Separation and control of the elution order of N-t-butyloxycarbonyl amino acids D/L isomers by reversed-phase HPLC using cyclodextrins as chiral selectors for the mobile phase.

The enantiomeric resolution of N-t-butyloxycarbonyl (N-t-Boc) amino acids D/L isomers by reversed-phase HPLC was investigated using cyclodextrins (CD's) as chiral selectors for the mobile phase. The use of a low pH (pH<4) for the mobile phase enabled the enantioseparation of N-t-Boc amino acids. The opposite elution order of D/L isomers was observed when hydroxypropyl-derivatized beta-CD was used instead of native beta-CD. A computer simulation of the enantioseparation showed that the ratio of the retention factors of the chiral selector and the sample determined the elution order and the resolution. When the retention factor of the chiral selector is smaller than that of the sample, an isomer having larger complex formation constant eluted faster. However, when the chiral selector had a larger retention factor than the sample, an opposite elution order of the isomers was obtained. The large difference in the retention factors between the chiral selector and the sample led to good enantiomeric separation.     

High‐performance liquid chromatographic separations of stereoisomers of chiral basic agrochemicals with polysaccharide‐based chiral columns and polar organic mobile phases

The separation of the stereoisomers of 23 chiral basic agrochemicals was studied on six different polysaccharide‐based chiral columns in high‐performance liquid chromatography with various polar organic mobile phases. Along with the successful separation of analyte stereoisomers, emphasis was placed on the effect of the chiral selector and mobile phase composition on the elution order of stereoisomers. The interesting phenomenon of reversal of enantiomer/stereoisomer elution order function of the polysaccharide backbone (cellulose or amylose), type of derivative (carbamate or benzoate), nature, and position of the substituent(s) in the phenylcarbamate moiety (methyl or chloro) and the nature of the mobile phase was observed. For several of the analytes containing two chiral centers all four stereoisomers were resolved with at least one chiral selector/mobile phase combination.     

Microcolumn LC enantioseparation of chiral compounds using diol silica gel functionalized with vancomycin crystalline degradation products

Vancomycin crystalline degradation products (CDPs) have been introduced as one of the newest and most interesting derivatives of vancomycin for enantiomer separation of a wide variety of chiral compounds. In this attempt, a chiral stationary phase (CSP) has been prepared using diol silica gel based on vancomycin CDPs which led to a new chiral selector with new functionality of functional groups on a microcolumn LC. Different kinds of mobile phases were examined to realize the behavior of the chiral selector in separation of atropine, fluoxetine, amlodipine, mandelic acid, alanine, and phenylalanine which were separated successfully on this column. Good results were obtained by using a polar mobile phase containing water, methanol, and acid additives for separation of chiral acidic compounds and amino acid samples. Considerable results were obtained for analysis of basic compounds by using polar organic mobile phase (POP) containing methanol, acid and base additives. These results can be associated with the presence of the carboxylic acid groups present in new CSP by using a diol silica gel.     

Enantioseparation of selected chiral sulfoxides in high‐performance liquid chromatography with polysaccharide‐based chiral selectors in polar organic mobile phases with emphasis on enantiomer elution order

The separation of the enantiomers of 17 chiral sulfoxides was studied on polysaccharide‐based chiral columns in polar organic mobile phases. Enantiomer elution order (EEO) was the primary objective in this study. Two of the six chiral columns, especially those based on amylose tris(3,5‐dimethylphenylcarbamate) and cellulose tris(4‐chloro‐3‐methylphenylcarbamate) (Lux Cellulose‐4) proved to be most successful in the separation of the enantiomers of the studied sulfoxides. Interesting examples of EEO reversal were observed depending on the chiral selector or the composition of the mobile phase. For instance, the R‐(+) enantiomer of lansoprazole eluted before the S‐(?) enantiomer on Lux Cellulose‐1 in both methanol or ethanol as the mobile phase, while the elution order was opposite in the same eluents on amylose tris(3,5‐dimethylphenylcarbamate) with the S‐(?) enantiomer eluting before the R‐(+) enantiomer. The R‐(+) enantiomer of omeprazole eluted first on Lux Amylose‐2 in methanol but it was second when acetonitrile was used as the mobile phase with the same chiral selector. Several other examples of reversal in EEO were observed in this study. An interesting example of the separation of four stereoisomers of phenaminophos sulfoxide containing chiral sulfur and phosphor atoms is also reported here.     

Effects of Temperature and Mobile Phase Condition on Chiral Recognition of Poly(l-phenylalanine) Chiral Stationary Phase

Characteristics of the chiral stationary phase with poly(l-phenylalanine) peptide selector, which was in ??-helical state, was reported. Since environmental factors affect peptide conformation, the changes in enantioselectivity were examined depending on column temperature and mobile phase conditions (ionic strength, pH, mobile phase composition). Column temperature and pH drastically affected the enantioselectivity. Based on these changes, the relation between chiral recognition and secondary structure of the peptide selector was discussed. The column stability during sequential analysis under different separation conditions was also evaluated.     

Recent progress in enantiomer separation by capillary electrochromatography

Enantiomer separation by electrochromatography (CEC) can be performed in three modes: (i) open-tubular capillary electrochromatography (o-CEC), in which the chiral selector is physically adsorbed coated, and thermally immobilized or covalently attached to the internal capillary wall; (ii) packed capillary electrochromatography (p-CEC), in which the capillary is either filled with chiral modified silica particles or with an achiral packing material, and a chiral selector is added to the mobile phase; and (iii) monolithic (rod)-capillary electrochromatography (rod-CEC) in which the chiral stationary phase (CSP) consists of a single piece of porous solid. We present an overview on methods and new trends in the field of electrochromatographic enantiomer separation such as CEC with either nonaqueous mobile phases or stationary phases with incorporated permanent charges, or with packing beds consisting of nonporous silica particles or particles with very small internal diameters.     

Enantioseparation of selected N-tert.-butyloxycarbonyl amino acids in high-performance liquid chromatography and capillary electrophoresis with a teicoplanin chiral selector

Enantioseparation of N-tert.-butyloxycarbonyl amino acids (N-t-Boc-Aas) with teicoplanin chiral selector was performed in two different separation systems: A teicoplanin-based chiral stationary phase (CSP-TE) was used in reversed-phase HPLC, and the same chiral selector (CS) was added into a background electrolyte (BGE) in HPCE. The enantioselective interaction with the same CSP/CS can be influenced by several factors, such as mobile phase/background electrolyte composition: the buffer concentration, pH, the CS concentration, the presence of organic modifiers. In addition, the charge of the chiral selector related to the charge of the analyte and to EOF are important variables in CE. The effect of these parameters on enantioselectivity and enantioseparation of selected N-t-Boc-Aas was studied. The presence of a sufficient concentration (1% solution) of a triethylamine acetate buffer in the mobile phase was shown to be essential for enantioseparation of these blocked amino acids in HPLC. A certain concentration of teicoplanin aggregates (along with teicoplanin molecules) in the BGE is required to obtain enantioseparation of N-t-Boc-Aas in HPCE.     

Enantioseparation of Chiral Antimycotic Drugs by HPLC with Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order

The separation of enantiomers of 10 chiral antimycotic drugs was studied on polysaccharide-based chiral columns with polar organic mobile phases. The emphasis was placed on some interesting examples of enantiomer elution order reversal observed depending on the chemistry of the chiral selector, separation temperature, major component, as well as the minor additive in the mobile phase. In particular, it was found that the elution order of enantiomers of chiral drug terconazole was opposite on cellulose- and amylose-based columns with the same pendant group. The affinity pattern of enantiomers of another chiral drug bifonazole was opposite towards to two amylose-based chiral selectors with different pendant groups. The affinity pattern of terconazole enantiomers also changed on some columns when the alcohol-based mobile phase was replaced with acetonitrile. An interesting effect of the minor acidic (formic acid) additives to the mobile phase on the affinity pattern of terconazole enantiomers was observed on Cellulose-2 and Cellulose-4 columns. In addition, a reversal of elution order of bifonazole enantiomers was observed on Amylose-2 column by variation of a separation temperature.

     

Chemically L-prolinamide-modified monolithic silica column for enantiomeric separation of dansyl amino acids and hydroxy acids by capillary electrochromatography and mu-high performance liquid chromatography

A silica-based chiral monolithic column prepared by sol-gel process and chemical modification of chiral selector was used for enantioseparation of dansyl amino acids and hydroxy acids by capillary electrochromatography (CEC) and mu-high-performance liquid chromatography (mu-HPLC). L-Prolinamide was modified as a chiral selector. The chiral stationary phase (CSP), the chiral complex of Cu(II) with L-prolinamide, provides an anodic electroosmotic flow (EOF) in CEC. The EOF was found to be dependent on applied electric field strength, the pH, and the composition of mobile phases. Scanning electron micrograph showed that monolithic columns have the morphology of continuous skeleton and large through-pore. D-Enantiomers migrated before L-enantiomers except for dansyl-(Dns)-DL-Ser. The separation efficiencies of up to 17600 (D) and 13,200 plates m(-1) (L) were achieved for the separation of DL-indole-3-lactic acid.     

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.     

Synthesis and enantiomer separation of a modified tris(2,2′-bipyridine)ruthenium(II) complex

The chiral [5-(4-hydroxybutyl)-5′-methyl-2,2′-bipyridine]-bis(2,2′-bipyridine)-ruthenium(II)-bis(hexafluoroantimonate) complex 3 was prepared and characterized by different NMR techniques and successfully separated into enantiomers by electrokinetic chromatography using anionic carboxymethyl-β-cyclodextrin as chiral mobile phase additive (CMPA). The optimum separation conditions were obtained with 40 mM borate buffer at pH 9.5 and 7.5 mg/mL of the chiral selector at 20°C.     

Enantioseparation of Chiral Antimycotic Drugs by HPLC with Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order

The separation of enantiomers of 10 chiral antimycotic drugs was studied on polysaccharide-based chiral columns with polar organic mobile phases. The emphasis was placed on some interesting examples of enantiomer elution order reversal observed depending on the chemistry of the chiral selector, separation temperature, major component, as well as the minor additive in the mobile phase. In particular, it was found that the elution order of enantiomers of chiral drug terconazole was opposite on cellulose- and amylose-based columns with the same pendant group. The affinity pattern of enantiomers of another chiral drug bifonazole was opposite towards to two amylose-based chiral selectors with different pendant groups. The affinity pattern of terconazole enantiomers also changed on some columns when the alcohol-based mobile phase was replaced with acetonitrile. An interesting effect of the minor acidic (formic acid) additives to the mobile phase on the affinity pattern of terconazole enantiomers was observed on Cellulose-2 and Cellulose-4 columns. In addition, a reversal of elution order of bifonazole enantiomers was observed on Amylose-2 column by variation of a separation temperature.     

Preparation and evaluation of a chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether having a phenolic hydroxy group for enantiomer separation of amino compounds

In order to develop a chiral stationary phase (CSP), which has even higher separation ability than the corresponding commercially available crown ether based CSP (OA-8000 having a pseudo-18-crown-6 ether with an OMe group as a selector), chemically bonded type CSP having a phenolic OH group on a crown ring was developed. Normal mobile phases with or without acid additive can be used with this OH type CSP in contrast to the conventional OMe type CSP which has a neutral chiral selector. Enantiomers of 25 out of 27 amino compounds, including 20 amino acids, 5 amino alcohols, and 2 lipophilic amines, were efficiently separated on a column with this CSP. Nine amino compounds out of 27 were separated with better separation factors than the corresponding OMe type CSP. It is noteworthy that the chromatography on this CSP exhibited excellent enantiomer-separations for amines and amino alcohols when triethyl amine was used as an additive in the mobile phase. Comparison of enantiomer separation ability on this OH type of CSP and on the OMe type of CSP and correlation between the enantioselectivity in chiral chromatography and that of the corresponding model compounds in solution imply that the chiral separation arose from chiral recognition in host guest interactions.     

Novel approach to the study of the chiral discrimination mechanism in a series of dansyl amino acids.

With poly(octadecylsiloxane) as the liquid chromatographic stationary phase, phosphate buffer as the mobile phase, a series of D- and L-dansyl amino acids as solutes, and beta-cyclodextrin as the chiral selector, a study was conducted of the hydrophobic effect on both the solute complexation with the chiral selector and chiral discrimination mechanisms by varying the sucrose concentration in the mobile phase and the column temperature. The number of sucrose molecules excluded during the solute complexation with the chiral selector proved to be a good marker of the solute inclusion in the cavity. Gibbs Helmotz parameters delta(deltaH) and delta(deltaS) between D- and L-enantiomers were determined from plots of the logarithm of the intrinsic selectivity versus the reciprocal of the temperature. The results obtained predicted that the enantioselectivity was related to the bulkiness of the solute. This numerical approach is a valuable tool in the exploration of the steric effects implied in the formation of the host-guest complex.     

HPLC separation of the enantiomers of racemic benzergoline derivatives on a chiral binaphthalene covalently bonded to silica gel

A novel chiral stationary phase (CSP) derived from the atropisomeric enantiomer S-3,3′-dicarboxy-2,2′-dihydroxy-1,1′-binaphthyl (S-DDBN) has been synthesized and its use for the separation of enantiomers demonstrated. The chiral selector is covalently bonded to amino-functionalized silica gel, thus enabling the use of alcohols as mobile phases. Good chiral discrimination was obtained for the pharmacologically interesting class of benzergoline derivatives which act as selective dopamine D₁ receptor agonists. This paper reports the successful separtion of fifteen structurally related benzergoline racemates with separation factors up to 3.5. The influence of small differences in molecular structure on chiral discrimination was examined.     

Preparation of polar group derivative β-cyclodextrin bonded hydride silica chiral stationary phases and their chromatography separation performances

Three new polar group-substituted β-cyclodextrin derivatives were synthetized and they covalent bonded to hydride silica to obtain chiral stationary phases. Their separation results of 35 chiral pyrrolidine compounds were also presented and discussed.     

On the use of dispersed nanoparticles modified with single layer beta-cyclodextrin as chiral selecor to enhance enantioseparation of clenbuterol with capillary electrophoresis

A new strategy for chiral separation by capillary electrophoresis employing modified-nanoparticles as chiral selector is described for clenbuterol analysis. Nanoparticles modified with β-cyclodextrin (β-CD) form a large surface area platform to serve as a pseudostationary chiral phase, which can be applied for the enhancement of the enantioseparation. The application of four kinds of nanoparticles was investigated (multi-walled nanotubes (MWNTs), polystyrene (PS), TiO₂ and Al₂O₃) modified with single layer β-CD as chiral selector in the enantioseparation of clenbuterol by capillary electrophoresis (CE). Successful clenbuterol enantioseparation could be achieved with the β-CD-modified MWNTs as chiral selector. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) confirmed the β-CD modification of the nanoparticles. The effects of nanoparticles, surfactant, chiral selector (β-CD) and run buffer were studied in relation to the enantiomeric separation of clenbuterol. This study opens attractive perspectives for the use of modified nanoparticles for chiral separational purposes in CE.     

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.     

Chiral separation of beta-adrenergic antagonists, profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides using teicoplanin as the chiral selector in capillary liquid chromatography

Three groups of structurally diverse chiral compounds were used to study the interaction mechanism responsible for stereoselective recognition with teicoplanin as chiral selector in capillary liquid chromatography. Teicoplanin-based chiral stationary phase (CSP) was used. The effect of the variation of mobile phase composition on retention and enantioselective separation was studied. The mobile phase composition suitable for enantioresolution of the various chiral compounds differed according to the interaction forces needed for chiral recognition. Mobile phases with high buffer portion (70-90 vol.%) were preferred for separation of enantiomers of profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides that require hydrophobic interactions, inclusion and pi-pi interactions for stereoselective recognition with teicoplanin. Higher concentration triethylamine in the buffer (0.5-1.0%) increased resolution of these acids. On the other hand, H-bonding and electrostatic interactions are important in stereoselective interaction mechanism of beta-adrenergic antagonists with teicoplanin. These interaction types predominate in the reversed phase separation mode with high organic modifier content (95% methanol) and in polar organic mobile phases. For this reason beta-adrenergic antagonists were best enantioresolved in the polar organic mode. The mobile phase composed of methanol/acetic acid/triethylamine, 100/0.01/0.01 (v/v/v), provided enantioresolution values of all the studied beta-adrenergic antagonists in the range 1.1-1.9. Addition of teicoplanin to the mobile phase, which was suitable for enantioseparation of certain compounds on the CSP, was also investigated. This system was used to dispose of nonstereoselective interactions of analytes with silica gel support that often participate in the interaction with CSPs. Very low concentration of teicoplanin in the mobile phase (0.1 mM) resulted in enantioselective separation of 2,2- and 2,4-chlorophenoxypropionic acids.     

Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Amylose tris(5-chloro-2-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSP) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the nature of silica, the particle size and pore diameter, the chiral selector loading onto silica, the mobile phase composition and pH, as well as separation variables such as a linear flow rate of the mobile phase, applied voltage in CEC, etc. on the separation of enantiomers was studied. It was found that CSPs based on amylose tris(5-chloro-2-methylphenylcarbamate) can be used for preparation of stable capillary columns for enantioseparations by nano-LC and CEC in combination with polar organic and aqueous–organic mobile phases. Higher peak efficiency was observed in CEC than in nano-LC.