Pirkle-type Chiral Stationary Phase Research Articles

Enantioselective Determination of Hydroxy Amino Acids in Japanese Traditional Amber Rice Vinegars

A fully-automated two-dimensional (2D) HPLC system for the determination of serine (Ser), threonine (Thr) and allo-threonine (aThr) enantiomers was developed. The system was validated and applied to the determination of the target hydroxy amino acid enantiomers in vinegar samples including the Japanese traditional amber rice vinegar. The Japanese traditional amber rice vinegar is one of the typical natural fermented products containing high levels of D-amino acids, and the clarification of the amounts of hydroxy amino acid enantiomers is expected. For the on-line 2D-HPLC system, a narrowbore ODS column (Singularity RP18, 1.0 x 250 mm) was used in the first dimension, and an originally-designed Pirkle-type chiral stationary phase (Singularity CSP-013S, 1.5 x 250 mm) was utilized in the second dimension. The target hydroxy amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence (ex. 470 nm, em. 530 nm). The concentrations and the %D values were compared among a grain vinegar, a widely-distributed black vinegar and Japanese traditional amber rice vinegars (fermented/aged 1 or 5 years by microorganisms in an earthenware jar). The amounts of hydroxy amino acid enantiomers were low (0.21-0.38 μmol/mL, only L-forms) in the grain vinegar, and relatively high amounts of the L-forms were observed (1.05-1.61 μmol/mL) in the widely-distributed black vinegar. On the other hand, clear peaks of the D-forms (0.16-0.86 μmol/mL for D-Ser, 0.02-0.11 μmol/mL for D-aThr) as well as high amounts of the L-forms (2.27-2.51 μmol/mL for L-Ser, 1.85-2.10 μmol/mL for L-Thr) were observed in the Japanese traditional amber rice vinegars. The amounts of D-Ser and D-aThr clearly increased with increasing the fermentation/aging period. This is the first report showing the profiles of the hydroxy amino acid enantiomers simultaneously, and further investigations using various fermented food/beverage samples are expected.

Development and evaluation of Pirkle-type chiral stationary phase for flash chromatography

Liquid chromatography is the most applied methodology for enantioseparation in preparative and semi-preparative scale; however, flash chromatography is seldom the first choice. This work proposes a new sustainable method to achieve pure enantiomers in mg scale. Herein, the functionalization of silica for flash chromatography columns with a suitable chiral selector, for subsequent quantitative enantioseparation of chiral compounds, is described. Accordingly, the Whelk-O®1 chiral selector was bonded to flash silica and packed into a reused solid phase extraction cartridge. For the evaluation of the enantioselective performance of the flash column, the enantiomers of a chiral derivative of xanthone were quantitatively enantioseparated with an average recovery of 70% and an enantiomer ratio (e.r.) of 99% and 97% for each enantiomer. Evaluation with the anti-inflammatory drug naproxen was also performed, resulting in an average recovery of 95% and 89% and 95% e.r. for each enantiomer. The flash column showed high stability and load ability, versatility, and good reproducibility.

Development of an off-line heart cutting two-dimensional HPLC system for enantioselective analysis of serine, threonine and allo-threonine in human physiological fluids

A highly-selective two-dimensional high-performance liquid chromatographic (2D-HPLC, off-line heart cutting mode) system was developed for the determination of serine (Ser), threonine (Thr) and allo-threonine (aThr) enantiomers in human physiological fluids. Ser, Thr and aThr have a hydroxy group in their side chains, and the development of a simultaneous analytical method with a practically sufficient enantio/chemo-selectivity has been required to clarify their amounts in human physiological fluids. The amino acids in the samples were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and were isolated by a reversed-phase column (Singularity RP18, 1.0 x 250 mm) in the first dimension. After the target amino acids were collected, the fractions were manually introduced into an enantioselective column in the second dimension and were detected by their fluorescence. For the second dimension, a Pirkle-type chiral stationary phase (Singularity CSP-013S, 1.5 x 250 mm) was used. The resolution values of the enantiomers obtained by the Singularity CSP-013S column were 7.64 for Ser, 7.58 for Thr and 4.71 for aThr by using the mixture of methanol and acetonitrile containing formic acid as the mobile phases. The developed method was validated and applied to human plasma and urine. In the plasma, the obtained %d values (the percentage of d-form to total amino acid) were 1.7 for Ser, and trace levels of d-aThr and d-Thr were observed. In the urine, the %d values were 48.0 for Ser, 1.6 for Thr and 8.0 for aThr (calculated using d-aThr and l-Thr).

Enantiomeric Separation and Molecular Modelling of Bioactive 4-Aryl-3,4-dihydropyrimidin-2(1H)-one Ester Derivatives on Teicoplanin-Based Chiral Stationary Phase

The enantiomeric separation of 15 racemic 4-aryl-3,4-dihydropyrimidin-2(1H)-one (DHP) alkoxycarbonyl esters, some of which proved to be highly active as A2B adenosine receptor antagonists, was carried out by HPLC on ChirobioticTM TAG, a chiral stationary phase (CSP) bearing teicoplanin aglycone (TAG) as the chiral selector. The racemic compounds were separated under polar organic (PO) conditions. Preliminarily, the same selectands were investigated on three different Pirkle-type CSPs in normal-phase (NP) conditions. A baseline separation was successfully obtained on TAG-based CSPs for the majority of compounds, some of which achieved high enantioselectivity ratios (α > 2) in contrast with the smaller α values (1–1.5) and the lack of baseline resolution observed with the Pirkle-type CSPs. In particular, the racemic tetrazole-fused DHP ester derivatives, namely compounds 8 and 9, were separated on TAG-based HPLC columns with noteworthy α values (8.8 and 6.0, respectively), demonstrating the potential of the method for preparative purposes. A competition experiment, carried out with a racemic analyte (6) by adding N-acetyl-d-alanine (NADA) to the mobile phase, suggested that H-bonding interactions involved in the recognition of the natural dipeptide ligand d-Ala-d-Ala into the TAG cleft should be critical for enantioselective recognition of 4-aryl DHPs by TAG. The X-ray crystal structure of TAG was elucidated at a 0.77 Å resolution, whereas the calculation of molecular descriptors of size, polar, and H-bond interactions, were complemented with molecular docking and molecular dynamics calculations, shedding light on repulsive (steric effects) and attractive (H-bond—polar and apolar) interactions between 4-aryl DHP selectands and TAG chiral selectors.

Design, preparation and application of a Pirkle-type chiral stationary phase for enantioseparation of some racemic organic acids and molecular dynamics studies

Design, preparation and application of a Pirkle-type chiral stationary phase for enantioseparation of some racemic organic acids and molecular dynamics studies

Pirkle-type chiral stationary phase on core–shell and fully porous particles: Are superficially porous particles always the better choice toward ultrafast high-performance enantioseparations?

Pirkle-type Whelk-O1 chiral stationary phase (CSP) was prepared on 2.6μm superficially porous particles (SPPs). The chromatographic behavior of columns packed with this new CSP was compared with that of columns packed respectively with 1.8 and 2.5μm Whelk-O1 fully porous particles (FPPs). In the comparison, both thermodynamic and kinetic aspects were considered. Contrary to initial expectations, chiral columns packed with 2.6μm SPPs were quasi-comparable to those packed with 2.5μm FPPs, apparently due to larger contributions to band broadening from both eddy dispersion and, especially for the second eluted enantiomer, adsorption–desorption kinetics. These findings raise the question if SPPs, in spite of the undeniable advantages of their morphology to speed up mass transfer, are always the best choice for high-efficient ultrafast chiral separations. The last part of the work focuses on the use of short columns (10mm long) and very high flow rates to realize the separation of the enantiomers of trans-stilbene oxide (TSO) in normal phase mode in less than 1s.

Enantiomeric purity determination of (l)-amino acids with pre-column derivatization and chiral stationary phase: Development and validation of the method

A simple, efficient and general HPLC method for the determination of enantiomeric purity of a series of (l)-amino acids was developed. In order to improve the detection sensitivity, pre-column derivatization was adopted and 7-chloro-4-nitrobenzoxadiazole (NBD-Cl) was selected as derivatization reagent. NBD-amino acid enantiomers were then enantioseparated on a Pirkle-type chiral stationary phase, Sumichiral OA-2500S (250mm×4.6mm, 5μm), using a mobile phase composed of acetonitrile–methanol (50:50, v/v) containing 5mmolL−1 citric acid at the flow rate of 0.5mLmin−1. The detection wavelength was 470nm. All the eleven pairs of tested amino acid enantiomers were well separated, and trace amounts of (d)-amino acids (0.5%) in the presence of a large excess of corresponding (l)-enantiomers could be quantified. The proposed method was validated in terms of selectivity, precision, linearity range, LOD, LOQ and accuracy, and then successfully applied to the determination of enantiomeric purity in bulk samples of (l)-amino acids.

Small Molecules as Chromatographic Tools for HPLC Enantiomeric Resolution: Pirkle-Type Chiral Stationary Phases Evolution

This review focuses on the evolution of Pirkle-type chiral stationary phases (CSPs), based on chiral recognition mechanism of small molecules and applications directly related with Medicinal Chemistry. Therefore, the strategies to plan these chiral selectors for enantioseparation of diverse therapeutic classes of chiral drugs and the understanding of the recognition mechanism are emphasized. The planning of Pirkle and co-workers to design different classes of CSPs was initially based on NMR studies, following the principle of reciprocity together with chromatographic results and studies of chiral recognition phenomena. All those features are described and critically discussed in this review. Finally, based on general principles established by Pirkle’s work it can be inferred that diverse chiral small molecules can be successfully used as chromatographic tools for enantiomeric resolution. In this context, several research groups were inspired on Pirkle’s design to develop new CSPs. Xanthone derivatives bonded to chiral groups were also exploited as selectors for CSPs and are briefly reported.

Enantioselective ultra-high and high performance liquid chromatography: A comparative study of columns based on the Whelk-O1 selector

This paper reports on the thermodynamic and kinetic evaluation of a new ultra-high performance liquid chromatography broad-spectrum Pirkle-type chiral stationary phase (CSP) for enantioselective applications (eUHPLC). The well-known Whelk-O1 selector was covalently immobilized onto 1.7-μm high-surface-area, porous spherical silica particles to produce a totally synthetic, covalently bonded CSP that was packed into 150mm, 100mm, 75mm and 50mm columns, either 4.6 or 3.0mm ID. A 100mm×4.6mm ID column was fully characterized from a kinetic and thermodynamic point of view, using as reference a conventional HPLC Whelk-O1 column, 250mm×4.6mm ID, based on 5-μm porous silica particles. On the eUHPLC column, van Deemter plots generated Hmin values of 3.53μm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (μinter) of 5.07mm/s, and Hmin of 4.26 and 4.17μm for the two enantiomers of acenaphthenol, at μinter of 4.85mm/s and 4.24mm/s, respectively. Resolution of 21 enantiomeric pairs including alcohols, epoxides, sulfoxides, phosphine oxides, benzodiazepines and 2-aryloxyproprionic esters used as herbicides, were obtained with significant advantages in terms of efficiency and analysis time. Speed gain factors were calculated for the different column geometries (150mm, 100mm, 75mm and 50mm, either 4.6 or 3.0mm ID), with respect to the standard HPLC column (250mm×4.6mm ID), and were as high as 13, in the case of the 50-mm-long column, affording sub-minute separations of enantiomers with excellent resolution factors. In particular, trans-stilbene oxide was resolved in only 10s, while a 50mm×3.0mm ID column was used as a compromise between reduced mobile phase consumption (less than 1mL per analysis) and smaller extra-column band-broadening effect. Given the relatively low viscosity in NP mode, and the excellent permeability of these eUHPLC columns, with backpressure values under 600bar for a wide range of flow rates, the use of standard HPLC hardware is possible. In this case, however, a significant loss in resolution is observed, compared to the UHPLC instrumentation, if no modifications are introduced in the HPLC apparatus to reduce extra-column variance. The excellent efficiency and selectivity, conjugated with the very high-throughput and the ultra-fast analysis time, prove the potentials of the eUHPLC Whelk-O1 columns in the development of enantioselective UHPLC methods.

Liquid chromatographic resolution of 3‐amino‐1,4‐benzodiazepin‐2‐one derivatives on various Pirkle‐type chiral stationary phases

The two enantiomers of N-acyl amide and N-ureide derivatives of 3-amino-5-phenyl-1,4-benzodiazepin-2-ones, which have been known to show anti-respiratory syncytial virus (RSV) activity, were resolved on seven different Pirkle-type chiral stationary phases (CSPs) with the use of 10% isopropyl alcohol in hexane as a mobile phase. Among the seven Pirkle-type CSPs, the one based on (S)-leucine derivative named as N-Phe-L-Leu was found to be most successful, the separation factors (α) and the resolutions (R(S) ) for seven analytes being in the range of 1.78-4.21 and 5.94-15.08, respectively. By resolving N-benzyloxycarbonyl derivatives of 3-amino-5-phenyl(or 5-methyl)-1,4-benzodiazepin-2-ones on Pirkle-type CSPs, the phenyl ring at the 5-position and the NH hydrogen at the 1-position of analytes were found to play an important role in the chiral recognition.

On the enantioselectivity of the mass transfer kinetics and the adsorption equilibrium of Naproxen on the chiral stationary phase ()-Whelk-O1 under reversed-phase conditions

The adsorption of the Naproxen enantiomers on a Pirkle-type chiral stationary phase (CSP) (R,R)-Whelk-O1 from 0.01M acetic acid solution in a methanol-water (85/15, v/v) mixture was studied using techniques of frontal analysis and elution chromatography. Adsorption was found to follow a model that assumes retention of a solute on two types of surface sites, enantioselective and nonselective. Some minor deviations from the classical bi-Langmuir model were found, but they were well accounted for by assuming the coexistence of two groups of enantioselective sites: sparse sites of strong affinity toward (R)-Naproxen and a more numerous type of low-energy sites exhibiting a somewhat different affinity toward the two enantiomers. Special consideration is paid to the adsorption of the methanol-water mixture on the surface of the CSP. The bonded organic layer exhibits hydrophobic properties, so the adsorbed layer is enriched in methanol throughout the whole range of mobile phase compositions. The study of the mass transfer kinetics was carried out by analyzing the dependence of the HETP on the flow velocity. It revealed the enantioselective character of the intraparticle transport processes. Other aspects of the column dynamics, such as axial dispersion, the external and internal mass transfer were also discussed.

On- and off column enantiomerization of 4,4′-bisquinolin-2-ones: A comparison of Auto-, DHPLcy2k and DCXplorer calculated thermodynamic data generated by dynamic high, performance liquid chromatography with theoretically calculated data

Fast semipreparative HPLC enantioseparation of four axially chiral biscarbostyrils (4,4′-bisquinoline-2-ones) using ULMO as a π-acidic Pirkle type chiral stationary phase leads to two racemizing pairs ( 1, 2; k obs 1.6 × 10 −4 and 3.0 × 10 −4 s −1 at 28 °C) and two stable ones ( 3, 4). 3 was stabilized by a crown ether linkage from pos. 6 to 6′, and 4 had sterically demanding bromo substituents in pos. 3 and 3′. On-column generated temperature-dependent chromatograms of 1 and 2 were fitted with Auto-DHPLCy2k and DCXplorer. For cpd 2 both programs delivered similar Δ G values of 90 and 93 kJ/mol, well comparable with the 99 kJ/mol calculated with the B3LYP/6-31G (d) procedure. At temperatures of high conversion DCXplorer delivered inconsistent series of rate constants for the more tailing and less resolved tetramethoxy derivative 1. We connect this problem with an almost impossible halfwidth calculation of tailing peak pairs which are weakly resolved. However, this problem could be observed only in the case of tetramethoxy derivative 1. Stochastic generated data of Auto-DHPLCy2k could be used at a lower percentage of conversion only while the theoretical plate model did not deliver useful data at temperatures of very low conversion but fitted well high conversion chromatogram series of 1 and 2.

Study of Elution Order in Chiral Separation of Organophosphonate Esters Using Tris-(3,5-dimethylphenyl Carbamate) Cellulose Chiral Stationary Phase by HPLC

It is known that the separation of several organic phosphonate esters using a tris(3,5-dimethylphenyl carbamate) cellulose (Chiralcel OD) column are better than the separation obtained using the Pirkle type chiral stationary phase (CSP), namely the N-(3,5-dinitrobenzoyl) leucine column. However, the elution order of the organic phosphonate esters enantiomers separated on tris(3,5-dimethylphenyl carbamate) cellulose chiral stationary phase is not known. The tandem use of these two CSPs together showed that the R-enantiomer had shorter retention time than S-enantiomer when tris(3,5-dimethylphenyl carbamate) cellulose chiral stationary phase was used.

Extending the use of “Inverted Chirality Columns Approach” for enantiomeric excess determination in absence of reference samples: Application to a water-soluble camptothecin derivative

The aim of the present study was to extend the use of the “Inverted Chirality Columns Approach (ICCA)” previously developed for the identification and quantitation of the trace enantiomer in highly enriched samples of the camptothecin (CPT) family of drugs to a novel water-soluble CPT derivative, namely namitecan (ST1968), currently undergoing phase I clinical trials as anticancer agent. Namitecan, identified from a series of hydrophilic 7-oxyiminomethyl derivatives, contains a free terminal amino group, which traditionally hampers the analysis under normal-phase HPLC conditions. Nevertheless, commercially available Pirkle-type chiral stationary phases (CSPs) available in both the enantiomeric forms (i.e., having the same bound selector with opposite configuration) mainly operate under normal-phase HPLC conditions. For this reason, namitecan was pre-column N-protected with isocyanates A–D and their sulfur analogues E–H to reduce its polarity by converting the amino group into a fragment compatible with the chiral recognition mechanism (i.e., ureido and thioureido groups). Once the optimal columns system and derivatizing agents were selected, an original enantioselective HPLC–MS/MS technique was developed on the Whelk-O1 CSPs.

Resolution of (±)‐β‐methylphenylethylamine by a novel chiral stationary phase for Pirkle‐type column chromatography

In this study, a new Pirkle-type chiral column stationary phase for resolution of beta-methylphenylethyl amine was described by using activated Sepharose 4B as a matrix, L-tyrosine as a spacer arm, and an aromatic amine derivative of L-glutamic acid as a ligand. The binding capacities of the stationary phase were determined at different pH values (pH = 6, 7, and 8) using buffer solutions as mobile phase, and enantiomeric excess (ee) was determined by HPLC equipped with chiral column. The ee was found to be 47%.

Absolute structure determination as a reference for the enantiomeric resolution of racemic mixtures of cyclophosphazenes via chiral high-performance liquid chromatography

Reversed-phase chiral high-performance liquid chromatography (HPLC) is a potentially powerful technique for the enantiomeric resolution of racemic mixtures, although the elution order of enantiomers is only relative and it is necessary to fully characterize reference systems for this method to provide absolute configurational information. The enantiomeric resolution of a series of racemic di-spiro cyclotriphosphazene derivatives, N3P3X2[O(CH2)(3)NH]2 (X = Cl, Ph, SPh, NHPh, OPh) [(1)-(5), respectively] was carried out by reversed-phase chiral HPLC on a commercially available Pirkle-type chiral stationary phase (R,R)-Whelk-01 using 85:15 (v/v) hexane-thf as the mobile phase. The absolute configurations of the resulting enantiomers of compounds (3) (X = SPh) and (5) (X = OPh) were determined unambiguously by X-ray crystallography. For both (3) and (5) it was found that the SS enantiomer eluted before the RR enantiomer, indicating a convenient method to determine the absolute configurations of enantiomers of this series of cyclophosphazene derivatives and providing the first set of enantiomeric reference compounds for cyclophosphazene derivatives. These structures demonstrate an interesting anomaly in that the pair of enantiomers of (3) crystallize in enantiomorphically paired space groups whilst, under the same conditions, the solid-state forms of the enantiomers of (5) form structures in Sohncke space groups that are not enantiomorphous.

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

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

Analysis and purification of alcohol-sensitive chiral compounds using 2,2,2-trifluoroethanol as a modifier in supercritical fluid chromatography

2,2,2-Trifluoroethanol (TFE) is evaluated as an alternative modifier for the analysis and purification of alcohol-sensitive chiral compounds using supercritical fluid chromatography (SFC). Four chiral compounds, selected for their sensitivity to alcohols, in addition to a variety of standard chiral compounds were analyzed by SFC using TFE with polysaccharide and Pirkle-type chiral stationary phases (CSPs) to produce selectivities ( α) and resolutions ( R s) as high as 1.4 and 7.2. A preparative isolation of 2-phenylglutaric anhydride was achieved using TFE as the mobile phase modifier to produce clean enantiomers.

Features of the adsorption of naproxen enantiomers on weak chiral anion-exchangers in nonlinear chromatography

The retention mechanism of the enantiomers of naproxen on a Pirkle-type chiral stationary phase (CSP) was studied. This CSP is made of a porous silica grafted with quinidine carbamate. It can interact with the weak organic electrolyte naproxen either by adsorbing it or by ion-exchange. Using frontal chromatography, we explored the adsorption equilibrium under such experimental conditions that naproxen dissociates or cannot dissociate. Under conditions preventing ionic dissociation, the adsorption isotherms were measured, the adsorption energy distributions determined, and the chromatographic profiles calculated. Three different types of the adsorption sites were found for both enantiomers. The density and the binding energy of these sites depend on the nature of the organic modifier. Different solute species, anions, neutral molecules, solvent-ion associates, and solute dimers can coexist in solution, giving rise to different forms of adsorption. This study showed the unexpected occurrence of secondary steps in the breakthrough profiles of S-naproxen in the adsorption mode at high concentrations. Being enantioselective, this phenomenon was assumed to result from the association of solute molecules involving a chiral selector moiety. A multisite Langmuir adsorption model was used to calculate band profiles. Although this model accounts excellently for the experimental adsorption isotherms, it does not explain all the features of the breakthrough profiles. A comparison between the calculated and experimental profiles allowed useful conclusions concerning the effects of the adsorbate–adsorbate and adsorbate–solvent interactions on the adsorption mechanism.

Surface plasmon resonance spectroscopic chiral discrimination using self-assembled leucine derivative monolayer

Pirkle-type chiral stationary phases (CSPs) showed excellent enantiomeric separation for amino acid derivatives by forming energetically different two transient diastereomeric π–π donor–acceptor complexes with two enantiomers. A CSP derived from N-(3,5-dinitrobenzoyl) leucine with a thiol ending group for immobilization on Au was synthesized and self-assembled on Au surface as chiral sensing layer. The monolayer characterized by spectroscopic and microscopic methods such as AFM, FTIR reflection absorption spectroscopy (FTIR-RAS) and cyclic voltammetry (CV). The enantiospecific detection onto CSP of the leucine derivative was studied by surface plasmon resonance (SPR). ( S)-CSP SAM showed high chiral differential detection for ( S)-analyte in a range of 1.0 × 10 −9 to 1.0 × 10 −4 M. In combination with the SPR method, the leucine derivative monolayer provided a reliable and simple experimental platform for enantiospecific detection.