Enantioseparation of chiral organochlorines on permethylated beta- and gamma-cyclodextrin, as well as 1:1 mixtures of them

Gas chromatography columns coated with 10% permethylated beta- and gamma-cyclodextrin in 85% dimethyl-15% diphenyl polysiloxane (beta- and gamma-PMCD, respectively) and 1:1 mixures are prepared and tested with regard to the enantioseparation of chiral chloropesticides. On the columns with the individual O-methylated cyclodextrins (O-tCDs), the enantiomers of aaeeee-hexachlorocyclohexane (alpha-HCH), e-aeee-1,3,4,5,6-pentachlorocyclohex-1-ene (beta-PCCH), and e-aaee-1,3,4,5,6-pentachlorocyclohex-1-ene (gamma-PCCH), cis- and trans-chlordane, and cis-heptachlor epoxide are separated on both columns, with the exception of the latter being separated only on beta-PMCD. On the column coated with 5% beta- and 5% gamma-PMCD, the resulting separation factor (a) is virtually 1/2 of the arithmetric mean of the elution-dependent separation factors on the individual O-tCDs. In case of reversed elution order on beta- and gamma-PMCD, the enantiomers are not resolved on the mixed columns as is the case with cis-chlordane. Likewise, the lower resolution of the gamma-PCCH enantiomers on the mixed columns prove the reversed elution order on beta- and gamma-PMCD without having enantioenriched standards available. On the column coated with 5% beta- and 5% gamma-PMCD, similar retention times to those observed on both 10% beta-PMCD and 10% gamma-PMCD are obtained. On the column coated with 10% beta- and 10% gamma-PMCD, significantly longer retention times are obtained compared with the columns that contain a total of 10% chiral stationary phase (CSP). This indicates that a relevant part of the interaction of the analytes with the chiral selector is non-enantioselective and, thus, only delays the elution of both enantiomers. Moreover, these non-enantioselective interactions prevent a direct comparison of CSPs with different amounts of the chiral selector. However, this is possible by using mixed phases of two CSPs with similar properties. Using this system, it is demonstrated that for the organochlorine compounds studied, no higher separation factor is observed on the mixed CSPs than on the individual O-tCD with the higher separation factor. Estimations allow a prediction that enantioseparations of organohalogen compounds can be achieved on columns coated with as little as 1% of the CSP.     

Enantioselective HPLC separation of bioactive C5-chiral 2-pyrazolines on lux amylose-2 and lux cellulose-2: Comparative and mechanistic approaches

Stereoselective analytical HPLC separations have been developed for a series of biologically active chiral 2-pyrazolines (1-22) to be used in monitoring their resolution reactions or to custom semipreparative HPLC separations prior to biological assessment of both enantiomers. Polysaccharide-based chiral stationary phases (CSPs), namely, Lux amylose-2 and cellulose-2, have been used. Both normal (n-hexane/ethanol) and polar organic (ethanol, methanol, acetonitrile, or mixtures thereof) elution modes were very beneficial for the achievement of baseline separations. The impact of various chemical moieties embedded in the structures of 2-pyrazolines 1-22 and the adopted stationary phases on chiral recognition has been investigated. A case of reversed order of elution following alterations in either stationary phase or elution mode has been observed. Our findings recommend that normal elution mode can be used for optimizing semipreparative HPLC methods whereas polar organic mobile phases (such as acetonitrile and ethanol) are more suited to stereoselective reactions monitoring, routine quality control work, or for pharmacological and toxicological assays. These results settle the implementation of polysaccharide-based CSPs using different elution modes and declare the practicality of such CSPs in stereoselective HPLC.     

True and false reversal of the elution order of barbiturates on a bonded cellulose-based chiral stationary phase

A set of racemic N-alkylated barbiturates were investigated for their direct enantioselective HPLC separation on a new chiral stationary phase (CSP) containing cellulose tris(3,5-dimethylphenylcarabamate) immobilized onto silica gel (Chiralpak IB) and its coated version (Chiralcel OD). They were online detected by UV and optical rotation detectors to trace the elution order of their enantiomers. Surprisingly, examples of false and true reversal of the elution order of enantiomers of barbiturates were observed and reported.     

Enantioseparation of vesamicol and novel vesamicol analogs by high-performance liquid chromatography on different chiral stationary phases

High-performance liquid chromatography enantioseparation of vesamicol and six novel azaspirovesamicols (amino alcohols) was accomplished on different chiral stationary phases (CSPs) by using an optical rotation based chiral detector for identification of the resolved enantiomers. The Pirkle-type column Reprosil Chiral-NR was found to be most suitable for chiral resolution in normal phase (NP) mode; all compounds could be enantioseparated successfully. Also the cellulose-based column Reprosil Chiral-OM showed appropriate separation properties by using NP conditions. The amylose-type column Reprosil Chiral-AM-RP was most suitable for enantioseparation in reversed phase (RP) mode; five out of seven compounds were resolved. This CSP showed a considerably higher capability for chiral recognition of vesamicol derivatives in RP mode than the corresponding cellulose-based column Reprosil Chiral-OM-RP. Enantioseparation with the teicoplanin aglycone-based column Reprosil Chiral-AA was successful under polar ionic mobile phase conditions.     

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.     

Enantiomers of New Synthetic Pyrrolylphenylethanoneamine Mono-Amino Oxidase Inhibitor Compounds: Analytical and Semipreparative HPLC Separations, and Chiroptical Characteristics

The polysaccharide chiral stationary phases (CSPs) Chiralcel OD and Chiralpak AD, and the brush-type (R,R)-Whelk-01 chiral stationary phases have been evaluated to separate new synthetic pyrrolylphenylethanoneamine racemic compounds, potentially monoamine oxidase (MAO) inhibitors, under various mobile phase compositions, using various temperatures. The enantioseparation was evaluated by comparing the (R,R)-Whelk-01 column performance with those of Chiralpak AD and Chiralcel OD. Significant differences were observed in their chiral recognition, as revealed from their retention, selectivity, resolution and elution order. Performances of the Chiralpak AD column were superior to those of the Chiralcel OD and (R,R)-Whelk-01 columns. Some of the racemic compounds were resolved by semipreparative chromatography on Chiralpak AD column in order to study the chiroptical proprieties of the single 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.

     

Development and validation of HPLC methods for enantioseparation of mirtazapine enantiomers at analytical and semipreparative scale using polysaccharide chiral stationary phases

Novel HPLC methods were developed for the analytical and semipreparative resolution of new antidepressant drug mirtazapine enantiomers. At analytical scale, the separation of the mirtazapine enantiomers was investigated using both cellulose and amylose tris(3,5-dimethylphenylcarbamate) (CDMPC and ADMPC) chiral stationary phases under normal-phases and polar organic modes. Good baseline enantioseparation was achieved using cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases under both normal-phases and polar organic modes. Furthermore, the elution order of mirtazapine enantiomic pairs was found reversed by changing the stationary phase from the amylose-based ADMPC–CSPs to its cellulose-based counterpart, CDMPC–CSPs. The validation of the analytical methods including linearity, limit of detection (LOD), limit of quantification (LOQ), recovery and precision, together with the semipreparative resolution of mirtazapine racemate were carried out using cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases and methanol as mobile phase without any basic additives under polar organic mode. At analytical scale, the elution times of both enantiomers were less than 6 min at normal temperature and 1.0 ml/min, with the separation factor () 1.99 and the resolution factor (Rs) 3.56. Then, the analytical methods were scaled up to semipreparative loading to obtain small quantities of both mirtazapine enantiomers. At semipreparative scale, about 16 mg/h enantiomers could be isolated and elution times of both enantiomers were less than 10 min at 2.0 ml/min. To increase the throughput, the technique of boxcar injections was used. One enantiomer ((−)-(R)-mirtazapine) was isolated with purity of >99.9% e.e. and >98.0% yield and another ((+)-(S)-mirtazapine) was isolated with purity of >97.0% e.e. and >99.0% yield. In addition, optical rotation and circular dichroism (CD) spectroscopy of both mirtazapine enantiomers isolated were also investigated.     

Chromatographic evaluation of poly (trans-1,2-cyclohexanediyl-bis acrylamide) as a chiral stationary phase for HPLC

Chiral stationary phases (CSPs) based on polymeric (R,R)- or (S,S)-1,2-diaminocyclohexane (DACH) derivatives are synthesized. When bonded to 5 microm porous spherical silica gel, the poly (trans-1,2-cyclohexanediyl-bis acrylamide) based poly-cyclic amine polymer (P-CAP) stationary phases is proved to be effective chiral stationary phases that could be used in the normal-phase mode, polar organic mode and with halogenated solvents mobile phases, if desired. Since these are entirely synthetic CSPs, the elution order of all enantiomers can be reversed between the (R,R) P-CAP and (S,S) P-CAP columns. Because of the high loading of chiral selectors, the columns exhibit very high sample capacities. Thus, P-CAP columns are useful for preparative and semi-preparative enantiomeric separations. The application of these CSPs and optimization of their separations are discussed.     

HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide‐based chiral columns

The separation of enantiomers of five chiral dihydropyridine derivatives was studied on five different polysaccharide‐based chiral HPLC columns with various normal‐phase (NP), polar organic, and reversed‐phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on enantiomer elution order (EEO) with various columns and mobile phase composition. The interesting phenomenon of reversal of EEO, recently reported in the case of amlodipine (AML) depending on the concentration of formic acid in acetonitrile, was also confirmed with NP eluents. Under RP conditions at relatively low water content, the EEO of AML could also be reverted by varying the concentration of formic acid in the mobile phase. However, at higher water content the same parameter did not affect the EEO, but only induced gradual decrease in resolution up to complete co‐elution of enantiomers. Additionally, in organic‐aqueous mobile phases retention factors decreased with increasing water content but only up to 20% (v/v), while above this concentration the expected typical RP behavior was observed. The presence of the commonly used additive diethylamine in the mobile phase seems important for observing a reversal in EEO with increasing concentration of formic acid. The reversal of the EEO was characteristic of AML only and was not observed for any of other dihydropyridines included in this study.     

Chiral discrimination studies of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid by high-performance liquid chromatography and NMR spectroscopy

Chiral discrimination studies using (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) as a chiral selector were performed by high-performance liquid chromatography (HPLC) and NMR spectroscopy. The enantiomers of alanine (Ala) or alanine methyl ester (Ala-ME) were well separated on the chiral stationary phases (CSPs) derived from (+)-18-C-6-TA by HPLC. The chiral selector, (+)-18-C-6-TA, used in the CSP was also applied for the chiral discrimination of the Ala and Ala-ME enantiomers, and it discriminated these enantiomers successfully by NMR spectroscopy. The chemical shift differences (Delta Delta delta) of the alpha-proton of these enantiomers in the presence of an equimolecular solution of 18-C-6-TA were observed to be 0.10 ppm for Ala in methanol-d4 containing 10 mM H2SO4 and 0.11 ppm for Ala-ME in methanol-d4. The observed NMR results agreed with the chromatographic data on the (+)-18-C-6-TA-derived CSP by HPLC in terms of both the elution order and solvents effects.     

Enantiomeric separation of chiral pesticides by high performance liquid chromatography on cellulose tris-3,5-dimethyl carbamate stationary phase under reversed phase conditions

Twenty chiral pesticides were tested, of which seven samples were directly separated by HPLC using cellulose tris-3,5-dimethyl carbamate (CDMPC) chiral stationary phase under RP conditions. The influence of mobile phase composition and column temperatures from 0 degrees C to 40 degrees C on the separations were investigated. The mobile phases were methanol/water or ACN/water at a flow rate of 0.8 mL/min with UV detection at 230 or 210 nm. Epoxiconazole, terallethrin, benalaxyl, and diclofopmethyl were observed to obtain the baseline separation under suitable conditions and other pesticides pyriproxyfen, lactofen, and quizalofop-ethyl were separated partially. The retention factors (k) and selectivity factor (alpha) for the enantiomers of most investigated pesticides decreased upon increasing the temperature except for the selectivity factors (alpha) of pyriproxyfen in methanol/water. The ln alpha - 1/T plots for racemic chiral pesticides were linear at the range of 0-40 except for that of pyriproxyfen enantiomers in methanol/water and the chiral separations were controlled by enthalpy. Better separations were not always at low temperature. The elution orders of the eluting enantiomers were determined by a circular dichroism (CD) detector.     

大分子键合型手性固定相的研究进展

高效液相色谱（HPLC）被广泛认为是分离制备光学纯单一对映体最有效的方法。在高效液相色谱手性拆分中,手性固定相的性能直接影响到色谱柱的手性分离能力。在众多手性固定相中,键合型手性固定相具有溶剂耐受性好,分离模式灵活等优点,是很重要的一大类手性固定相。本文主要针对大分子键合型手性固定相,包括多糖衍生物键合型手性固定相、蛋...     

Direct chiral resolution and its application to the determination of fungicide benalaxyl in soil and water by high-performance liquid chromatography

A simple chiral high-performance liquid chromatography (HPLC) method with ultraviolet (UV) and circular dichroism (CD) detection was developed and validated for measuring benalaxyl enantiomers using (R,R) Whelk-O 1 column. The effects of mobile phase composition and column temperature on the entioseparation were investigated. A CD detector was used to determine the elution order of the enantiomers. Excellent resolution was easily obtained using n-hexane-polar organic alcohols mobile phase. The chiral recognition mechanism was also discussed. Based on the developed chiral HPLC method, enantioselective analysis methods for this fungicide in environment matrix (soil and water) were developed and validated. Good linearities were obtained over the concentration range of 0.25-25 mg L⁻¹ for both enantiomers. Liquid-liquid extraction and solid phase extraction (SPE) were used for the enrichment and cleanup of soil and water samples. Recoveries for the two enantiomers were 79-91% at 0.02, 0.04 and 0.2 mg kg⁻¹ levels from soil, and 89-101% at 0.0025, 0.01 and 0.05 mg L⁻¹ levels from water. Run-to-run and day-to-day assay precisions were below 10% for both enantiomers at concentrations of 0.5, 1 and 5 mg L⁻¹. Individual detection limits of the two enantiomers were both 2 ng. Limits of detection (LOD) were 0.004 mg kg⁻¹ in soil and 0.001 mg L⁻¹ in water.     

Chiral analysis of milnacipran by a nonchiral HPLC--circular dichroism: improvement of the linearity of dichroic response by temperature control

The determination of the enantiomeric excess (e.e.) of a basic drug has been investigated in LC using a nonchiral stationary phase and a circular dichroism (CD) detector in order to avoid expensive chiral columns. The CD detector records both dichroic (Deltaepsilon) and UV (epsilon) signals at the same wavelength and calculates the anisotropy factor (g=Deltaepsilon/epsilon), which is linearly related to the e.e. The enantiomeric and chemical composition of a chiral drug can be simultaneously determined on a nonchiral HPLC support. However, the g factor from the CD signal is temperature dependent. Indeed, the temperature has an influence on the stability of the CD signal and the linear regression between g factor and the e.e. of 1R,2S-enantiomer. So, a decrease in temperature gives rise to an improvement of the above-mentioned linearity correlation. After optimization of chromatographic parameters (porous graphitic carbon-based column, methanol/ phosphate buffer as mobile phase) and selection of CD wavelength, a linear regression of g factor versus e.e. of 1R,2S-enantiomer was obtained at temperature-controlled CD detection and an LOQ of 94% was found. The enantiomeric composition of milnacipran was determined with good accuracy.     

Application of 3 μm particle-based amylose-derived chiral stationary phases for the enantioseparation of potential histone deacetylase inhibitors

In this work, we report on the difference in performance of the two 3 μm particle-based Chiralpak IA-3 and Chiralpak AD-3 chiral stationary phases (CSPs) in the direct resolution of four racemic cinnamyl 2-aminoanilides, endowed with histone deacetylase inhibitory activity. The 3 μm CSPs were explored to determine if they could provide an effective resolution of enantiomers in presence of alcoholic eluents such as pure methanol, ethanol and 2-propanol. Temperature variable enantioselective HPLC and subsequent van't Hoff analysis were performed. In most of cases the van't Hoff plots were found to show a non-linear behaviour. The knowledge of the enantiomeric elution order associated with the data coming from enantioselective HPLC permitted to advance some hypothesis about the groups involved in chiral recognition mechanism.     

A new application of stopped-flow chiral HPLC: inversion of enantiomer elution order

A newly developed procedure to reverse the enantiomer elution order of compounds resolved on chiral stationary phases (CSPs) for HPLC is presented. The optimized analytical protocol is based on the effect of temperature on enantioselectivity and does not involve any changing in mobile phase composition or type of CSP. In essence, the approach entails variable temperature chromatography at two temperatures. The enantiomer separation is performed at a low column temperature, with stopping the flow prior to elution of the less retained enantiomer. Then, the column temperature is changed with the peaks trapped inside the column, followed by elution with the same mobile phase in reverse direction. Under these conditions, the more pronounced loss in free energy of binding for the more strongly bound enantiomer results in an inversion of the elution order. This procedure may be applied to each enantiomer pair that is separated by chiral HPLC under an appreciable enthalpy-control.     

键合型聚丙烯酰胺衍生物手性固定相的制备与手性识别性能

高效液相色谱(HPLC)被广泛认为是分离制备光学纯单一对映体的最有效方法。在高效液相色谱手性拆分中,手性固定相(CSP)的性能直接影响到色谱柱的手性分离能力。在众多手性固定相中,键合型手性固定相具有溶剂耐受性好,分离模式灵活等优点,已经发展成为一类重要的手性固定相。本文通过两步化学反应合成了新型的光学活性丙烯酰胺衍生物--(S)-1-丙烯酰-2-(N-苯基甲酰胺基)吡咯烷((S)-APACP),采用核磁共振氢谱表征了(S)-APACP的化学结构;通过3步化学反应制备了键合型聚丙烯酰胺衍生物手性固定相,采用热重分析法表征了聚合物的键合量,采用HPLC评价了键合型手性固定相的识别能力,分析了影响其手性识别能力的因素。研究结果表明,APACP聚合物成功地键合到硅胶表面制备了具有良好溶剂耐受性的键合型手性固定相,其聚合物键合量为10.2%~11.8%,该键合型手性固定相对若干种对映体显示了较好的手性识别能力。     

Selectivity tuning in chiral dual column gas chromatography

The enantioselective tuning of two columns coupled in series is investigated in chiral high-resolution gas chromatography. Two columns with opposite enantioselectivities (Chirasil-L-Val and Chirasil-D-Val) are coupled in series via a T connector, and the relative retention of enantiomers chromatographed on the system is changed by varying the individual carrier gas flow rates in the coupled columns. The flow-rate ratio necessary for the required selectivity is calculated on the basis of the measured retention factors on the individual columns. The performance of this method for adjusting selectivity is studied by the separation of enantiomers of the N-TFA-O-methyl esters of six amino acids. It is demonstrated that the change of the coupling point carrier gas pressure, at the constant inlet and outlet pressures, may change the enantioselectivity of the given column series to such an extent that the enantiomer elution order may be reversed.     

Stereospecific analysis of lorazepam in plasma by chiral column chromatography with a circular dichroism-based detector

The chiral separation of lorazepam was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of lorazepam enantiomers was obtained on the column of beta-cyclodextrin derivative immobilized silica gel under reversed-phase conditions. The enantiomeric separation and identification of lorazepam were succeeded by CD detector. The method described allows the quantitation of the stereoisomers of lorazepam in human plasma following the administration of a therapeutic dose of the racemic drug. Chiroptical detection is useful for the pharmacokinetic study of chiral drugs.