Enantioseparation Of Amino Acids Research Articles

Maltodextrin-modified graphene oxide composite membrane applied to the enantioseparation of amino acids

The separation of enantiomers using chiral membranes has garnered much research interest. In this study, the enantioseparation of amino acids using chiral membranes, namely graphene oxide-ethylenediamine-maltodextrin (GO-EDA-MD) and GO-EDA-hydroxypropyl-MD (GO-EDA-HP-MD), was evaluated. HP-MD and MD were investigated as chiral selectors due to their inherent chirality. Various characterization techniques, including atomic force microscopy, Fourier transform infrared spectrometry, field emission scanning electron microscopy, water contact angle analysis, tensile properties, and thermal gravimetric analysis were employed to analyze the membrane structures. The evaluation of enantioseparation performance was conducted by employing tryptophan, phenylalanine, and tyrosine enantiomers. Optimal conditions for enantiomer separation were achived using a GO-EDA-HP-MD chiral composite (1.75 wt%), a feed concentration of 10 mg/L for each enantiomer, a separation time of 15 min, and a membrane effective surface area of 1.0 cm2. Also, the bovine serum albumin rejection was 90.0 %, and the water flux reached 37.1 L m−2 h−1. The highest enantiomeric excess (ee.%) values were 46.33 %, 76.97 %, and 73.04 % for tryptophan, phenylalanine, and tyrosine, respectively. The impact of voltage on ee.% and substance flux was also explored. This membrane was able to separate enantiomers successfully.

Click preparation of triazole-bridged teicoplanin-bound chiral stationary phases for efficient separating amino acid enantiomers

Enantioseparation of amino acids is considered as a challenging task due to the extreme structural similarity of their enantiomers. Herein, teicoplanin was modified with different chemical equivalents of azide groups and attached to silica particles by employing Click Chemistry for resolution of chiral amino acids for the first time. Interestingly, teicoplanin modified with 5-fold the chemical equivalent of azide groups (TK-2 CSP) exhibited superior amino acid separation ability compared to two other columns: one modified with only 1-fold the chemical equivalent of azide groups (TK-1 CSP), and the other modified with excess azide groups (TK-3 CSP). Additionally, the TK-2 CSP exhibited superior enantioselectivity when separating amino acids containing hydrophobic alkyl side chains in comparison to other teicoplanin-based CSPs. The TK-2 CSP column allows the baseline separation of 7 native amino acids. Molecular docking demonstrates that effective enantioseparation arises from distinct patterns of interaction between the host and guest molecules. Moreover, (p-methyl) phenylcarbaminoylated-teicoplanin CSP (TK-4, TK-5 CSP) were prepared by post-modification from TK-1 CSP and TK-2 CSP to isolate Fmoc-modified amino acids. This work explores the impact of various modification methods on the enantioseparation effects of host molecules and paves the way for expanding the potential applications of teicoplanin and macrocyclic glycopeptide molecules.

Analysis and Enantioseparation of Amino Acids by Liquid Chromatography.

Enantioseparation studies of proteinogenic, non-proteinogenic, and dansyl amino acids are described herein by using liquid chromatographic techniques, i.e., HPLC and TLC. A researcher who wants to perform amino acid (AA) analysis or separate enantiomers of AAs by HPLC or TLC can follow the method. Figures included represent the actual experiments.Synthesis and application of chiral derivatizing reagents (CDRs) based on cyanuric chloride (CC) and difluorodinitrobenzene (DFDNB) have been described for AA analysis and enantioseparation by indirect approach. The methods represent pre-column derivatization of AAs and represent a good and less expensive substitute of AA analyzer. The application of commercial "Chiralplate" and use of erythromycin and L-tartaric acid have been described as chiral selector either as impregnating reagent in the stationary phase or as an additive in the mobile phase for direct enantioseparation by TLC. Application of the homemade TLC plates has also been described; the methods are successful in obtaining the native enantiomer as well.

Chiral ligand exchange countercurrent chromatography: Enantioseparation of amino acids.

This work deals with the enantioseparation of α-amino acids by chiral ligand exchange high-speed countercurrent chromatography using N-n-dodecyl-l-hydroxyproline as a chiral ligand and copper(II) as a transition metal ion. A biphasic solvent system composed of n-hexane/n-butanol/aqueous phase with different volume ratios was selected for each α-amino acid. The enantioseparation conditions were optimized by enantioselective liquid-liquid extractions, in which the main influence factors, including type of chiral ligand, concentration of chiral ligand and transition metal ion, separation temperature, and pH of the aqueous phase, were investigated for racemic phenylalanine. Altogether, we tried to enantioseparate 15 racemic α-amino acids by the analytical countercurrent chromatography, of which only five of them could be successfully enantioseparated. Different elution sequence for phenylalanine enantiomer was observed compared with traditional liquid chromatography and the proposed interactions between chiral ligand, transition metal ion (Cu2+ ), and enantiomer are discussed.

Enantioseparation of amino acids by micellar capillary electrophoresis using binary chiral selectors and determination of D-glutamic acid and D-aspartic acid in rice wine

ABSTRACTA new chiral capillary electrophoresis (CE) analytical method for the determination of six pairs of amino acid enantiomer-derivatized precapillary with 9-fluorenylmethoxycarbonyl chloride (FMOC) was developed. CE separation parameters such as the pH of background electrolyte, the boric acid concentration, the addition of D-fructose and isopropanol, and the effect of new binary chiral selectors on the electropherograms were investigated. Precapillary and in-capillary derivatization was compared as well. The results showed that enantiomeric separations were obtained with enantio-resolution (Rs) ranged from 2.61 to 9.99 for the FMOC-derivatized amino acid enantiomers except alanine with Rs 1.06. Precapillary derivatization method provides overall better Rs for nearly all the targets except FMOC-aspartate with efficiency up to 1.309 × 106 plates and limit of detection (LOD) down to 2.5 μM. This method was successfully applied to analyze the concentration of the D/L-glutamic acid and D/L-aspartate in seven rice wine samples.

Selector screening for enantioseparation of DL-α-methyl phenylglycine amide by liquid-liquid extraction.

Enantioseparation through liquid extraction technology is an emerging field, e.g., enantioseparations of amino acids (and derivatives thereof), amino alcohols, amines, and carboxylic acids have been reported. Often, when a new selector is developed, the versatility of substrate scope is investigated. From an industrial point of view, the problem is typically approached the other way around, and for a target racemate, a selector needs to be found in order to accomplish the desired enantioseparation. This study presents such a screening approach for the separation of the enantiomers of DL-α-methyl phenylglycine amide (DL-α-MPGA), a model amide racemate with high industrial relevance. Chiral selectors that were reported for other classes of racemates were investigated, i.e., several macrocyclic selectors and Pd-BINAP complexes. It appeared very challenging to obtain both high extraction yields and good enantioselectivity for most selectors, but Pd-BINAP-based selectors performed well, with enantioselectivities up to 7.4 with an extraction yield of the desired enantiomer of 95.8%. These high enantioselectivities were obtained using dichloromethane as solvent. Using less volatile chlorobenzene or 1-chloropentane, reasonable selectivities of up to 1.7 were measured, making these the best alternative solvents for dichloromethane.

Dress-up chiral columns for the enantioseparation of amino acids based on fluorous separation

In this paper, we report a new type of chiral high-performance liquid chromatography (HPLC) column--a so-called dress-up chiral column--featuring a chiral stationary phase adsorbed reversibly in a commercial fluorous HPLC column through fluorous interactions. We synthesized perfluroalkylated proline derivatives as chiral stationary phase compounds and then adsorbed them reversibly in the fluorous HPLC column through the pumping of their solutions. By using this dress-up chiral column and fluorophobic elution of an aqueous copper(II) sulfate/MeOH mixture, we could enantioseparate seven racemic amino acids within 40 min. When we washed the dress-up chiral column with fluorophilic tetrahydrofuran or MeOH, the adsorbed chiral stationary phase compounds desorbed from the column, completely destroying its enantioseparation ability. The relative standard deviation of the retention times, the number of theoretical plates, and the resolution for each of four preparations of the dress-up columns were all less than or equal to 9.53% in 20-times repeated analysis, and were all less than or equal to 18.7% in four different preparations, respectively.

Enantioseparations of amino acids by capillary array electrophoresis with 532 nm laser induced fluorescence detection

Capillary array electrophoresis (CAE) is a promising technique for multiple enantiomeric separations. Carboxytetramethylrhodamine succinimidyl ester (TAMRA SE), a rhodamine-core fluorescent probe, has rarely been applied as an original precolumn derivatization reagent for chiral amino acid (AA) analysis so far. For these purposes, high-throughput enantiomeric separations of 12 TAMRA SE-AAs by a home-made 532nm CAE-LIF scanner are presented. The effect of cyclodextrins (CDs) and a variety of organic modifiers was quickly investigated. Baseline separations were achieved in 100mM Tris–borate buffer (pH 10.0) containing 2mM β-CD and 10mM hexamethylenediamine (HDA). Multiple determination of the enantiomeric excess (ee) in non-racemic mixtures of alanine is successfully presented.

Simulated moving bed enantioseparation of amino acids employing memory effect-constrained chromatography columns

Teicoplanin aglycone-based chromatography columns (Chirobiotic TAG) enable amino acid enantioseparation with aqueous mobile phases, which perfectly accommodates the distinct hydrophilicity of most amino acids. Therefore, this stationary phase constitutes a promising option in particular for preparative-scale separations that require high feed concentrations for economic operation. However, detailed studies revealed a solute-related memory effect when this column is subjected to high loadings of amino acids, conditions that prevail in SMB operation. High loadings yield an activation of the column as indicated by increased retention times when comparing finite injection chromatograms obtained before and after the column had been subjected to a concentrated amino acid feed. This effect can be slowly reversed by flushing the column with solvent devoid of amino acid. Obviously, the activation of the stationary phase needs to be accounted for in the determination of adsorption isotherms that are used for SMB design. In this work we introduce a perturbation method adapted specifically to capture the stationary phase behaviour at SMB-like conditions. The adsorption isotherms obtained from this method indeed allowed for accurate SMB design of a methionine enantioseparation as judged by the very good agreement of experimentally obtained and model-predicted purities. Furthermore, SMB operation over 3 days with constant purities (besides deviations originating from a dip in temperature) was accomplished indicating that the adsorption behaviour in the activated state is indeed time invariant and stable long-term SMB operation with these columns is principally feasible.

Cover Picture: Electrophoresis 23‐24'2010

AbstractIssue no. 23–24 is a regular issue with an “Emphasis on Proteomics” and comprises 20 contributions assembled into 5 distinct parts. Part I is on innovation in proteomics and has 9 research articles describing novel proteomics methodologies and applications. Part II is on innovative assays and applications, and contains 5 full research articles on in‐capillary CE assay, electrophoretically mediated microanalysis, genotyping, SNP, STR and CE of polyphenols. Two research articles on preconcentration of peptides and melamine are included in Part III. Part IV has two articles on enantioseparations of amino acids by ligand exchange CE and on sensitized phosphorescence in the chiral separation of bupropion by CE. Part V is on open tubular CEC with 2 articles describing novel wall coated stationary phases.Featured articles include:Generation and characterization of a specific polyclonal antibody against the mouse serotonin receptor 1A: A state of the art‐recommendation how to characterize antibody specificity (10.1002/elps.201000374))Electrophoretically‐mediated microanalysis assay for sirtuin enzymes (10.1002/elps.201000336))

GC separation of amino acid enantiomers via derivatization with heptafluorobutyl chloroformate and Chirasil‐L‐Val column

Heptafluorobutyl chloroformate (HFBCF), a recently introduced derivatization reagent, was examined in enantioseparation of amino acids (AAs) by GC. Twenty proteinogenic AAs, plus ornithine, cystine and 4-fluorophenylalanine (internal standard) were treated with the reagent and separation properties of the derivatives were assessed on a Chirasil-Val capillary column. Nineteen AA enantiomers were efficiently separated in 43 min except proline, arginine and cystine. The HFBCF derivatives of the studied DL-AAs show improved separation over other chloroformate-based derivatives hitherto reported. A combination of the improved and faster separation with a simple derivatization protocol, involving an immediate one-step reaction-extraction in two-phase aqueous-organic medium, and low elution temperatures extend application of HFBCF to chiral AA analysis.

Enantioseparation of amino acids, α‐hydroxy acids, and dipeptides by ligand‐exchange CEC using silica‐based chiral stationary phases

This work deals with the application of silica-based ligand-exchange chiral stationary phases (CSPs) for the enantioseparation of underivatized amino acids, alpha-hydroxy acids, and dipeptides with packed CEC. Two different possibilities of preparing silica-based CSPs are presented. One phase contains L-4-hydroxyproline chemically bonded via a spacer to 3 mum silica material. The other approach makes use of N-decyl-L-4-hydroxyproline dynamically coated on a reversed-phase packed capillary. Dynamical coating of reversed-phase material represents a simple alternative to prepare CSP. A comparison of the chemically bonded phase with the dynamically coated CSP by means of resolution of complex-forming analytes is presented. The chemically bonded phase was found to be superior to the dynamically coated phase in terms of resolution of amino acids and dipeptides. However, the dynamically coated CSP was found to be especially suitable for the separation of alpha-hydroxy acids. Both techniques are applicable for enantiomer purity tests.

Adsorption behavior of a teicoplanin aglycone bonded stationary phase under harsh overload conditions

Silica-bonded teicoplanin aglycone allows enantioseparation of amino acids by reversed-phase liquid chromatography with a low organic solvent content. However, a reversible change in the adsorption behavior leading to a retention time shift (RTS) was observed when a preparative scale column was treated with harsh preparative chromatography-like conditions between finite-injection HPLC runs conducted under exactly the same conditions. This behavior was observed for all five investigated aliphatic and aromatic amino acids. In all cases, the retention times were prolonged after the overload conditions and the RTS was more pronounced for the later eluting d-enantiomer. We defined a standardized method for measuring the RTS and performed a systematic investigation on the influence of experimental conditions (type and concentration of pH modifier and organic modifier, temperature, pH) on the RTS. In this way a solvent composition – 90/10 50 mM NH 4Ac pH 5.8/MeOH – was identified that yielded no observable shift in retention time after overload conditions for both enantiomers. In order to treat the observed phenomenon on a mechanistic level, we applied band profile analysis based on the stochastic theory of chromatography and identified two different enantioselective sites. When the band profile analysis was performed on elution profiles obtained from runs with prolonged retention time after harsh overload conditions, the retention time shift could be attributed to both differentiable types of adsorption sites. One site was found to make both, enantioselective and non-selective contributions.

HPLC separation of amino acid enantiomers and small peptides on macrocyclic antibiotic‐based chiral stationary phases: A review

The search for new and effective chiral selectors capable of separating a wide variety of enantiomeric compounds is an ongoing process. In the past decade, macrocyclic antibiotics have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance by means of HPLC, TLC and electrophoresis. More chiral analytes have been resolved through the use of glycopeptides than with all the other macrocyclic antibiotics combined (ansamycins, thiostrepton, aminoglycosides, etc.). The glycopeptides avoparcin, teicoplanin, ristocetin A and vancomycin have been extensively used as chiral selectors in the form of chiral bonded phases in HPLC, and HPLC stationary phases based on these glycopeptides have been commercialized. Teicoplanin, vancomycin, their analogs and ristocetin A seem to be the most useful glycopeptide HPLC bonded phases for the enantioseparation of proteins and unusal native and derivatized amino acids. In fact, the macrocyclic glycopeptides are to some extent complementary to one another: where partial enantioresolution is obtained with one glycopeptide, there is a high probability that baseline or better separation can be obtained with another. This review sets out to characterize the physicochemical properties of these antibiotics and their application in the enantioseparations of amino acids. The mechanism of separation, the sequence of elution of the stereoisomers and the relation to the absolute configuration are also discussed.

Enantioseparation of amino acids by co-extractants with di(2-ethylhexyl)phosphoric acid and tartaric acid derivatives

A co-extraction method using di(2-ethylhexyl)phosphoric acid (D2EHPA) with each of the two tartaric acid derivatives, O, O′-dibenzoyl-(2 S,3 S)-tartaric acid [(+)-DBTA] and O, O′-dibenzoyl-(2 S,3 S)-4-toluoyl-tartaric acid [(+)-DTTA] was developed for the enantioseparation of four amino acids: racemic tryptophan ( rac-Trp), racemic phenylalanine ( rac-Phe), racemic p-hydroxyphenylglycine ( rac-Hpg) and racemic tyrosine ( rac-Tyr). The extractants were prepared in various proportions and diluted in n-octanol. The influence of the extractant composition, concentration of amino acid enantiomers, molecular structure of the solutes and extractive equilibrium temperature were studied. The maximum enantioselectivities of the four amino acids were 4.02, 1.37, 1.52 and 2.06 for Trp, Phe, Hpg and Tyr, respectively. The effects of the molecular structure of the solutes and amino acids along with the equilibrium temperature on the distribution ratio and enantioselectivity were examined, which is helpful for optimizing the extraction systems and realize the large-scale production of pure enantiomer.

Suitability of teicoplanin–aglycone bonded stationary phase for simulated moving bed enantioseparation of racemic amino acids employing composition-constrained eluents

The suitability of a teicoplanin–aglycone based chiral stationary phase for the simulated moving bed (SMB) enantioseparation of amino acids under enzyme-compatible conditions was shown following a procedure that is based solely on model-based simulations and HPLC experiments. A set of eight amino acids could be separated employing aqueous solvent containing only 10% (v/v) methanol, five of them with baseline resolution. The impact of type and concentration of organic modifier and pH modifier and pH on the separation characteristics of racemic methionine was investigated. Invariant elution profiles of repetitive adsorption/desorption of large amounts of methionine representing SMB-like conditions suggest stable adsorption behavior. Competitive loading capacity (20 mg of methionine per g of chiral stationary phase (CSP)) and SMB productivity (1 g of d-methionine per g of CSP per day) were predicted. The applied transport-dispersive model based on a competitive Bi-Langmuir isotherm was validated and its parameter estimated by model-based experimental analysis.

The enantioseparation of amino acids on a teicoplanin chiral stationary phase using non-aqueous mobile phases after pre-column derivatization with sulfur-containing reagents: the considerations of mobile phase composition and analyte structure variation on resolution enhancement

Amino acids were derivatized with sulfur-containing reagents in alkaline medium and enantioresolved by HPLC on a teicoplanin chiral stationary phase. Much better resolution was achieved using methanol-based mobile phase compared with elution with acetonitrile-based solvent mixture. The value of selectivity factor for many derivatives examined in the study can be easily several tenths of 100 or even exceed 100 in some cases. This magnitude of resolution is suitable for the preparative-scale application of isolating alpha-amino acid enantiomers in the derivatized form using a short column. The resolution is believed to be a result of the hydrogen bonding and steric hindrance by the nitrogen and sulfur atoms from the isothiocyanatyl fragment of the reagent, respectively, and is enhanced as the fragment is structurally phenylated (e.g. 2,4-difluorophenyl isothiocyanate and others examined in this study). The enhancement is even more significant if the aromatic moiety of the reagent becomes more acidic through halogenation with chlorine or fluorine for the stronger pi-pi interaction. However, this type of enhancement is offset to some extent and sometimes obscured by a chlorinated reagent that is relatively large in size as compared with the fluorinated one. Judging from the chromatographic data and the elution profile, the mechanisms leading to the resolutions are believed to be different under the elution of polar-organic mobile phases (i.e. methanol- and acetonitrile-based mobile phases).

Enantioseparation of Amino Acids on a Chiral Stationary Phase Derived from L-α-Norleucinyl- and Pyrrolidinyl-disubstituted Cyanuric Chloride by High-Performance Liquid Chromatography

A silica-based chiral stationary phase (CSP) derived from L-α-norleucinyl- and pyrrolidinyl-disubstituted cyanuric chloride was prepared for the enantioseparation of methyl esters of N-(3,5-dinitrobenzoyl) amino acids by high-performance liquid chromatography. The chromatographic results show that effective enantioseparation of methyl esters of N-(3,5-dinitrobenzoyl)amino acids, except for proline, was achieved on this chiral stationary phase. The chromatographic resolution of racemic n-propyl ester of N-(3,5-dinitrobenzoyl)valine on CSP-B is better than that of racemic methyl ester of N-(3,5-dinitrobenzoyl)valine on CSP-B or CSP-A reported previously (J. Chromatogr. A, 676 (1994) 303). The comparison of the chromatographic results obtained in this study with those on CSP-A reported previously reveals that steric effect, instead of hydrophobic interaction, between the n-butyl group attached to the chiral center of the chiral selector and the alkyl group attached to the chiral center of the chiral selectand plays a significant role in chiral discrimination. The increase in the selectivity factor of methyl esters of N-(3,5-dinitrobenzoyl)amino acids with bulky alkyl groups was examined on CSP-B.

Enantioseparation of amino acids and drugs by CEC, pressure supported CEC, and micro-hPLC using a teicoplanin aglycone stationary phase

Chiral separation of amino acids, drugs with amino alcohol structure, and other drugs has been investigated by capillary electrochromatography (CEC), pressure supported CEC, and micro-HPLC on a teicoplanin aglycone stationary phase. The teicoplanin aglycone lacks the D-glucosamine and the D-mannose moieties and shows different separation behavior compared to a teicoplanin stationary phase. The selector is bonded to 3.5-μm silica gel and separations are carried out either in the reversed phase or the polar organic mode. This chiral stationary phase (CSP) shows high enantioselectivity especially for amino acids, e. g. for DOPA a Rs value of 10.5 was achieved. Although the chiral recognition ability for drugs is lower than that of intact teicoplanin, out of 22 β-blockers investigated, 16 are baseline separated by CEC.

Enantioseparation of amino acids on a polysaccharide-based chiral stationary phase

Sulfonic acids have been shown to be more effective than the commonly used trifluoroacetic acid (TFA) in the chiral resolution of underivatized aromatic amino acids on an amylosic column. Sulfonic acid additives give a more UV transparent mobile phase, possibly allowing the detection of non-aromatic analytes. Work presented demonstrates that through the combination of sulfonic acid mobile phase additives, amine mobile phase additives and solvent modifier variations, the enantiomers of 20 of 25 probe amino acids are fully resolved, four are partially resolved with only one failing to be separated on a common amylosic column.