in the mobile phase is favored process compared with interaction with the stationary phases (RP or covered by permethylated cyclodextrin), the shortest retention time and the best selectivity is observed at low temperature.     

Determination of association constants between enantiomers of orciprenaline and methyl-beta-cyclodextrin as chiral selector by capillary zone electrophoresis using a partial filling technique

The principles for the determination of conditional association constants of enantiomers by capillary zone electrophoresis employing a partial filling technique (PFT) using methyl-beta-cyclodextrin as chiral selector is presented. Orciprenaline was used as a model compound. Partial filling is a separation technique, where different lengths of the chiral selector solution are introduced into the capillary to a final zone length shorter than the effective length of the capillary, prior to application of the solutes. Lengthening of the separation zone results in improving enantioresolution in addition to decreasing electrophoretic mobility of the enantiomers, because of longer interaction time between the solute and chiral selector. The degree of the reduction in electromobility depends on the affinity of the solute to the chiral selector, i.e. strength of the complex formed between the solute and cyclodextrin. The decrease in the electrophoretic mobility with increasing length of the separation zone is used for determination of the association constant. The association constants of the enantiomers of orciprenaline and the chiral selector were evaluated from the slope of the plot, observed electrophoretic mobility versus the ratio between the length of the separation zone and the effective length of the capillary. It was found that the association constants were independent of the chiral selector concentration. The mean values were 110 M(-1) and 160 M(-1) for respective enantiomer. Constants obtained by a conventional CE technique were in good agreement with those from the PFT experiments. The highest enantioselectivityy was obtained when about 50% of the solute was distributed to the selector phase.     

Effects of Temperature and Mobile Phase Condition on Chiral Recognition of Poly(l-phenylalanine) Chiral Stationary Phase

Characteristics of the chiral stationary phase with poly(l-phenylalanine) peptide selector, which was in ??-helical state, was reported. Since environmental factors affect peptide conformation, the changes in enantioselectivity were examined depending on column temperature and mobile phase conditions (ionic strength, pH, mobile phase composition). Column temperature and pH drastically affected the enantioselectivity. Based on these changes, the relation between chiral recognition and secondary structure of the peptide selector was discussed. The column stability during sequential analysis under different separation conditions was also evaluated.     

Determination of the Optical Purity of Fungicides of the Triazole Series

The influence of the nature of the chiral selector and the composition of the mobile phase on the enantioselectivity of the separation of two fungicides of the triazole series by high-performance liquid chromatography was studied. The optimum conditions were selected for the separation of enantiomers with the use of cellulose tris-(3,5-dimethylphenylcarbamate) as the chiral selector (Chiralcel OD column) and hexane–isopropanol mixtures as the mobile phase. Samples of technical diniconazole (China) were analyzed.     

Enantiomeric Resolution of Amino Acids by Reversed Phase High Performance Liquid Chromatography Using a New Chiral Mobile Phase

Abstract

The synthesis of a new chiral agent, (R,R) (-)N, N'-trans-1, 2-dihexylcyclohexanediamine, for the chromatographic resolution of racemates is reported. Highly selective separations of D- and L-isomers of free and Dns-amino acids were accomplished on a reversed-phase column using in the mobile phase a Cu(II) complex of the above chiral selector. The procedure was extended to resolve diastereomeric derivatives, which were obtained by reaction of an optically active amine with o-phthaldeyde in the presence of N-acetyl-L-cysteine.     

Separation and control of the elution order of N-t-butyloxycarbonyl amino acids D/L isomers by reversed-phase HPLC using cyclodextrins as chiral selectors for the mobile phase.

The enantiomeric resolution of N-t-butyloxycarbonyl (N-t-Boc) amino acids D/L isomers by reversed-phase HPLC was investigated using cyclodextrins (CD's) as chiral selectors for the mobile phase. The use of a low pH (pH<4) for the mobile phase enabled the enantioseparation of N-t-Boc amino acids. The opposite elution order of D/L isomers was observed when hydroxypropyl-derivatized beta-CD was used instead of native beta-CD. A computer simulation of the enantioseparation showed that the ratio of the retention factors of the chiral selector and the sample determined the elution order and the resolution. When the retention factor of the chiral selector is smaller than that of the sample, an isomer having larger complex formation constant eluted faster. However, when the chiral selector had a larger retention factor than the sample, an opposite elution order of the isomers was obtained. The large difference in the retention factors between the chiral selector and the sample led to good enantiomeric separation.     

Thin-layer chromatography separation of enantiomers of verapamil using macrocyclic antibiotic as a chiral selector

Silica gel thin-layer chromatography plates impregnated with macrocyclic antibiotic, vancomycin, as chiral selector were prepared and used for the resolution of (+/-)-verapamil. A mobile phase system of acetonitrile-methanol-water (15:2.5:2.5, v/v) was worked out systematically. The effects of chiral selector, temperature and pH on resolution were also studied. The spots were detected with iodine vapors and the detection limit was found to be 0.074 microg of each enantiomers.     

Enantioseparation of Chiral Antimycotic Drugs by HPLC with Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order

The separation of enantiomers of 10 chiral antimycotic drugs was studied on polysaccharide-based chiral columns with polar organic mobile phases. The emphasis was placed on some interesting examples of enantiomer elution order reversal observed depending on the chemistry of the chiral selector, separation temperature, major component, as well as the minor additive in the mobile phase. In particular, it was found that the elution order of enantiomers of chiral drug terconazole was opposite on cellulose- and amylose-based columns with the same pendant group. The affinity pattern of enantiomers of another chiral drug bifonazole was opposite towards to two amylose-based chiral selectors with different pendant groups. The affinity pattern of terconazole enantiomers also changed on some columns when the alcohol-based mobile phase was replaced with acetonitrile. An interesting effect of the minor acidic (formic acid) additives to the mobile phase on the affinity pattern of terconazole enantiomers was observed on Cellulose-2 and Cellulose-4 columns. In addition, a reversal of elution order of bifonazole enantiomers was observed on Amylose-2 column by variation of a separation temperature.     

Optimization of the LC enantioseparation of chiral pharmaceuticals using cellulose tris(4‐chloro‐3‐methylphenylcarbamate) as chiral selector and polar non‐aqueous mobile phases

The resolving power of a new commercial polysaccharide‐based chiral stationary phase, Sepapak‐4, with cellulose tris(4‐chloro‐3‐methylphenylcarbamate) coated on silica microparticles as chiral selector, was evaluated toward the enantioseparation of ten basic drugs with widely different structures and hydrophobic properties, using ACN as the main component of the mobile phase. A multivariate approach (experimental design) was used to screen the factors (temperature, n‐hexane content, acidic and basic additives) likely to influence enantioresolution. Then, the optimization was performed using a face‐centered central composite design. Complete enantioseparation could be obtained for almost all tested chiral compounds, demonstrating the high chiral discrimination ability of this chiral stationary phase using polar organic mobile phases made up of ACN and containing an acidic additive (TFA or formic acid), 0.1% diethylamine and n‐hexane. These results clearly illustrate the key role of the nature of the acidic additive in the mobile phase.     

The HPLC Enantioresolution of Selected Native or Derivatized Amino Acids on Teicoplanin Bonded Chiral Stationary Phase Using a Methanol‐Based Mobile Phase

The facile HPLC enantiomeric resolution of a variety of selected native or derivatized amino acids is carried out on the glycopeptide antibiotic teicoplanin bonded chiral stationary phase using a methanol‐based mobile phase and found very sensitive to the structural variations. This mobile phase is mainly composed of methanol. Organic additives such as acetic acid and triethylamine are introduced to the mobile phase in small percentages to control the analyte's retention time. Additive of low viscosity such as ethyl ether or petroleum ether is incorporated in the mobile phase as well to improve the resolution. Further increasing its percentage in the mobile phase deteriorates the resolution slightly; however, it extends the retention scale of enantiomers. The change in enantioselectivity is found to be insignificant under these circumstances. The hydrogen bonding and π‐π complexation in the hydrophobic pocket of teicoplanin chiral selector is believed to be the mechanism mainly responsible for the enantioresolution observed in this report.     

Enantioseparation of selected N-tert.-butyloxycarbonyl amino acids in high-performance liquid chromatography and capillary electrophoresis with a teicoplanin chiral selector

Enantioseparation of N-tert.-butyloxycarbonyl amino acids (N-t-Boc-Aas) with teicoplanin chiral selector was performed in two different separation systems: A teicoplanin-based chiral stationary phase (CSP-TE) was used in reversed-phase HPLC, and the same chiral selector (CS) was added into a background electrolyte (BGE) in HPCE. The enantioselective interaction with the same CSP/CS can be influenced by several factors, such as mobile phase/background electrolyte composition: the buffer concentration, pH, the CS concentration, the presence of organic modifiers. In addition, the charge of the chiral selector related to the charge of the analyte and to EOF are important variables in CE. The effect of these parameters on enantioselectivity and enantioseparation of selected N-t-Boc-Aas was studied. The presence of a sufficient concentration (1% solution) of a triethylamine acetate buffer in the mobile phase was shown to be essential for enantioseparation of these blocked amino acids in HPLC. A certain concentration of teicoplanin aggregates (along with teicoplanin molecules) in the BGE is required to obtain enantioseparation of N-t-Boc-Aas in HPCE.     

Enantioseparation of Chiral Antimycotic Drugs by HPLC with Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order

The separation of enantiomers of 10 chiral antimycotic drugs was studied on polysaccharide-based chiral columns with polar organic mobile phases. The emphasis was placed on some interesting examples of enantiomer elution order reversal observed depending on the chemistry of the chiral selector, separation temperature, major component, as well as the minor additive in the mobile phase. In particular, it was found that the elution order of enantiomers of chiral drug terconazole was opposite on cellulose- and amylose-based columns with the same pendant group. The affinity pattern of enantiomers of another chiral drug bifonazole was opposite towards to two amylose-based chiral selectors with different pendant groups. The affinity pattern of terconazole enantiomers also changed on some columns when the alcohol-based mobile phase was replaced with acetonitrile. An interesting effect of the minor acidic (formic acid) additives to the mobile phase on the affinity pattern of terconazole enantiomers was observed on Cellulose-2 and Cellulose-4 columns. In addition, a reversal of elution order of bifonazole enantiomers was observed on Amylose-2 column by variation of a separation temperature.

     

异喹啉酸衍生物刷型手性键合固定相的制备及在联萘酚衍生物拆分中的应用

在(S)-THIQCA环上引入π 酸基团, 制备了一种新型的刷型手性固定相(CSP), 并用于联萘酚及其衍生物的拆分, 探讨了改性剂对色谱行为的影响.     

High‐performance liquid chromatographic separations of stereoisomers of chiral basic agrochemicals with polysaccharide‐based chiral columns and polar organic mobile phases

The separation of the stereoisomers of 23 chiral basic agrochemicals was studied on six different polysaccharide‐based chiral columns in high‐performance liquid chromatography with various polar organic mobile phases. Along with the successful separation of analyte stereoisomers, emphasis was placed on the effect of the chiral selector and mobile phase composition on the elution order of stereoisomers. The interesting phenomenon of reversal of enantiomer/stereoisomer elution order function of the polysaccharide backbone (cellulose or amylose), type of derivative (carbamate or benzoate), nature, and position of the substituent(s) in the phenylcarbamate moiety (methyl or chloro) and the nature of the mobile phase was observed. For several of the analytes containing two chiral centers all four stereoisomers were resolved with at least one chiral selector/mobile phase combination.     

How mobile phase composition and column temperature affect enantiomer elution order of liquid crystals on amylose tris(3-chloro-5-methylphenylcarbamate) as chiral selector

A comprehensive study into the effects of mobile phase composition and column temperature on enantiomer elution order was conducted with a set of chiral rod-like liquid crystalline materials. The analytes were structurally similar and comprised variances such as length of terminal alkyl chain, presence of chlorine, number of phenyl rings, and type of chiral center. Experiments were carried out in polar organic and reversed-phase modes using amylose tris(3-chloro-5-methylphenylcarbamate) immobilized on silica gel as the chiral stationary phase. For all liquid crystals, reversal of elution order of enantiomers was observed based on type of used cosolvent and/or its content in the mobile phase; for some of the liquid crystals a temperature-induced reversal was also observed. Both linear and nonlinear dependencies of natural logarithm of enantioselectivity on temperature were found. Tested mobile phases comprised pure organic solvents and binary and tertiary mixtures of acetonitrile with organic solvents and/or water. Effect of acidic/basic mobile phase additives was also tested. Effect of structure of chiral selector is briefly discussed.     

Displacement study on a vancomycin-based stationary phase using N-acetyl-D-alanine as a competing agent

The analysis of the binding data of D,L-dansyl amino acids on a vancomycin stationary phase is investigated in relation to the addition of N-acetyl-D-alanine in the mobile phase. This eluent additive acts as a specific competing agent for the aglycone pocket of the immobilized chiral selector. A model taking into account both stereoselective and nonstereoselective interactions between the solutes and the stationary phase is used to fit the experimental data. From the results, the theoretical approach is considered to be adequate to describe the competing agent dependence on solute retention. To the best of our knowledge, this report constitutes the first example of a displacement study on a macrocyclic antibiotic stationary phase. This work shows that dansyl amino acids bind to the active aglycone pocket of the selector and that this interaction is enantioselective. The results also demonstrate that additional enantioselective sites at the vancomycin surface are involved in the chiral discrimination of these solutes.     

Chiral separation of basic drugs by capillary electrophoresis with carboxymethylcyclodextrins

Capillary electrophoresis (CE) with carboxymethylated beta- or gamma-cyclodextrins was used to achieve the rapid enantiomeric separation of a set of basic drugs. The enantiomers of 12 chiral amino-containing pharmaceutical compounds belonging to various therapeutic categories were analyzed by CE using an uncoated 60 cm x 75 microm I.D. silica capillary. Several experimental parameters such as the nature, concentration and pH of the buffer, nature and concentration of the anionic cyclodextrin and temperature were studied in order to optimize the enantiomeric separation. The variation of the solute partition coefficient for the chiral selector, the enantioselectivity and resolution factors are used to assess the quality of the chiral separation. It is shown that the solute affinity for the chiral selector is not related to its enantioresolution factor. None of the two cyclodextrin selectors used was able to separate the whole set of basic drugs.