Preparation and evaluation of a doubly tethered chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A new doubly tethered chiral stationary phase (CSP) was prepared to enhance CSP stability as well as to take advantage of the tertiary amide linkage by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to bis(3-aminopropyl)silica gel. The new CSP was quite effective in the resolution of alpha-amino acids, beta-amino acids, amino alcohols, and amines and the chromatographic resolution behaviors of the new CSP were exactly consistent with those of the corresponding singly tethered CSP containing N-CH3 tertiary amide linkage. Direct comparison between the stabilities of the new CSP and the corresponding singly tethered CSP containing N-CH3 tertiary amide linkage under harsh chromatographic conditions reveals that the new CSP is more stable than the latter.     

Preparation and application of a chiral stationary phase based on (+)-(1 8-crown-6)-2,3,11,12-tetracarboxylic acid without extra free aminopropyl groups on silica surface

A crown ether-based chiral stationary phase (CSP) without extra aminopropyl groups on the surface of silica gel was newly prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel. The new CSP was applied to the resolution of various racemic alpha-amino acids, amines, and amino alcohols. The chiral recognition efficiency of the new CSP was generally superior to that of the original CSP containing unreacted residual aminopropyl groups on the surface of silica gel in terms of the separation (alpha) and the resolution factors (Rs). The retention behaviors of analytes on the new CSP with the variation of the content of organic and acidic modifiers in aqueous mobile phase were consistent with those on the original CSP in the resolution of alpha-amino acids, but somewhat different in the resolution of racemic amines and amino alcohols from those on the original CSP and the difference was rationalized by the lipophilicity difference of the two CSPs. The effect of the column temperature on the chromatographic resolution behaviors on the new CSP was consistent with that on the original CSP.     

High performance liquid chromatographic separation of dipeptide and tripeptide enantiomers using a chiral crown ether stationary phase

A chiral stationary phase (CSP) based on (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was evaluated for the direct resolution of the enantiomers of dipeptides and tripeptides. The type and concentration of the acid and the methanol content were optimized with regard to retention time and resolution using Ala-Phe as model peptide. A mobile phase consisting of 10 mM sulfuric acid in 70% aqueous methanol was applied to the separation of a set of 16 structurally diverse dipeptides and tripeptides. Generally, the configuration of the amino acid at the N-terminus determined the enantiomer elution order. With a few exceptions the LL- and LD-enantiomers interacted stronger with the CSP compared to the corresponding DD- or DL-enantiomers. The experimental conditions also allowed the simultaneous separation of all four stereoisomers of Ala-Phe. Addition of ammonium sulfate generally reduced retention times and enantiomer resolution. Addition of triethylamine as modifier led to an overall increase of the retention times while the resolution did not show a general trend, increasing in the case of Ala-Ala but decreasing in the case of Ala-Phe.     

Comparison of enantiomer separation on two chiral stationary phases derived from (+)-18-crown-6-2,3,11,12-tetracarboxylic acid of the same chiral selector

Liquid chromatographic comparisons for enantiomer resolution of α-amino acids and chiral primary amino compounds were made using chiral stationary phases (CSPs) prepared by covalently bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) of the same chiral selector. The resolution of all α-amino acids on CSP 1 developed in our group was found to be better than that on CSP 2 reported by Machida et al. All α-amino acids examined in this study were well enantioseparated on CSP 1 (α=1.22–2.47), while four analytes were not resolved or all the other analytes were poorly resolved on CSP 2 than on CSP 1. However, in resolving the primary amino compounds without a carbonyl group, CSP 1 was comparable with CSP 2. Although (+)-18-C-6-TA of the same chiral selector was used to prepare CSP 1 and CSP 2, this study showed that different connecting methods for the CSPs might influence their ability to resolve the analytes depending on their structures related to the chiral recognition mechanism.     

Liquid chromatographic resolution of 1-aryl-1,2,3,4-tetrahydroisoquinolines on a chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A liquid chromatographic chiral stationary phase (CSP) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied for the first time to the resolution of biologically important 1-aryl-1,2,3,4-tetrahydroisoquinolines. The unusual resolution of cyclic secondary amino compounds on a chiral crown ether-based CSP was quite successful with the use of a mixture of methanol-acetonitrile-triethylamine at a ratio of 30/70/0.5 (v/v/v) as a mobile phase. From the chromatographic behaviours for the resolution of seven 1-aryl-1,2,3,4-tetrahydroisoquinolines, the steric bulkiness of the 1-phenyl ring at the chiral center of analytes was concluded to play an important role in the chiral recognition.     

Preparation of a new crown ether-based chiral stationary phase containing thioester linkage for the liquid chromatographic separation of enantiomers

A new chiral stationary phase (CSP) containing thioester linkages was prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to mercaptopropylsilica gel. The chiral recognition ability of the new CSP was found to be greater than that of the previously reported CSP containing amide linkages in the resolution of the various α-amino acids that were tested, except for that of Met, Ser and Thr. In the resolution of racemic amines and amino alcohols, the new CSP was always better than the one containing amide linkages in terms of the separation factors (α) and the resolutions (R_S). Given the identical elution orders on the two CSPs, it was concluded that the chiral recognition mechanism is not affected by the change of the linkage type. In addition, the new CSP was found to be quite stable under the acidic mobile phase conditions that were utilized, indicating that the thioester linkage is useful as a tethering group.     

Liquid chromatographic resolution of beta-amino acids on CSPs based on optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6

Two chiral stationary phases (CSPs) based on optically active (3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 covalently bonded to silica gel were utilized for the first time for the resolution of racemic β-amino acids using high performance liquid chromatography. All of the 10 β-amino acids tested were resolved on the CSP containing residual silanol-protecting n-octyl groups, while only five β-amino acids were resolved on the CSP containing residual silanol groups. The superiority of the CSP containing residual silanol-protecting n-octyl groups and the characteristic retention behaviors of the two enantiomers on the CSP were rationalized to stem from the removal of the residual silanol groups, which can otherwise induce the non-enantioselective retention of the analytes, and the improved lipophilicity of the CSP. The elution orders of the two enantiomers of β-amino acids were identical on the two CSPs and, consequently, it was concluded that the two CSPs were concluded to utilize identical chiral recognition mechanisms. The different elution orders of the analytes were proposed to be attributed to the presence or absence of π-π interactions between the CSP and analytes.     

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.     

Liquid chromatographic separation of the enantiomers of beta-amino acids on a ligand exchange chiral stationary phase

A liquid chromatographic ligand exchange chiral stationary phase (CSP) derived from (S)-leucinol was applied in the separation of the enantiomers of 12 beta-amino acids. The resolution was quite successful especially for the enantiomers of beta-amino acids containing aromatic functional group in the side chain. The chromatographic resolution behaviors were dependent on the organic modifier and Cu(II) concentration in aqueous mobile phase and the column temperature.     

HPLC enantioseparation of β2‐homoamino acids using crown ether‐based chiral stationary phase

RP high‐performance liquid chromatographic methods were developed for the enantioseparation of eleven unusual β²‐homoamino acids. The underivatized analytes were separated on a chiral stationary phase containing (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as chiral selector. The effects of organic (alcoholic) and acidic modifiers, the mobile phase composition and temperature on the separation were investigated. The structures of the substituents in the α‐position of the analytes substantially influenced the retention and resolution. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers.     

Liquid chromatographic resolution of racemic rasagiline and its analogues on a chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

A liquid chromatographic chiral stationary phase based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was applied to the resolution of 15 analytes, including racemic rasagiline, a chiral drug for the treatment of Parkinson's disease, and its analogues. The composition of mobile phase was optimized to be ethanol/acetonitrile/acetic acid/triethylamine (80:20:0.2:0.3, v/v/v/v) by evaluating the chromatographic results for the resolution of five selected analytes under various mobile phase conditions. Under the optimized mobile phase conditions, racemic rasagiline was resolved quite well with a separation factor of 1.48 and resolution of 2.71 and its 14 analogues were also resolved reasonably well with separation factors of 1.06–1.54 and resolutions of 0.54–2.11. Among 15 analytes, racemic rasagiline was resolved best except for just one analyte. The analyte structure–enantioselectivity relationship indicated that racemic rasagiline has the most appropriate structural characteristics for resolution on the chiral stationary phase.     

High-performance liquid chromatographic enantioseparation of beta-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of 14 unnatural beta-amino acids, including several beta-3-homo-amino acids on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid bonded to 3-aminopropyl silica gel as chiral selector. The effects of the organic and the acidic modifiers and the mobile phase composition on the separation were investigated. The natures and positions of the substituents on the aromatic ring substantially influenced the retention and enantioseparation. The elution sequence in most cases was determined and the R enantiomers were eluteted before the S enantiomers.     

The effect of analyte lipophilicity on the resolution of α-amino acids on a HPLC chiral stationary phase based on crown ether

The effect of analyte lipophilicity on the resolution of α-amino acids on a chiral stationary phase based on chiral crown ether has been examined by the chromatographic resolution trends for the resolution of a homologous series of five α-amino acids with an alkyl group of different length at the chiral center. The retention factors (k₁ and k₂) for the two enantiomers and the separation factors (α) were found to depend on the lipophilicity of the α-amino acid. In general, the retention factors increased as the organic modifier content in the mobile phase increased, the degree of the enhancement of retention factors being dependent on the analyte lipophilicity. The separation factors also increased as the analyte lipophilicity and the organic modifier content in the mobile phase increased. Possible rationales for these behaviors have been proposed.     

Separation of the enantiomers of pyrethroic acids and their esters by high-performance liquid chromatography on a polysaccharide-derived chiral stationary phase

Summary The liquid-chromatographic separation of the enantiomers of pyrethroic acids and their esters has been investigated on a polysaccharide-derived chiral stationary phase (CSP), Chiralpak AS. Good separation of the enantiomers of underivatized pyrethroic acids was achieved on the column, and the enantiomers of pyrethroic acid methyl and ethyl ester derivatives were also resolved.     

Preparation of a new doubly tethered chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and its application

A new doubly tethered chiral stationary phase (CSP) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was prepared by attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the corresponding singly tethered CSP, which was previously developed in our laboratory. The new doubly tethered CSP was applied successfully to the resolution of various racemic alpha-amino acids, amines and amino alcohols containing a primary amino group. In most cases, the chiral recognition efficiency of the new doubly tethered CSP was superior to that of the corresponding singly tethered one in the resolution of alpha-amino acids, amines and amino alcohols. In the resolution of some racemic primary amino compounds, the new doubly tethered and the corresponding singly tethered CSPs were complementary with each other. The chiral resolution behaviors on the new doubly tethered CSP were examined with the variation of the type and content of organic and acidic modifiers in aqueous mobile phase and the column temperature. The chiral resolution behaviors on the new doubly tethered CSP were generally quite similar to those on the corresponding singly tethered CSP. The stability of the new doubly tethered CSP was greater than that of the corresponding singly tethered CSP.     

Effect of the residual silanol group protection on the liquid chromatographic resolution of α‐amino acids and proton pump inhibitors on a ligand exchange chiral stationary phase

A new ligand exchange chiral stationary phase (new CSP) containing residual silanol group‐protecting n‐octyl groups on the silica surface was prepared by treating a ligand exchange CSP (original CSP) based on sodium N‐[(R)‐2‐hydroxy‐1‐phenylethyl]‐N‐undecylaminoacetate bonded to silica gel with excess n‐octyltriethoxysilane. The new and original CSPs containing an identical amount of chiral selector were applied to the resolution of α‐amino acids and proton pump inhibitors (PPIs) including omeprazole, pantoprazole, lansoprazole, and rabeprazole. The separation factors (α) and resolutions (R_S) were greater on the new CSP than on the original CSP except for the resolution of asparagine. The trends of the retention factors (k₁) for the resolution of α‐amino acids on the new and original CSPs with the variation of the organic modifier content in aqueous mobile phase were opposite to those for the resolution of PPIs. Removal of the nonenantioselective interactions between the residual silanol groups and the analytes and the improved lipophilicity of the new CSP were proposed to be responsible for the improved chiral recognition ability of the new CSP and the different retention behaviors of the enantiomers between the new and original CSPs.     

Preparation and application of a new modified liquid chromatographic chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

As an effort to improve the chiral recognition efficiency of a previously reported chiral stationary phase (CSP) based on (+)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid, a new CSP was prepared by simply replacing the amide N-H hydrogens of the tethering groups of the old CSP with methyl groups. The new CSP was superior to the old one in the resolution of racemic primary amines. However, in the resolution of alpha-amino acids and amino alcohols, the new and the old CSPs were complementary with each other. The elution orders on the new CSP were sometimes opposite to those on the old one. Consequently, the chiral recognition mechanism on the new CSP was presumed to be different from that on the old one. The chiral recognition behavior of the new CSP were investigated with four selected analytes and found to be dependent to some extent on the content of organic and acidic modifiers in aqueous mobile phase and the column temperature.     

High-performance liquid chromatographic enantioseparation of beta-3-homo-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase

High-performance liquid chromatographic methods were developed for the separation of the enantiomers of thirteen unusual beta-3-homo-amino acids and three of its ethyl esters on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as chiral selector. The effects of the mobile phase composition and the acidic modifiers on the separation were investigated. The structures of the substituents in beta-position substantially influenced the retention and enantioseparation. The influence of ionic strength on the enantioseparation was established experimentally. The elution sequence was determined in all cases.     

Effect of analyte lipophilicity on the resolution of α‐ and β‐amino acids on liquid chromatographic ligand exchange chiral stationary phases

Separation of the two enantiomers of racemic α‐ and β‐amino acids on two ligand exchange chiral stationary phases (CSPs) prepared previously by covalently bonding sodium N‐((S)‐1‐hydroxymethy‐3‐methylbutyl)‐N‐undecylaminoacetate or sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate on silica gel was studied with variation of the organic modifier (methanol) concentration in the aqueous mobile phase. In particular, the variation of retention factors with changing organic modifier concentration in the aqueous mobile phase was found to be strongly dependent on both the analyte lipophilicity and the stationary phase lipophilicity. In general, the retention factors of relatively lipophilic analytes on relatively lipophilic CSPs tend to increase with increasing organic modifier concentration in the aqueous mobile phases while those of less lipophilic or hydrophilic analytes tend to increase. However, only highly lipophilic analytes show decreasing retention factors with increasing organic modifier concentration in the aqueous mobile phase on less lipophilic CSPs. The contrasting retention behaviors on the two CSPs were rationalized by the balance of the two competing interactions, viz. hydrophilic interaction of analytes with polar aqueous mobile phase and the lipophilic interaction of analytes with the stationary phase.