Influence of the nature and substitution of chiral 2,3-epoxy alcohol derivatives on the enantiomeric elution order on chiralcel OD column

A number of 2,3-epoxy alcohol derivatives (1–16), obtained either as racemates or through the Sharpless asymmetric epoxidation reaction, were studied on a Chiralcel OD column. Nearly all compounds exhibit good enantioselective resolution on this chiral support. The order of elution of enantiomers is reversed between nerol and geraniol compounds. For 2,3-epoxy alcohols bearing a remote alkoxy (or silyloxy) group, the order of the enantiomeric elution alternates with the number n (n = 1–3) of methylenic groups present between the epoxide ring and the terminal OR (R = p − BrBn or OSitBuPh₂) functionality. In the case of trans 2,3-epoxy alcohols for the same number n, the order of elution is reversed when changing the terminal group −OSi to −OR. The latter group greatly improves the separation of the two enantiomers. Chirality 10:804–807, 1998. © 1998 Wiley-Liss, Inc.     

Preparative chiral chromatography and chiroptical characterization of enantiomers of omeprazole and related benzimidazoles

To chiroptically characterize the enantiomers of omeprazole and some structurally related benzimidazoles with circular dichroism (CD), preparative chiral liquid chromatography was utilized for the isolation of the pure enantiomers. A limited analytical column screen was performed identifying Kromasil-CHI-TBB and the amylose-based phases Chiralpak AD and AS as possible chiral stationary phases (CSPs) for the preparative scale separation of the enantiomers of the different benzimidazoles. Optimization of the chromatographic conditions with respect to retention, enantioseparation, and resolution was achieved by variation of the mobile phase constituents as well as of temperature. Because of the lability of the compound in slightly acidic media, supercritical fluid chromatography (SFC) could not be applied for a preparative scale separation of the enantiomers. The separation of omeprazole was optimized to give high throughput (2.6 kg racemate/kg CSP/day) and high enantiomeric excess of the obtained isomers. The absolute configurations of the pure enantiomers of rabeprazole, lansoprazole, and pantoprazole were determined from the strong correlation to the CD spectrum of (+)-(R)-omeprazole. For all the compounds, the (+)-enantiomers displayed similar chiroptical features as (+)-(R)-omeprazole and were thus assigned the (R)- configuration. Elution order of the optical isomers was monitored by injecting racemic solutions spiked with one of the isomers and also by an on-line laser polarimeter. Both the type of CSP and also the mobile phase constituents had a strong effect on elution order of the enantiomers.     

Structural features affecting chiral resolution of cannabimimetic enantiomers by amylose 3,5-dimethylphenylcarbamate chiral stationary phase

The effect of structural features of six pairs of enantiomers of cannabimimetic compounds on their chromatographic resolution on an amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase was studied using various compositions of n-hexane with 2-propanol and ethanol. Structural analysis by molecular mechanics was also performed to verify that the 3D conformation within this family of compounds was preserved with substitution. The homologous enantiomeric pairs showed better resolution when there was an additional OH group near the chiral centers (position 7 on the cannabinoid structure). Better resolution was observed also for the enantiomeric pair that had the smaller alkyl side chain. These differences indicated that the additional OH group contributed to a better discrimination of the enantiomers by the chiral sites of the stationary phase and that the bulkier alkyl side chain reduced it. The chromatographic resolution of two enantiomeric pairs of nonclassical cannabinoids HU-249 and HU-250, HU-255 and HU-256, was compared both in ethanol and 2-propanol. Both enantiomeric pairs showed relatively high resolution and selectivity, but the rigid benzofuran analogs (HU-249 and HU-250) exhibited better resolution using 2-propanol, in spite of the flexibility of the open chain analog (HU-255 and HU-256) and its additional OH group. The elution order of all the cannabinoids was (+)/(?) using both solvents. Unusual solvent effects were displayed by one enantiomeric pair, Δ⁶-THC, which was resolved easily using 2-propanol, but whose elution order reversed with 1% ethanol in the mobile phase. Partial separation was obtained at 5% ethanol [elution order (+)/(?)] and full separation was obtained at 0.5% ethanol [elution order (?)/(+)]. © 1995 Wiley-Liss, Inc.     

Dual-column high-performance liquid chromatographic urinary organic acid profiling

Two-dimensional high-performance liquid chromatography (HPLC) is increasingly used for the separation and identification of compounds in biological matrices. Conventional two-dimensional HPLC involves either heart-cutting or column-switching techniques. These techniques work well but are very time consuming when the analysis involves many compounds and requires more than a few minutes to completely elute from the column. We have modified the column-switching technique by utilizing two columns in series, an Aminex HPX-87H organic acid column followed by a 15-cm 5-μm C₁₈ analytical column. Both columns are compatible with the isocratic pH 2.5 H₂SO₄ mobile phase employed in the organic acid profiling. The dual-column system affords better resolution of urinary organic acids than does either column separately. Reversing the column order does not dramatically affect the elution pattern (peak shape, peak height, and R_f values are approximately the same). The dualcolumn HPLC configuration works well as a rapid means of screening urinary carboxylic acids prior to subsequent definitive analyses of abnormal samples.     

Effect of Basic and Acidic Additives on the Separation of Some Basic Drug Enantiomers on Polysaccharide‐Based Chiral Columns With Acetonitrile as Mobile Phase

The separation of enantiomers of 16 basic drugs was studied using polysaccharide‐based chiral selectors and acetonitrile as mobile phase with emphasis on the role of basic and acidic additives on the separation and elution order of enantiomers. Out of the studied chiral selectors, amylose phenylcarbamate‐based ones more often showed a chiral recognition ability compared to cellulose phenylcarbamate derivatives. An interesting effect was observed with formic acid as additive on enantiomer resolution and enantiomer elution order for some basic drugs. Thus, for instance, the enantioseparation of several β‐blockers (atenolol, sotalol, toliprolol) improved not only by the addition of a more conventional basic additive to the mobile phase, but also by the addition of an acidic additive. Moreover, an opposite elution order of enantiomers was observed depending on the nature of the additive (basic or acidic) in the mobile phase. Chirality 27:228–234, 2015. © 2015 Wiley Periodicals, Inc.     

A borate column for the separation of ribo and deoxyribonucleosides

A method is described for the separation of mixtures of the naturally occurring ribo- and deoxyribonucleosides by chromatography on columns of Dowex 50 eluted with ammonium borate. Nucleotides, ribonucleosides, and nucleotide-containing complex sugars are not retained on the column under these conditions, permitting unambiguous separation of these compounds from the deoxyribonucleosides, which are easily separated from each other by this chromatography. The elution positions of the nucleic acid bases allow partial to complete resolution from the corresponding deoxynucleosides. This column has either preparative or analytical utility for the procurement of nucleoside fractions free of the common contaminants.     

Supercritical fluid chromatography comparison of the poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1'-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

High-performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides

The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.     

Direct high-performance liquid chromatographic separation of the enantiomers of venlafaxine and 11 analogs using amylose-derived chiral stationary phases

A direct liquid chromatographic enantioselective separation of venlafaxine and 11 analogs was obtained in the normal phase mode using Chiralpak AD. For some compounds, a comparison between the enantioseparation using coated and immobilized amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak AD and Chiralpak IA, respectively) was made. The best separations were achieved on Chiralpak AD with ethanol as alcoholic modifier in a mobile phase made basic by DEA addition: separation factor ranges between 2.08 and 1.15 and resolution factor between 7.0 and 1.0. Using the same CSP and 2-propanol doped with TFA as acidic modifier, 10 compounds were enantioseparated with separation factor ranging between 1.40 and 1.04 and resolution factor between 3.1 and 0.3. The use of ethanol as alcoholic modifier also has the advantage of better solubility of the compounds in the mobile phase. The nature of the substituent (electron donating or withdrawing) affects in general the separation factor. A memory effect that involves a long equilibration time of the CSP is present when switching from an acidic mobile phase to a basic one.     

Direct coupled column separation and determination of the diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine.

A reversed-phase coupled column separation (CCS) system for the analysis of two diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine is described. After direct injection of urine samples (50 microliters) the glucuronides were isolated by complex formation on a terbium(III) loaded strong cation exchanger at alkaline pH. The solutes were eluted from the precolumn by an acidic mobile phase, enriched and separated on Hypercarb (porous graphitic carbon) as analytical column with 0.1 M acetic acid pH 2.8 and 30% acetonitrile as mobile phase. The calibration graph was linear (r2 = 0.9999) and the detection limits were in the low picomole (UV) or femtomole (fluorescence) range. Optimization of the analytical column revealed that elution order and selectivity for the glucuronides were dependent on the buffer agent and temperature used. By appropriate choice of mobile phase conditions all four diastereomers could be separated.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.     

Evaluation of the performance of protein separation in figure-8 centrifugal counter-current chromatography

The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column.     

Theory-guided efficient strategy to maximize speed and resolution in rapid gradient LC-MS/MS bioanalysis

Reversed-phase gradient LC-MS/MS bioanalytical methods of 5-100% organic solvent in a 1-3 min gradient time are common in today's bioanalytical laboratory. The goal of this work was to develop a theory-guided systematic strategy for maximizing resolution and speed in rapid gradient LC-MS/MS bioanalysis. We studied the effect of gradient time (t(G)), initial and final eluent strength (% B=% organic), and flow rate (F) on the separation of multiple critical pairs (R(s)) and peak capacity (n(c)) in a gradient elution of a mixture of five structurally related compounds. By optimizing the gradient time t(G), the initial and final percentages of the organic component of the mobile phase, comparable resolution and peak capacity could be achieved in a shorter run time. More importantly, we demonstrated that higher flow rates improved resolution, peak capacity and reduced run time in rapid gradient separations on a 5 μm particle column. A straightforward mathematical explanation of the phenomenon was provided applying basic resolution equations in gradient elution theory. A systematic approach to execute a rapid gradient LC-MS/MS bioanalytical method to shorten run time and improve resolution is proposed, taking into consideration not only the analytes of interest but also potential matrix effects from the dosing vehicle and biological matrix.     

Enantioselective chromatography and absolute configuration of N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines: potential sigma1 ligands

We describe the preparation of racemic N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines, potential sigma1 ligands, and their resolution via chiral HPLC. In order to obtain enantiopure compounds, direct chromatographic methods of separation using chiral stationary phases were investigated. Different methods suitable for both analytical and semipreparative purposes are proposed. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD and OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). On the basis of the preliminary chromatographic results, the resolution of compound 1 was transferred onto a Chiralcel OD semipreparative column. The enantiomers were obtained in high enantiomeric excess. The configurational assignment was performed by circular dichroism. Computational analysis was used to explore the enantioselective recognition process of compound 1 with the Chiralcel OD stationary phase.     

Simultaneous extraction and separation of liquiritin,glycyrrhizic acid,and glabridin from licorice root with analytical and preparative chromatography

Simultaneous extraction and separation of liquiritin, glycyrrhizic acid, and glabridin from licorice were developed by liquidliquid extraction with liquid chromatography separation. By utilizing different extraction solvents, procedures, and times, the optimum extraction conditions were established. The extracts of licorice were separated and determined using a C₁₈ column with a mobile phase consisting of acetonitrile-water (containing 1.0% acetic acid) with a gradient elution of 0∼10 min from 20:80 to 60:40 (v/v). Preparative columns with different packing sizes were investigated to isolate the three compounds from the extracts of licorice. The 12 μm chromatographic column showed better separation for the three compounds from licorice. 0.29 mg/g for liquiritin, 1.43 mg/g for glycyrrhizic acid, and 0.07 mg/g for glabridin were obtained and the recoveries were 80.8, 89.7, and 72.5%, respectively.     

Chromatographic comparison of atenolol separation in reaction media on cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase using ultra fast liquid chromatography

Because chiral liquid chromatography (LC) could become a powerful tool to estimate racemic atenolol quantity, excellent enantiomeric separation should be produced during data acquisition for satisfactory observation of atenolol concentrations throughout the racemic resolution processes. Selection of chiral LC column and analytical protocol that fulfill demands of the ultra fast LC analysis is essential. This article describes the characteristics of atenolol chromatographic separation that resulted from different resolution media and analytical protocols with the use of a Chiralcel® OD column. The chromatograms showed quite different characteristics of the separation process. The single enantiomer and racemic atenolol could be recognized by the Chiralcel® OD column in less than 20 min. Symmetrical peaks were obtained; however, several protocols produced peaks with wide bases and slanted baselines. Observations showed that efficient enantioresolution of racemic atenolol was obtained at slow mobile phase flow rate, decreased concentration of amine‐type modifier but increased alcohol content in mobile phase and highest ultraviolet detection wavelength were required. The optimal ultra fast LC protocol enables to reduce and eliminate the peaks of either the atenolol solvent or the buffers and provided the highest peak intensities of both atenolol enantiomers. Chirality 24:356–367, 2012. © 2012 Wiley Periodicals, Inc.     

Simultaneous LC/ESI‐MS Separation Method for the Enantioseparation of Some New Anticonvulsant Drugs

A sensitive and specific method for the simultaneous determination of the enantiomeric purity of 2,6‐dimethylphenoxyacetyl derivatives as trans or cis racemic and enantiomeric forms with 2‐ or 4‐aminocyclohexanol moiety ( 1 , 2 , 3 , 6 ) and their amine analogs ( 8 , 9 ) was developed. The compounds studied are known for their anticonvulsant activity and the most interesting pharmacological results were those for (±)‐trans‐2‐(2,6‐dimethylphenoxy)‐N‐(2‐hydroxycyclohexyl)acetamide ( 1 ) as well as (±)‐trans‐2‐[(2,6‐dimethylphenoxy)ethyl]aminocyclohexanol ( 8 ). The analytical method for determining the enantiomeric purity of the compounds studied is based on direct separation of the analytes using a chiral stationary phase (Chiralpak AS column). The mass spectrometric analysis was done on a coupled liquid chromatograph–mass spectrometer system with an electrospray ionization source (LC/ESI‐MS). For the compounds 1 , 8 , and 9 , the method allows an excellent separation of enantiomers, with a resolution higher than 3.2, and a tailing factor of less than 1.67 with a final enantiomer purity better than 97.5%. Chirality 26:144–149, 2014. © 2014 Wiley Periodicals, Inc.     

Elucidation of the enantioselective recognition mechanism of a penicillin G acylase-based chiral stationary phase towards a series of 2-aryloxy-2-arylacetic acids

A series of structurally related 2-aryloxy-2-arylacetic acids (1-3, 5-16) together with a thioisostere derivative (4) have been synthesized and characterized by GC-MS and 1H NMR. The designed compounds were analyzed on a Penicillin G Acylase chiral stationary phase (PGA-CSP) and the influence of the structure variations on retention and enantioselectivity was investigated. The chromatographic study includes the direct separation of the enantiomers of the synthesized compounds and the determination of the elution order of selected racemic mixtures. 10 out of 16 racemates were separated; high chromatographic enantioseparation factors (alpha > 2) were achieved for some compounds. For the enantiomers of four compounds whose absolute configuration was known (1, 3, 12, 16), the elution order was R:S with the exception of 2-(4-chloro-phenoxy)phenylacetic acid (1), for which the elution order was reversed. Preliminary molecular modeling studies suggest that both polar and charge-transfer interactions as well as steric effects play an important role in determining the retention factors and the enantioselectivities observed.     

Preparation of milligrams of pure enantiomers using analytical-scale high-performance liquid chromatography

Pure enantiomers of an agrochemical process intermediate, (RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)-pentan-3-one ( 1 ), have been prepared on the milligram scale under overload chromatographic conditions on an analytical chiral column (250 × 4.6 mm i.d.). The effects of variation of temperature and mobile phase composition on retention factor, separation factor, and peak resolution have been investigated. Effects of flow rate, enantiomer ratio, sample concentration, and column load on productivity are also studied. Seven milligrams of the less retained (+)-enantiomer and 5 mg of the (?)-enantiomer were obtained from a single injection of 21 mg of (RS)- 1 . © 1994 Wiley-Liss, Inc.