High-performance liquid chromatographic enantioseparation of (R,S)-fluoxetine using Marfey's reagent and (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as chiral derivatizing reagents along with direct thin-layer chromatographic resolution and isolation of enantiomers using L-tartaric acid as mobile phase additive

Chiral assay of enantiomers of fluoxetine was achieved in pharmaceutical formulations using direct and indirect methods. L-tartaric acid was used as a mobile phase additive in thin-layer chromatography; the enantiomers were separated and isolated and were used to determine the elution order in HPLC. (R,S)-flouxetine was derivatized with (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester [(S)-NIFE], Marfey's reagent and 1-fluoro-2,4-dinitrophenyl-L-methionine amide (FDNP-L-Met-NH?. The diastereomers were separated using RP-HPLC. The effect of flow rate and TFA concentration on resolution was studied. The diastereomers obtained by derivatization with FDNP-L-Met-NH? were also separated by RP-TLC.     

Optical resolution methods

Despite the large number of elaborate enantioselective syntheses for the preparation of a single enantiomer to achieve industrial and scientific goals, the separation and purification of enantiomers (components of racemic compounds) is also necessary. Hence, we present the most often used thought-provoking modern methods based on momentous recognitions (e.g. spontaneous resolution, induced crystallization, resolution by formation of diastereomers, resolution by formation of non-covalent diastereomers, resolution by diastereomeric salt formation, resolution by diastereomeric complex formation, "half equivalent" methods of resolution, separation by crystallization, separation by distillation, separation by supercritical fluid extraction, resolution with mixtures of resolving agents, resolution with a derivative of the target compound, enantioselective chromatography, resolution by formation of covalent diastereomers, resolution by substrate selective reaction, kinetic resolution without enzymes, kinetic resolution by enzyme catalysis, hydrolytic and redox enzymes, kinetic and thermodynamic control, resolutions combined with 2nd order asymmetric transformations, enrichment of partially resolved mixtures, role of the solvent and methods of optimization in the separation of diastereoisomers, non-linear effects and selected examples of resolution on an industrial scale).     

Chiral separation of selected proline derivatives using a polysaccharide type stationary phase by high-performance liquid chromatography

Proline derivatives, such as Boc-proline, Boc-2-methylproline, Boc-2-methylproline benzyl ester and Boc-2-methyl-4-hydroxy-proline benzyl ester, have been widely used as a building block leading to a variety of pharmaceutical compounds. Therefore, there is a wide interest in the chiral separation of these compounds. High-performance liquid chromatography (HPLC) methods were developed using a Chiralpak AD-H column to separate enantiomers of these proline derivatives. The effect of mobile phase composition and column temperature was studied. For the proline derivatives studied in this work, good resolution was achieved using a mobile phase composition of hexane, ethanol and 0.1% TFA. For prolines containing carboxyl or hydroxy group, resolution was changed dramatically corresponding to changes as little as 1% of ethanol in the mobile phase, suggesting that the dominant chiral recognition is from hydrogen bonding interactions. On the other hand, for prolines containing a benzyl ester instead of hydroxy group next to the chiral center, resolution was not affected as significantly with the changes of ethanol content in the mobile phase, indicating a different leading chiral recognition mechanism, such as inclusion, steric effect, or possible pi-pi interaction. Linearity, precision and limit of detection were also measured for Boc-2-methylproline and Boc-2-methylproline benzyl ester.     

Thin-layer chromatographic enantiomeric resolution via ligand exchange

A thin-layer chromatographic technique for the separation of proteinogenic and non-proteinogenic amino acids, dipeptides and alpha-hydroxy acids is described. Other examples are given from the field of alpha-methyl, N-alkyl and halogenated amino acids. The separation of the enantiomers is achieved, without derivatization, by means of ligand exchange on a reversed-phase silica gel as stationary phase, which is covered with a chiral selector (proline derivative). The resolution is so good that the respective enantiomers can be determined at trace levels (greater than or equal to 0.25%). The proposed method is simple, inexpensive and needs no sophisticated instruments.     

Synthesis of fluorescent label, DBD-beta-proline, and the resolution efficiency for chiral amines by reversed-phase chromatography

DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD-H, with n-hexane-EtOH-TFA-diethylamine (70:30:0.1:0.1) as the mobile phase. The dl-forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD-d(or l)-proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD-d(or l)-beta-proline were higher than those derived from DBD-d(or l)-proline, e.g. dl-phenylalanine methylester (dl-PAME), 2.23 vs 1.37; (R)(S)-1-phenylethylamine [(R)(S)-PEA], 2.09 vs 1.13; and (R)(S)-1-(1-naphthyl)ethylamines [(R)(S)-NEA], 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers.     

New chiral crown ether stationary phase for the liquid chromatographic resolution of alpha-amino acid enantiomers

A new chiral stationary phase (CSP) for the liquid chromatographic separation of enantiomers was prepared by bonding a novel enantiopure (diphenyl-substituted 1,1'-binaphthyl) crown ether to 5 microm silica gel. The resulting CSP was applied to the separation of the enantiomers of various natural and unnatural alpha-amino acids. All alpha-amino acids tested were resolved very well on the new CSP, with the exception of proline, which does not contain a primary amino group. The resolution of alpha-amino acid enantiomers on this new CSP was found to be dependent on the type and amounts of organic and acidic modifiers, and on column temperature.     

Molecularly imprinted monolithic stationary phases for liquid chromatographic separation of enantiomers and diastereomers

The method for preparation of molecularly imprinted monolithic stationary phase has been improved to achieve liquid chromatographic separation of enantiomers and diastereomers. By adopting low polar porogenic solvents of toluene and dodecanol and optimal polymerization conditions, the molecularly imprinted monolithic stationary phases with good flow-through properties and high resolution were prepared. Enantiomers of amino acid derivatives and diastereomers of cinchona alkaloids were completely resolved using the monolithic stationary phases. The influence of porogenic composition, monomer-template ratio and polymerization conditions on the chromatographic performance was investigated. Some chromatographic conditions such as the composition of the mobile phase and the temperature were characterized. Scanning electron microscopy showed that the molecularly imprinted monolithic stationary phase has a large through-pore structure to allow the mobile phase to flow through the column at very low backpressure. Accelerated separations of enantiomers and diastereomers were therefore achieved at elevated flow rates. Finally, the chiral recognition performance of the prepared stationary phase in aqueous media was investigated. Hydrophobic interaction, and ionic and/or hydrogen bonding interactions were proposed to be responsible for the recognition mechanism.     

Cellulose Derivatives Used as Chiral Stationary Phases in Capillary Gas Chromatography

Abstract

In this work, we present the first separation of enantiomers in gas chromatography (GC) using a fused‐silica capillary column containing cellulose triacetate, cellulose triphenylcarbamate, or cellulose tris(3,5‐dimethylphenylcarbamate) as the new chiral stationary phase. The separated solutes included alcohols, amine, ketone, ether, ester, and amino acid. Their column efficiency, polarity, and chiral selectivity were studied. The retention mechanism was discussed. The results showed that those derivatives had relatively high chiral recognition abilities and can be used as the chiral stationary phases in GC.     

New chiral crown ether stationary phase for the liquid chromatographic resolution of α-amino acid enantiomers

A new chiral stationary phase (CSP) for the liquid chromatographic separation of enantiomers was prepared by bonding a novel enantiopure (diphenyl-substituted 1,1′-binaphthyl) crown ether to 5 μm silica gel. The resulting CSP was applied to the separation of the enantiomers of various natural and unnatural α-amino acids. All α-amino acids tested were resolved very well on the new CSP, with the exception of proline, which does not contain a primary amino group. The resolution of α-amino acid enantiomers on this new CSP was found to be dependent on the type and amounts of organic and acidic modifiers, and on column temperature.     

高效液相色谱法分离2-(4-羟基苯氧基)丙酸酯的对映体

采用高效液相色谱法在手性柱上分离芳氧基苯氧基丙酸类除草剂中间体(±)-2-(4-羟基苯氧基)丙酸酯的对映体。实验结果表明,在三苯甲酸纤维素酯的手性柱上,以无水乙醇为流动相能较好地分离(±)-2-(4-羟基苯氧基)丙酸乙酯和甲酯,其分离因子α值分别为1.44和1.29;同时还发现在三苯甲酸纤维素酯的手性柱上2-取代芳氧基或芳基丙酸酯结构中的酯基团的大小对其对映体的分离有明显的影响,其中以乙基为最佳。并通过对照试验证实了2-(4-羟基苯氧基)丙酸乙酯的右旋体先流出,其左旋体后流出。     

反相液相色谱法分离2-(4-羟基苯氧基)丙酸酯对映体

合成了三-(4-甲基苯甲酸)纤维素酯(MCTB)手性固定相，用反相高效液相色谱法在该手性固定相上对2-(4-羟基苯氧基)丙酸酯对映体进行拆分．实验结果表明，在三-(4-甲基苯甲酸)纤维素酯手性固定相上，以甲醇与水的体积分数为75：25做流动相能较好的拆分2-(4-羟基苯氧基)丙酸甲酯、乙酯和丁酯对映体，其分离因子分别为1．38、1．49、0．98；同时还发现2-(4-羟基苯氧基)丙酸酯中酯基团的大小对其对映体的分离也有明显的影响，其中以乙酯的拆分效果最佳。     

Use of enantioselective liquid chromatography for preparation of pure atenolol enantiomers

The study reported here shows a practicable preparation of pure atenolol enantiomers using enantioselective liquid chromatography. The successful separation of enantiomers of the final atenolol and the intermediate ester and the good peak shapes could not have been obtained without diethylamine as a component of the mobile phase. That makes difficult the recycling of the three-component mobile phase, an unavoidable step in simulated moving bed chromatography separation technology. The only suitable methodology for preparation of atenolol enantiomers proved to be synthesis from its N-benzyl-N-isopropyl precursor and the chiral stationary phase Chiralpak AD was found to be very convenient for preparative separation of these enantiomers. The enantiomeric purities and recovery of separated enantiomers of this N-benzyl-N-isopropyl precursor were very high, allowing high enantiomeric purities of the final products, ee's 99.3% for S- and 99.0% for R-atenolol. The chromatographic separation parameters, as well as solubility of racemate in the mobile phase, are good bases for the further examination of possible scale-up resolution of compound 6.     

Stereoisomeric flavor compounds. Part LXV: Preparative resolution of the enantiomers of chiral dihydrofuranones by recycling chromatography

Preparative resolution of the enantiomers of several chiral dihydrofuranones, known to be important flavor compounds, has been achieved by combining the use of bonded β-cyclo-dextrin as chiral stationary phase with closed-loop recycling chromatography. The purity of the separated enantiomers has been determined by chirospecific capillary GC analysis, using heptakis(2,3-di-O-methyl-6-TBDMS)-β-cyclodextrin as the chiral stationary phase.     

Improved Synthesis of Cyclic α-Hydrazino Acids of Five- to Nine-Membered Rings and Optical Resolution of 5,6,7-Membered Ring Hydrazino Acids

Synthesis of cyclic α-hydrazino acids of five- to nine-membered rings has been described. Di-tert-butyl or dibenzyl malonate was used as starting materials instead of diethyl malonate, which was used in our first report. Deprotection of tert-butyl or benzyl ester of the final compounds was much easier than that of ethyl or methyl esters. Overall yield of these acids were 39, 50, 47, 52, and 51%, respectively. These acids were then converted to the diastereomers either via the formation of peptides with L-phenylalanine methyl ester or via the formation of esters (for five- to seven-membered rings) with L-2-phenylalaninol. All diastereomers were separated except the nine-membered ring by flash chromatography. Hydrolysis of diastereomeric esters generated the optically pure five-, six- and seven-membered cyclic α-hydrazino acids. In this process, both the enantiomers have been isolated and the chiral auxiliary L-2-phenylalaninol was recovered. Absolute stereochemistry was determined from x-ray crystallographic analysis.     

Synthesis and evaluation of new fluorescent derivatization reagents for resolution of chiral amines by RP-HPLC

New fluorescent chiral derivatization reagents (i.e., DBD-trans-4-hydroxy-l-proline, DBD-cis-4-hydroxy-l-proline, DBD-cis-4-hydroxy-d-proline, and DBD-trans-3-hydroxy-l-proline) were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole with corresponding hydroxy-prolines. These reagents reacted with chiral amine to produce a couple of diastereomers. The labeling efficiently proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and pyridine as the activation reagents. The reaction conditions are mild and no racemization occurred during both the reagent synthesis and the diastereomer formation (<0.4%). The resulting diastereomers fluoresce at around 560 nm (excitation at around 450 nm). Good linearity of the calibration curves was obtained in the range of 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was evaluated in terms of separation factor (α) and resolution value (Rs). DBD-trans-4-hydroxy-l-proline was efficient for the resolution of dl-phenylalanine methylester; while DBD-cis-4-hydroxy-d(or l)-proline was excellent for the separation of 1-(1-naphtyl)ethylamines, as comparing with trans-4-hydroxy isomer. The reagents of cis-isomer seemed to be predominant for the resolution of hydrophobic enantiomers. On the other hand, trans-isomers were suitable for the separation of the racemic amines containing ester in the structure. With respect to the position of OH group, the effect seems to be less, judging from the results of DBD-trans-4-hydroxy-l-proline and DBD-trans-3-hydroxy-l-proline toward phenylalanine methylester. The results suggest that the separation is dependent upon both structures of the amines and the reagent used. Thus, the stereostructure, hydrophobicity and hydrogen bonding of the diastereomer, etc. seem to be affecting the separation.     

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the clinical reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C8 column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.     

Separation of amino acid enantiomers VIA chiral derivatization and non-chiral gas chromatography

Two GC-MS methods for the enantioselective separation of the 20 proteinogenic amino acids are compared. Ethyl chloroformate and 2-chloropropanol were used to derivatize amino acid enantiomers. The diastereomers formed were separated on a non-chiral column by capillary gas chromatography. The separation performances were compared to those obtained when using non-chiral derivatization on a chiral column.     

Activated carbamate reagent as chiral derivatizing agent for liquid chromatographic optical resolution of enantiomeric amino compounds

Summary A chiral derivatization reagent having activated succinimido carbamate moieties were developed for the optical resolution of enantiomeric amines by high-performance liquid chromatography. Succinimido R-(+)- or S-(–)-1-phenylethyl and R-(+)- or S-(–)-1-(-naphthyl)-ethyl carbamates were synthesized by the reaction of optically active phenylethyl and naphthylethyl amines with discuccinimido carbonate (DSC). These reagents reacted with both primary and secondary amine enantiomers such as amino acids and -amino alcohols to give the corresponding diastereomeric urea derivatives. These diastereomers were efficiently separated by reversed-phase liquid chromatography and detected by their absorption or the fluorescence of the chromophores. The chiral derivatization procedure was applied to the separation and determination of enantiomeric propranolol in serum.     

Practical synthesis of L-erythro- and L-threo-4-fluoroglutamic acids using aminoacylase

Enantiomerically pure L-erythro- and L-threo-4-fluoroglutamic acids 1a and 1b were conveniently prepared. The key steps in this synthesis relied upon separation of diastereomers of N-chloroacetyl-4-fluoroglutamic acid 5-methyl ester 7 by recrystallization and enzymatic resolution of enantiomers of the resulting 7(a+c) and 7(b+d) by aminoacylase. Protection of the γ-carboxyl group as a methyl ester was found to be crucial for this enzymatic reaction.     

Effect of amine mobile phase additives on chiral subcritical fluid chromatography using polysaccharide stationary phases

Increased retention and selectivity in the subcritical fluid chromatography (SFC) of various amine compounds on polysaccharide chiral stationary phases (CSP) was observed upon incorporation of cyclic amines into the modifier. The retention increases are most pronounced with 2-propanol and are almost absent when methanol is used as modifier. This suggests that the effect may arise from a restriction to the modifier access to the binding site required to effect elution. The effect of the amine additives in SFC does not remain after their removal from the mobile phase. Findings were applied to the development of a 5 min separation of amphetamine and methamphetamine enantiomers.