Chiral Stationary Phase HPLC Research Articles

Supramolecular Systems-Based HPLC for Chiral Separation of Beta-Adrenergics and Beta-Adrenolytics in Drug Discovery Schemes

Increasing amount of data considering polymorphism, splice variants and various affinity states of beta-adrenoceptors has resulted in a new range of opportunities for enantiopure beta-adrenergic and beta-adrenolytic drug discovery and continuous development of reliable high-throughput screening procedures enabling tissue specific pharmacological evaluation of these drugs. Design and fast pharmacological profiling of single enantiomeric molecules combining beta-adrenoceptor affinity with other pharmacophores is also still challenging ability. As the use of chiral stationary phases in HPLC has particularly benefited from results of supramolecular chemistry, this review summarises recent achievements provided by this technique in deciphering of enatiorecognition phenomena affecting pharmacological selectivity of beta-adrenergics and beta-adrenolytics and modifying the efficiency of currently proposed beta-adrenoceptor-targeted therapies. Detailed characteristic of chiral separation performance of these drugs in the range of available supramolecular HPLC systems has also been presented.

Liquid Chromatographic Resolution of N-(3,5-Dinitrobenzoyl)-α-amino Acids on a New Chiral Stationary Phase: the First Liquid Chromatographic Utilization of a Double-Ureide Pocket for the Recognition of Chiral Carboxylate Anions

An HPLC chiral stationary phase (CSP) which has only two ureide functional groups was prepared starting from (1S,2S)-1,2-diaminocyclohexane. The CSP was successful in the resolution of various N-(3,5- dinitrobenzoyl)-α-amino acids, the separation (α) and the resolution factors (RS) being within the range of 1.11-1.35 and 2.19-5.17, respectively with the use of 20% 2-propanol in hexane containing 0.1% trifluoroacetic acid as a mobile phase. However, ethyl esters of N-(3,5-dinitrobenzoyl)-α-amino acids were not resolved or resolved with only marginal separation and resolution factors on the CSP under the identical mobile phase condition. From these results, the complexation of the carboxylate anions of analytes inside the double-ureide pocket of the CSP was expected to play some important role for the chiral recognition. In contrast, N-(3,5- dinitrobenzoyl)-α-amino acid N-propylamides were resolved on the CSP with reasonable separation and resolution factors. Enantioselective hydrogen bonding interactions between analytes and the CSP were presumed to be responsible for these resolutions.

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

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

Enzyme‐Catalysed Synthesis and Absolute Configuration Assignments of cis‐Dihydrodiol Metabolites from 1,4‐Disubstituted Benzenes

A series of ten cis-dihydrodiol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and 1H NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.

Chemical immobilization of azido cellulose phenylcarbamate onto silica gel via Staudinger reaction and its application as a chiral stationary phase for HPLC

Azido cellulose phenylcarbamate (AzCPC) was synthesized regioselectively and chemically immobilized onto aminized silica gel to afford urea-bonded chiral stationary phases (CSPs). The obtained CSPs showed good enantioselectivity in HPLC towards various racemates using normal phase eluants (hexane/2-propanol, hexane/chloroform or hexane/2-propanol/chloroform).

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

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

Preparation and evaluation of a new synthetic polymeric chiral stationary phase for HPLC based on the trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide monomer

A new synthetic polymeric chiral stationary phase for liquid chromatography was prepared via free-radical-initiated polymerization of trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide. The new polymeric chiral stationary phase (CSP) showed enantioselectivity for many chiral compounds in multiple mobile phases. High stability and sample capacities were observed on this polymeric chiral stationary phase. Mobile phase components and additives affected chiral separation greatly. This new synthetic chiral stationary phase is complementary to two other related commercially available CSPs: the P-CAP and P-CAP-DP columns. Interactions between the chiral stationary phase and analytes that lead to retention and chiral recognition include hydrogen bonding, dipolar, and pi-pi interactions. Repulsive (steric) interactions also contribute to chiral recognition. Figure LC chromatograms showing the analytical (blue) and preparative (red) separations of N-(3,5-dinitrobenzoylleucine) enantiomers on a new synthetic polymeric chiral stationary phase.

A scalable and expedient method of preparing diastereomerically and enantiomerically enriched pseudonorephedrine from norephedrine

Norephedrine has been efficiently converted into the corresponding diastereomer pseudonorephedrine using a three step, one-pot reaction. The three step process involves treatment of norephedrine with di- tert-butyl dicarbonate (Boc 2O); cyclization by way of mesylate formation at the alcohol; and lithium hydroxide mediated hydrolysis of the oxazolidinone. The diastereomeric purity was determined by HPLC and the enantiomeric purity was determined by optical activity measurements and chiral stationary phase HPLC analysis of the pseudonorephedrine oxazolidinone derivatives.

Enantiomer separation on monolithic silica HPLC columns using chemically bonded methylated and methylated/acetylated 6-O-tert-butyldimethyl- silylated β-cyclodextrin

A new 2,3-methylated 3*-monoacetylated 6-O-tert-butyldimethylsilylated beta-CD derivative was synthesized and chemically bonded onto aminopropyl derivatized monolithic silica HPLC columns. In this CD derivative, only one of seven methyl groups in 3-position was substituted by an acetyl group. Its applicability as a chiral stationary phase for HPLC was tested and compared with exclusively 2,3-methylated 6-O-tert-butyldimethylsilylated beta-CD immobilized onto aminopropyl-modified monoliths. Thirty-two chiral compounds from different chemical classes and different functionalities were tested under RP conditions. Fourteen compounds were resolved into their enantiomers by methylated 6-O-tert-butyldimethylsilylated beta-CD. By use of methylated/acetylated 6-O-tert-butyldimethylsilylated beta-CD as the chiral stationary phase 7 analytes were successfully stereodifferentiated.

Enantioseparation by Sonochromatography

Although chiral separation has been one of the main topics of chromatographic practice for over twenty-five years, it still presents many difficulties. In this work, the ultrasonic dependence of chiral resolution was investigated at various temperatures to improve resolution and reduce analysis time. The chiral resolution Was performed on recently commercialized two HPLC chiral stationary phases (CSP 1 and CSP 2) with the analogues of racemic N-acylnaphthylethylamines (la-d) and racemic amino acid derivatives (2a-c, 3a-c) as analytes. The CSP 1 was prepared from a (R)-N-(3,5-dinitrobenzoyl)phenylglycinol and the CSP 2 was prepared from a (S)-N-3,5-(dinitrobenzoyl) leucine. From the comparison of the chromatographic results under sonic condition with those under non-sonic condition, we found that the ultrasound decreased the elution time in chiral chromatography at all temperatures and improved the enantioselectivity at high temperature (45, 50, 60 °C).

Enantioseparation using alkoxyphenylcarbamates of cellulose and amylose as chiral stationary phase for high‐performance liquid chromatography

Cellulose and amylose phenylcarbamates having one or two alkoxy groups on a phenyl ring were synthesized, and their chiral recognition abilities as chiral stationary phases for HPLC were evaluated. Compared to the 4-methoxyphenylcarbamates of cellulose and amylose, which are known to show a poor chiral recognition, the 3-methoxyphenylcarbamates exhibited much higher chiral recognitions. For cellulose derivatives, as the bulkiness of the 3-alkoxy group increased, the chiral recognition ability increased. On the other hand, for the amylose derivatives, a clear relation between the chiral recognition and the bulkiness of the alkoxy group was not observed, and the 3-methoxy, ethoxy, and isopropoxyphenylcarbamates showed relatively high chiral recognitions. The introduction of two methoxy groups to the meta-positions decreased the chiral recognition ability. In order to discuss the relationship between the structure and chiral recognition ability of the alkoxyphenylcarbamates, their molecular models were constructed.

Preparation and application of HPLC chiral stationary phases based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

Preparation of liquid chromatographic chiral stationary phases (CSPs) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and their application are reviewed. The various methods of connecting (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel covalently or dynamically are demonstrated. The CSPs based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid have been very successful for the resolution of various primary amino compounds with the use of an aqueous mobile phase containing organic and acidic modifiers. In addition, the resolution of secondary amino compounds including beta-blockers and N-(3,5-dinitrobenzoyl)-alpha-amino acids has been demonstrated on a CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid with a non-aqueous mobile phase.

Performance of brush‐type HPLC chiral stationary phases with tertiary amide in the connecting tether

The replacement of the N-H hydrogen of the secondary amide-tethered Pirkle-concept N-(3,5-dinitrobenzoyl)-L-leucine derived chiral stationary phase with various pi-basic or aliphatic groups improved the chiral discrimination ability of new chiral stationary phases, based on the leucine- or alanine-derived chiral selector, for the enantiomers of various racemic neutral analytes with amide functional groups. Retention times decreased while separation and resolution factors increased, thus proving the role of pi-donor aromatic unit as an electron-rich shield in the front of a silica surface. In general, chiral stationary phase (CSP) 5 with the 3,5-dimethylphenyl unit showed best performance, while CSP 3, with phenyl unit, and CSP 7, with 1-naphthyl unit in the tertiary amide connecting tether, were less efficient.

Unusual resolution of N-(3,5-dinitrobenzoyl)-α-amino acids on a chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

While HPLC chiral stationary phases (CSPs) based on chiral crown ethers have been known useful for the resolution of only racemic primary amino compounds or some secondary amino compounds, in this study, we first demonstrated that the CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid is also useful for the resolution of N-benzoyl-α-amino acids, which do not contain a primary or secondary amino group. Especially, N-(3,5-dinitrobenzoyl)-α-amino acids were resolved better than corresponding N-(3-nitrobenzoyl)- or N-benzoyl-α-amino acids, the separation ( α) and the resolution factors ( R S) for the resolution of eight N-(3,5-dinitrobenzoyl)-α-amino acids being in the range of 1.06–1.81 and 0.54–2.81, respectively. The optimum mobile phase condition was the mixture of acetic acid–triethylamine–acetonitrile with the ratio of 0.05/0.25/100 (v/v/v).

Comparison, Applications, Advantages, and Limitations of Immobilized and Coated Amylose Tris‐(3,5‐Dimethylphenylcarbamate) Chiral Stationary Phases in HPLC

A direct high performance liquid chromatographic enantioselective separation of a set of racemic acidic drugs on the new immobilized and conventional coated amylose tris‐(3,5‐dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) was studied using n‐hexane and 2‐propanaol (80∶20 v/v), containing TFA (0.1%) as mobile phase. The separation and elution order of the enantiomers on both columns under the same conditions were compared. The effect of the immobilization of the amylose tris‐(3,5‐dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA) on the chiral recognition ability was noted, as the coated phase (Chiralpak AD) possesses a higher resolving power than the immobilized one (Chiralpak IA). A few racemates, which were not or poorly resolved on the immobilized Chiralpak IA were most efficiently resolved on the coated Chiralpak AD. However, the immobilized phase withstands the prohibited HPLC solvents such as dichloromethane, ethyl acetate, tetrahydrofuran, and others when used as eluents or as a dissolving agent for the analyte itself. The versatility of the immobilized Chiralpak IA in monitoring reactions performed in dichloromethane using direct analysis techniques without further purification, workup, or removal of dichloromethane, was studied on a representative example consisting of the lipase‐catalyzed enantioselective esterification of flurbiprofen, with n‐butanol in dichloromethane as organic solvent.

Comparison, Applications, Advantages, and Limitations of Immobilized and Coated Amylose Tris‐(3,5‐Dimethylphenylcarbamate) Chiral Stationary Phases in HPLC

A direct high performance liquid chromatographic enantioselective separation of a set of racemic acidic drugs on the new immobilized and conventionally coated amylose tris‐(3,5‐dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) was studied using n‐hexane and 2‐propanaol (80∶20 v/v) containing TFA (0.1%) as mobile phase. The separation and elution order of the enantiomers on both columns under the same conditions were compared. The effect of the immobilization of the amylose tris‐(3,5‐dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA), on the chiral recognition ability was noted, as the coated phase (Chiralpak AD) possesses a higher resolving power than the immobilized one (Chiralpak IA). A few racemates, which were not resolved or were poorly resolved on the immobilized Chiralpak IA were most efficiently resolved on the coated Chiralpak AD. However, the immobilized phase withstands the prohibited HPLC solvents such as dichloromethane, ethyl acetate, tetrahydrofuran, and others, when used as eluents or as a dissolving agent for the analyte itself. The versatility of the immobilized Chiralpak IA in monitoring reactions performed in dichloromethane using direct analysis techniques without further purification, workup, or removal of dichloromethane, was studied on a representative example consisting of the lipase‐catalyzed enantioselective esterification of flurbiprofen with n‐butanol in dichloromethane as organic solvent.

Effect of chiral additive on formation of cellulose triacetate chiral stationary phase in HPLC

Cellulose triacetate, an chiral polymer that has been widely used in chiral recognition in high performance liquid chromatography, and also used in membrane science such as reverse osmosis, microfiltrtion and ultrafiltration, was coated on silica gel as stationary phase for HPLC and the pores of membrane were created by chiral additive (+)-di-O,O′- p-toluyl- d-tartarric acid, (+)-1-phenyl-1,2-ethanediol and (+)- l-mandelic acid, respectively. The results show that those stationary phases not only can separate extensively racemates, but also can resolve the chiral additive. This is a report, for the first time, that the chiral additive was used in formation of chiral stationary phase of optically active polymer. This work indicates that some optically active polymers, which have been used for chiral resolution, could soon become very attractive as new chiral stationary phase having a predictable chiral selectivity advantage.

Development and Application of Crown Ether-based HPLC Chiral Stationary Phases

Crown ether-based HPLC chiral stationary phases (CSPs) have been successfully utilized in the resolution of various racemic compounds containing a primary amino group. Especially, CSPs based on chiral crown ethers incorporating chiral binaphthyl unit or tartaric acid unit and based on phenolic pseudo chiral crown ethers have shown high chiral recognition efficiency. In this account paper, a review on the development of crown etherbased HPLC CSPs, their structural characteristics and applications to the resolution of racemic compounds including chiral drugs containing a primary or secondary amino group with the variation of the type and the content of mobile phase components and with the variation of the column temperature is presented.

Liquid Chromatographic Resolution of Racemic 1,4-Benzodiazepin-2-ones on Pirkle-type Chiral Stationary Phases Based on (S)-Leucine

It has been well known that two enantiomers of chiral drugs often show different pharmacological activity. In this instance, exact determination of the enantiomeric composition of chiral drugs is essential for the development of chiral drugs and for the better insight into the in vivo distribution and metabolism of the two enantiomers. Among various methods, liquid chromatographic separation of enantiomers on HPLC chiral stationary phases (CSPs) has been demonstrated as very attractive means for the analysis of enantiomeric composition of chiral compounds including chiral drugs. A series of 3-substituted 1,4-benzodiazepin-2-ones such as camazepam, lorazepam, lormetazepam and oxazepam belongs to a class of widely used anxiolytics and/or tranquilizer. The pharmacological activity of a series of 3substituted 1,4-benzodiazepin-2-ones has been shown to be enantiodependent. Consequently some efforts have been devoted to the development of the liquid chromatographic separation of the two enantiomers of 3-substituted 1,4benzodiazepin-2-ones. Among others, Pirkle-type CSPs have been found to be quite useful in the resolution of racemic 3-substituted 1,4-benzodiazepin-2-ones. Especially, CSP 1 (Figure 1) derived from (S)-N-(3,5-dinitrobenzoyl)leucine has been most successfully utilized in the resolution 3-substituted 1,4-benzodiazepin-2-ones. As an improved CSP, we recently developed CSP 2 (Figure 1) by simply replacing the superfluous adsorption site, the N-H hydrogen of the connecting tether of CSP 1, with a phenyl group. CSP 2 has shown greater enantioselectivities than CSP 1 for the enantiomers of π-acidic or π-basic derivatives of a-amino acids. However, CSP 2 has not been applied in the resolution of racemic 3-substituted 1,4-benzodiazepin-2ones. In this study, as an effort to extend the use of CSP 2, we wish to elucidate that CSP 2 is also useful in the resolution of 3-substituted 1,4-benzodiazepin-2-ones and wish to compare the chiral recognition efficiency of CSP 2 with that of CSP 1. 3-Substituted 1,4-benzodiazepin-2-ones (3 and 4) used in this study (Figure 1) were prepared via the reported procedure. For the purpose of comparison, 3-substituted 1,4-benzodiazepin-2-ones (3 and 4) prepared in our laboratory were resolved on CSP 1 and CSP 2 under an identical chromatographic condition and the resolution results were summarized in Table 1. The representative chromatograms on CSP 2 are shown in Figure 2. As shown in Table 1 and Figure 2, the resolutions of 3substituted 1,4-benzodiazepin-2-ones (3 and 4) are excellent on both CSP 1 and CSP 2. Interestingly, the retention factors (k) on CSP 2 are always smaller than those on CSP 1. Even when the content of isopropyl alcohol in mobile phase was varied within the range of 3% to 20%, the retention factors (k) on CSP 2 were still always smaller than those on CSP 1. This means that CSP 2 is better than CSP 1 in terms of reducing analytical time and saving solvent. The smaller retention factors (k) on CSP 2 might be rationalized by considering the chiral recognition mechanism. A chiral recognition mechanism for the resolution of 3substituted 1,4-benzodiazepin-2-ones on CSP 1 was

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 μm 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.