High-performance ligand-exchange chromatography of amino acids on chiral stationary phases

Three chiral stationary phases, obtained by grafting silica gel with (-)-trans-1,2-cyclohexanediamine, were studied for the resolution of α-amino acids by ligand-exchange chromatography. The packings were prepared by bonding the chiral ligand to silica gel via different hydrocarbon spacers. Separation of the optical isomers was accomplished by eluents containing a constant concentration of copper(II) acetate (0.05mM). The elution sequence of amino acids was found to be dependent on the grafting reaction selected to prepare the chiral packings.     

Chiral separation of halogenated amino acids by ligand-exchange capillary electrophoresis

The chiral separation of halogenated amino acids by ligand-exchange CE is described. Halogenated amino acids attracted increasing interest in recent years because of their physiological activities. Different chiral selectors, as there are L-4-hydroxyproline, L-histidine, and N-alkyl derivatives of L-4-hydroxyproline in form of their copper(II) complexes, are compared for their chiral recognition ability for halogenated amino acids. The influence of various parameters, such as selector concentration, pH, organic modifier, and field strength, on the resolution was investigated. All halogenated amino acids investigated were baseline-separated under optimized conditions.     

S-trityl-(R)-cysteine, a powerful chiral selector for the analytical and preparative ligand-exchange chromatography of amino acids

S-trityl-(R)-cysteine [(R)-STC] is the new selector of a dynamically coated, chiral ligand-exchange stationary phase which proved to be highly effective in both analytical and preparative-scale separation of enantiomers of some natural and unnatural underivatized amino acids, with good separation and resolution factors. With the aim of identifying the best chromatographic conditions suitable for the preparative-scale separations, some parameters controlling retention, separation and resolution factors (such as the type and amount of cupric salt and the eluent pH) were investigated. The relatively easy removal of the Cu(II) ions renders this technique suitable for obtaining small amounts of enantiomerically pure samples for preliminary biological evaluations.     

Enantiomer separation of amino acids as their N-alkyloxycarbonyl alkylamide derivatives by chiral phase capillary GC

The enantiomers of amino acids were first converted into N-alkyloxycarbonyl 2,2,2-trifluoroethyl esters, and then into N-alkyloxycarbonyl alkylamides by nucleophilic substitution of the ester group with amines. The first reaction proceeds instantaneously, while the second substitution occurs smoothly with n-propylamine and isobutylamine. The final derivatives were produced for separation on a capillary column coated with Chirasil-Val by GC. Pro, which is difficult to separate completely as its N-perfluoroacyl alkyl ester derivative, showed complete separation of the enantiomeric pair. All amino acids examined in this study showed an increased separation factor.     

Improved separation of amino acids by thin-layer chromatography

Summary The separation of 35 amino acids on Avicel F layers was investigated and 6 solvent systems are recommended for use either singly or in combination in 2-dimensional chromatography. The mechanisms and limitations of these methods are discussed.     

The role of achiral sorbent matrix in chiral recognition of amino acid enantiomers in ligand-exchange chromatography

Summary As an alternative to the known three-point interaction model describing recognition of optical isomers by a chiral resolving agent, a new concept has been developed stating that two interaction points between the resolving agent and the enantiomers are also sufficient for achieving chiral recognition of the latter, provided that the diastereomeric adducts formed by the resolving agent with the enantiomers additionally interact with a non-chiral chromatographic sorbent. This concept is based on the results of ligand-exchange chromatography of -amino acid enantiomers with copper(II) complexes of chiral bifunctional ligands as the resolving agents in chromatographic systems.     

Enantiomeric separation using temperature-responsive chiral polymers composed of L-valine diamide derivatives in aqueous liquid chromatography

This paper describes enantiomer separation by aqueous liquid chromatography using chiral stationary phases (CSPs) in which temperature-responsive polymers derived from acryloyl-L-valine N-methylamide (1) and its N,N-dimethylamide analogue (2) were bound on silica gel supports. The linear polymers composed of monomer 1 and monomer 2 are temperature-responsive in solution and their aggregation and extension states related to water solubility are reversible at particular critical temperatures. During chromatography, enantioselectivity and retentivity for solute enantiomers were controlled by column temperature, which changes the aggregation and extension states of the chiral polymers depending upon their interior hydrophobic nature. Two different types of CSPs were made: a temperature-responsive linear polymer derived from 3-mercaptopropyl silica gel, and another polymer cross-linked with ethylene dimethacrylate from 3-methacryloyloxypropyl silica gel. The former CSP could separate racemic N-(3,5-dinitrobenzoyl(DNB))amino acid isopropyl esters. Retention of the amino acid derivatives was prolonged with an increase in column temperature. Enantioselectivity was also enhanced with temperature increase until the particular critical temperature. The latter, cross-linked CSP could not provide enantioselectivity for the amino acid derivatives in aqueous media, although the chiral valine diamide moieties were effective for enantiomer separation in non-aqueous media. The degree of hydrophobicity and volume of the bonded phase formed by the polymers on the support surface was determined by measuring the fluorescence of pyrene.     

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.     

Dynamic column-coating procedure for chiral ligand-exchange chromatography

Summary The preparation of HPLC columns for chiral ligand-exchange chromatography by dynamic adsorption of a histidine-based chiral selector (N-n-decyl-L-histidine, LNDH) on commercial reversed-phase, C₁₈ columns was investigated. The most suitable solvent for column preparation was sought through a study of the behaviour of LNDH on the reversed-phase column eluting with different methanol-water ratios at varying pH. It is shown that the use of an aqueous solvent instead of pure methanol is advantageous, despite the decrease in LNDH solubility, and that the solvent pH must be controlled. Open and closed-cycle procedures for flowing the selector solution through the column are compared through a study of chromatographic retention, selectivity and efficiency parameters of selected enantiomers. Even though no significant difference in column loading was found between the two procedures, the open cycle method gave rise to more efficient and selective chiral columns.     

Separation of some amino acids by supercritical fluid chromatography after a pre-derivatization step with classical reagents

Summary The separation of polar compounds by supercritical fluid chromatography is a difficult problem to solve. In this work, we have used a pre-derivatization method to obtain apolar or less polar compounds. Amino acids have been chosen as model polar compounds of biological interest, and 9-fluorenylmethyl chloroformate (FMOC) and (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) have been used as derivatizating reagents.With this procedure, the amino acids studied have been separated in less than 40 minutes with a good efficiency. Furthermore, with FLEC reagent, the enantiomeric separation was rapid in comparison with liquid chromatography techniques and the selectivities obtained were in the same order of magnitude.     

Degradingdehydroabietylisothiocyanate as a chiral derivatizing reagent for enantiomeric separations by capillary electrophoresis

Abietic acid is a naturally occurring enantiomeric diterpenic acid. Its absolute optical purity and very stable stereochemistry structure makes it an excellent starting material for preparing chiral derivatizing reagents for chromatographic or electrophoretic applications. This paper describes the synthesis and evaluation of a novel chiral derivatization reagent, i.e., degradingdehydroabietylisothiocyanate (DDHAIC) derived from dehydroabietic acid. Its applicability for the enantioseparation of racemic amino acids by CE was demonstrated. DDHAIC reacted readily with amino acids at an elevated temperature (70 degrees C). The resulting derivatives were highly stable and separable by MEKC. Separation of amino acid-DDHAIC diastereomers was achieved with a running buffer consisting of 50 mM Na(2)HPO(4) (pH 9.0), 18 mM SDS, and 25% v/v ACN. Under the conditions selected, diastereomers formed from ten pairs of tested amino acid enantiomers including D/L-Asn, D/L-Met, D/L-Leu, D/L-Phe, D/L-Trp, D/L-Ser, D/L-Val, D/L-Ala, D/L-Thr, and R/S-vigabatrin were well resolved. The resolution values were in the range of 0.95-8.9.     

Synthesis and application of dehydroabietylisothiocyante as a new chiral derivatizing agent for the enantiomeric separation of chiral compounds by capillary electrophoretic

A new chiral derivatizing reagent, dehydroabietylisothiocyante (DHAIC), was synthesized and used for the enantiomeric separation of chiral compounds in capillary electrophoresis (CE). The synthetic route to obtain DHAIC is described. The separation conditions for the chiral separation of several chiral compounds, such as protein amino acids and chiral drug DOPA were optimized. Best results for the chiral separation of DHAIC derivatized amino acids and DOPA were obtained in a running buffer consisted of 50 mM borate (pH 9.5), 5 mM sodium dodecyl sulphate (SDS) and 20% acetonitrile for amino acids and 60 mM Na₂HPO₄ (pH 8.0), 17 mM SDS and 25% acetonitrile for DOPA. Under the conditions studied, chiral separation of five amino acids including Ser, Val, Ala, Thr, Cys and a chiral drug DOPA as their diastereomeric DHAIC derivatives has been achieved by micellar electrokinetic chromatography (MEKC).     

Chiral ligand-exchange chromatography of amino acids using porous graphitic carbon coated with a dinaphthyl derivative of neamine

In this paper, we describe the preparation and the evaluation of a porous graphitic carbon (PGC) column coated with a new dinaphthyl derivative of neamine for chiral ligand-exchange (LE) chromatography. It was shown that the graphitic surface/dinaphthyl anchor system efficiently (1.15 μmol/m²) and stably (three months of intensive use) adsorbs the neamine template onto the chromatographic support. The resulting coated PGC stationary phase showed appreciable LE-based enantioselective properties towards several native amino acids. Chromatographic separation of methionine enantiomers using a dinaphtyl neamine-based ligand-exchange chiral stationary phase     

Enantioselective binding of amino acids and amino alcohols by self-assembled chiral basket-shaped receptors

Amino acid appended diphenylglycoluril-based chiral molecular receptors 2 and 3 have been prepared and their aggregation has been studied in water at various pH's and in chloroform. The binding of several biologically relevant guests with aromatic moieties to these aggregates has been studied with UV-Vis spectroscopy in competition experiments with 4-(4-nitrophenylazo)resorcinol (Magneson) and 2-(4-hydroxyphenylazo)benzoic acid (HABA) as probes. Aggregates of chiral host 2b showed binding of catecholamines and aromatic amino acids in an aqueous environment, as well as discrimination between amino acid enantiomers, and can be considered a mimic for adrenergic receptors.     

Enantioselective capillary electrophoresis-mass spectrometry of amino acids in cerebrospinal fluid using a chiral derivatizing agent and volatile surfactant

The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R² > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%–8.0% and 0.34%–0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs.     

Determination of amino acids in urine by gas chromatography

This report describes a convenient means of reducing the complex matrix which is responsible for interference during gas chromatographic determination of amino acids in urine. The pre-chromatographic clean-up employs the principle of solid phase extraction using bonded silica incorporating cation exchange groups. This approach avoids the detrimental effects on amino acid recoveries associated with resin-based cation exchangers. In spite of significant reduction in the complexity of chromatograms, only the high efficiency and resolving power offered by the analytical capillary column (e. g. fused silica open tubular, FSOT) is sufficient for quantitative and analysis of amino acids in urine. Reproducibility data from the complete procedure are determined, coefficients of variation (CV) for most amino acids being better than 5% with a mean recovery of 96%.     

Separation of racemic 2,4-dinitrophenyl amino acids on 9-O-(phenyloxycarbonyl)quinine-bonded carbon-clad zirconia in reversed-phase liquid chromatography

Zirconia is known to be one of the best materials for the chromatographic support due to its excellent chemical, thermal, and mechanical stability. In this work, we report preparation and use of 9-O-(phenyloxycarbonyl)quinine-bonded carbon-clad zirconia (QNCZ) as a chiral stationary phase (CSP) for separation of N-(2,4-dinitrophenyl) (DNP)-amino acids (AAs) enantiomers in reversed-phase liquid chromatography. Retention and enantioselectivity of the QNCZ CSP were compared with those of quinine 3-triethoxysilylpropylcarbamate-coated zirconia (QNZ) and quinine 3-triethoxysilylpropylcarbamate-bonded silica (QNS). The QNCZ CSP showed in general the better enantioselectivity for most of the amino acids studied.     

Resolution of sulphur-containing amino acids by chiral phase gas chromatography

Summary Methyl esters of the pentafluoropropionyl-amino acid derivatives of the tetrafunctional, sulphur-bridged, stereoisomeric lanthionines, cystathionines and -methyl-lanthionines were resolved on glass capillaries coated with the chiral stationary phase N-propionyl-L-valine-N-tert-butylamide-polysiloxane (Chirasil-Val) within 35min. Interestingly, L-cystathionine elutes before its D-enantiomer in contrast to the usual order of emergence on an L-phase. The method was applied to the polypeptide antibiotic nisin, which contains mesolanthionine and 2S,3S,6R-3-methyl-lanthionine.N-Pentafluoropropionyl-S-alkylthiocysteine methyl esters (R=methyl, ethyl, n- and iso-propyl, n- and sec-butyl, n-octyl, neo-pentyl, cyclohexyl-, benzyl-, tolyl-) were separated on Chirasil-Val within 30min. The identity of all derivatives was shown by combined gas chromatography-mass spectrometry.     

Reversal of the enantiomeric elution order of some aromatic amino acids using reversed-phase chromatographic supports coated with the teicoplanin chiral selector

In this paper, two chiral stationary phases were prepared by coating the surface of both C8 and C18 high-performance liquid chromatography (HPLC) supports with the teicoplanin chiral selector. The hydrophobic C11 acyl side chain, attached to the d-glucosamine group of teicoplanin, served as anchor moiety for the immobilization of the chiral selector on the apolar support material. The retention and enantioselectivity of these coated stationary phases were studied using some aromatic amino acids as probe solutes and an aqueous solution as mobile phase. It was found that the enantiomer elution order on the modified C8 and C18 stationary phases was reversed (l > d) relatively to that classically observed with a teicoplanin covalently immobilized on a silica support (d > l). Such a dynamic coating on the reversed-phase supports was found to be of interest since the apparent enantioselectivity was not significantly changed by the use during an extended period of time or following a long-term storage of the columns.