Use of chiral amino acid ester‐based ionic liquids as chiral selectors in CE

In this study, the applicability of a chiral ionic liquid (CIL) as the sole chiral selector in CE was investigated for the first time. In particular, five amino acid ester‐based CILs were synthesized and used as additives in the BGE in order to evaluate their chiral recognition ability. The performance of these CILs as the sole chiral selectors was evaluated by using 1,1′‐binaphthyl‐2,2‐diylhydrogenphosphate (BNP) as the analyte and by comparing the resolution values. Different parameters were examined, such as the alkyl group bulkiness and the configuration of the cation, the anion type of the CIL and its concentration, and the pH of the BGE, in order to optimize the separation of the enantiomers and to demonstrate the effect that each parameter has on the chiral‐recognition ability of the CIL. Baseline separation of BNP within 13 min was achieved by using a BGE of 100 mM Tris/10 mM sodium tetraboratedecahydrate (pH 8) and a chiral selector of 60 mM l ‐alanine tert butyl ester lactate. The run‐to‐run and batch‐to‐batch reproducibilities were also evaluated by computing the %RSD values of the EOF and the two enantiomer peaks. In both cases, very good reproducibilities were observed, since all %RSD values were below 1%.     

Chiral capillary electrophoretic separation of amino acids derivatized with 9-fluorenylmethylchloroformate using mixed chiral selectors of beta-cyclodextrin and sodium taurodeoxycholate

Chiral separation of 19 pairs of amino acid (AA) enantiomers derivatized with 9-fluorenylmethylchloroformate (FMOC) was successfully conducted by capillary electrophoresis using the mixture of beta-CD and sodium taurodeoxycholate (STDC) as selectors. Resolution was considerably superior to that obtained by using either beta-CD or STDC alone. After a systematic inspection, a buffer composed of 150 mM borate and 18% v/v isopropanol at pH 8.0, dissolved with 30 mM beta-CD and 30 mM STDC, was adopted and able to generate baseline resolution (>1.50) for 17 pairs of FMOC-AA enantiomers and somewhat lower resolution for arginine (1.36) and alanine (1.18), respectively. Experimental data revealed that the addition of the second selector did not increase the mobility difference between a pair of enantiomers (Delta mu = mu(D) - mu(L) and the number of theoretical plates (N), but decreased the summed apparent mobility of a pair of enantiomers (Sigma mu = mu(D) - mu(L)), which was mainly due to the decrease of the electroosmotic flow. The variation of Sigma mu was thus the major reason responsible for the improvement of chiral resolution in this study. The result demonstrated that not only the intrinsic selectivity of the selectors was the basis of the chiral separation, but also the non-chiral effect of the selectors, the change of the electroosmotic flow, was an important factor in enhancing the enantioseparation resolution. This study could probably help to explain the reasons for resolution improvement in some dual selectors systems, which are not very clear at present.     

A comparison of vancomycin and sulfated beta-cyclodextrin as chiral selectors for enantiomeric separations of selenoamino acids using capillary electrophoresis with UV absorbance detection

The enantiomeric separation of three selenoamino acids, D,L-selenomethionine, D,L-selenoethionine and D,L-selenocystine is described. Both sulfated beta-cyclodextrin and vancomycin have been successfully used to separate all enantiomers of the compounds with UV detection. Reproducible separations, in terms of peak area and migration time were obtained using sulfated beta-cyclodextrin with reversed polarity and UV detection. With vancomycin as a chiral selector, reversed polarity was found to be more reproducible than positive polarity in terms of peak migration times.     

On-column detection of multiphoton-excited fluorescence in CE using hyphenated cylindrical-square capillaries

Multiphoton-excited fluorescence (MPEF) is a complementary and useful mode of LIF detection in CE with advantages of ultra-low mass detectability and spectral excitability, but it is currently quite limited by its end-column configuration. In this article, we demonstrate a novel strategy of on-column schemes that can greatly facilitate MPEF detection in CE. FITC-labeled amine species were used as the model samples for the evaluation and comparison of those detection scenarios. By using the square capillary instead of the conventional cylindrical one, the on-column MPEF could be readily achieved, with detection sensitivity of 0.72 microM that was comparable with the end-column mode. However, this strategy unfavorably reduced separation efficiency. The theoretical plate number on averaging all the sample peaks was significantly decreased from 283,000 to 19,000/m. To minimize such an influence, a short square capillary acting as an on-column MPEF detection cell was then mounted to a long cylindrical capillary responsible for the CE separation. Results indicated that both high separation efficiency (240,000/m) and better detectability (0.42 microM) were realized simultaneously by using this binary-capillary configuration. Quantitative analysis was performed under the optimized detector configuration and revealed a linear dynamic range of 2 orders of magnitude, with mass detection limit down to the mid-yottomole level.     

Chiral separation of amino acids derivatized with fluoresceine-5isothiocyanate by capillary electrophoresis and laser-induced fluorescence detection using mixed selectors of beta-cyclodextrin and sodium taurocholate

Chiral separation of 20 pairs of amino acids derivatized with fluoresceine-5-isothiocyanate (FITC) by capillary electrophoresis and laser-induced fluorescence detection was studied using the mixture of beta-cyclodextrin (beta-CD) and sodium taurocholate (STC) as selector. Resolution was considerably superior to that obtained by using either beta-CD or STC alone. The molar ratio of beta-CD to STC of about 2:3 was found to be critical to achieve maximum separation. At this beta-CD-to-STC ratio, chiral separation occurred at really low total concentration of beta-CD and STC (<0.1 mM). Other impacting factors were investigated including the total concentration of beta-CD and STC, pH, and capillary conditioning procedure between two successive runs. Using a running buffer of 80 mM borate containing 20 mM beta-CD and 30 mM STC at pH 9.3, all of the 20 pairs of FITC-amino acid enantiomers were baseline resolved. The resolutions of the most pairs of the amino acid enantiomers (17 of 20) were higher than 3.0, only three pairs gave a resolution lower than 3.0 but higher than 1.90 (beta-phenylserine, pSer). The highest resolution reached 14.58 (Glu). Two derivatives of beta-CD, 2-hydroxypropyl-beta-CD (HP-beta-CD) and heptakis(2,6-di-O-methyl)-beta-CD (DM-beta-CD) were also explored. HP-beta-CD showed similar cooperative effect with STC, while DM-beta-CD together with STC led to poorer chiral separation.     

Quantitation of chiral amino acids from microalgae by MEKC and LIF detection

In this work, chiral and nonchiral MEKC methods have been combined with LIF detection (MEKC-LIF) to identify and quantify a group of D- and L-amino acids (D/L-aa) in different microalgae samples. The combination of the nonchiral and chiral-MEKC-LIF methods made the identification of the microalgae amino acids easier, previously derivatized with FITC, providing a double proof on the correct detection of these analytes. Three microalgae species, Spirulina platensis, Dunaliella salina, and Tetraselmis suecica, were compared in terms of their content in D-Arg, L-Arg, D-Lys, L-Lys, D-Ala, L-Ala, D-Glu, L-Glu, D-Asp, and L-Asp. Also, a comparison between two Spirulina platensis samples dried under different conditions (i.e., hot air or lyophilized) was carried out in order to investigate the effect of the thermal processing on the amino acid content. Moreover, two procedures for the extraction of amino acids from microalgae (i.e., a classical procedure and pressurized liquid extraction (PLE)) together with different conditions for amino acid derivatization were studied in order to increase the sensitivity of the whole analytical method. By using the selected chiral-MEKC-LIF conditions (100 mM sodium tetraborate, 30 mM SDS, and 20 mM beta-CD at pH 9.7) the main microalgae D/L-aa are separated in less than 25 min with efficiencies up to 840 000 plates/m and good sensitivity (i.e., 330 ng of D-Arg per gram of microalga could be detected by this procedure for an S/N of 3). Several D-aa were detected in all the microalgae, observing interesting differences in their D/L-aa profiles, what corroborates the usefulness of the chiral-MEKC-LIF approach to characterize different microalgae species as well as different microalgae drying processes. Moreover, the use of PLE can selectively extract different free amino acids from microalgae.     

Enantiomeric separation of N-protected amino acids by non-aqueous capillary electrophoresis with dimeric forms of quinine and quinidine derivatives serving as chiral selectors

A simple method for analysis of anatoxin-a in aqueous samples was developed using solid-phase microextraction (SPME) and high-performance liquid chromatography (HPLC) with fluorescence detection. Anatoxin-a was derivatized to a fluorogenic agent on the surface of the SPME fiber. In the method an SPME fiber was immersed for 30 min in the aqueous sample. The fluorogenic derivatizing reagent (4-fluoro-7-nitro-2,1,3-benzoxadiazole, 1.0 mg/ml in methanol) was dropped or sprayed onto the fiber containing extracted analytes. The fiber was then heated for 10 min in an empty vial at 70 degrees C in a waterbath to promote derivatization. The derivatives formed on the fiber were desorbed in a SPME-HPLC interface. The interface was filled with methanol-1 mM hydrochloric acid (7:3, v/v) before inserting of the fiber into the interface. For desorption, the fiber was inserted in the interface for 5 min. For anatoxin-a in an aqueous sample, the calibration curve showed linearity in the range of 50-1500 ng/ml and the limit of detection of anatoxin-a was 20 ng/ml. No interferences were found, and the time for analysis was 55 min for one sample.     

Direct UV detection of underivatized amino acids using capillary electrophoresis with online sweeping enrichment

This is an original report proposed a CE method for direct analysis of the underivatized amino acids using UV detection with relatively higher sensitivity, which was based on coordination interactions between amino acids and Cu (II) ions. In addition, an online sweeping preconcentration technique was easily combined to improve the detection sensitivity. Satisfying separations of the amino acids were obtained under optimized conditions: 50 mmol/L CuSO₄–0.05% HAc–H₂O (pH 4.5), and the separation voltage of 15 kV. The LODs for the analytes ranged from 0.1 to 0.5 μmol/L. The linearity of detection for all analytes was two orders of magnitude with the correlation coefficients greater than 0.99. The repeatability was displayed with an RSD less than 3% for migration time and peak height (n = 5). Moreover, some amino acids in real samples of human saliva and green tea were analyzed by this direct UV detection CE method with acceptable sensitivity.     

CE coupling with end-column electrochemiluminescence detection for chiral separation of disopyramide

CE with electrochemiluminescence (ECL) detection technique was successfully applied for the chiral separation of a kind of class IA antiarrhythmic racemic drug. To the best of our knowledge, this is the first report of ECL detection used in chiral CE. To get better detection sensitivity and good enantioresolution at the same time, the conditions of capillary inlet and outlet buffer were systematically optimized. Unlike the traditional chiral separation method, the buffers we used in the capillary inlet and outlet differed from each other in terms of buffer pH, ionic strength, type of BGE as well as buffer composition. Under the optimum conditions, baseline enantioseparation and highly sensitive detection of the enantiomers were achieved. Wide linear relationship of each enantiomer was achieved in the range of 5 x 10(-7) to 2 x 10(-5) mol/L with relative coefficients of 0.996 and 0.997, respectively. The detection limits were estimated to be 8 x 10(-8) and 1.0 x 10(-7) mol/L (S/N = 3) for the enantiomers, respectively. In addition, a successful application of this new method to the chiral separation of the racemic drug in spiked plasma samples confirmed the validity and applicability of the chiral CE-ECL method.     

High-speed chiral separations on a microchip with UV detection

Fast chiral separations of a variety of basic and acidic compounds could be realized on microfluidic quartz chips. A microchip electrophoresis instrument equipped with a linear imaging UV-detector was used. The usually applied but troublesome fluorescence tagging in order to enable fluorescence detection could be omitted. Using sulfated cyclodextrins as chiral selectors baseline separation of 19 compounds could be achieved in less than 1 min with high reproducibility. The relative standard deviation of migration time was below 7%. The fastest separation could be performed in 2.5 s which is to date the fastest separation of enantiomers reported. It was possible to apply microchip electrophoresis (MCE) for the determination of high enantiomeric excess (ee) values, as exemplarily shown for pseudoephedrin where 2% of the minor enantiomer could reliably be determined beside high amount of the other isomer. Successful separation of a mixture of 3 chiral drugs could be performed in a single run in less than 11 s utilizing a separation length of only 12 mm. These results show that MCE has great potential for fast chiral analysis and high-throughput screening.     

Chiral resolution of tryptophan derivatives by CE using canine serum albumin and bovine serum albumin as chiral selectors

This work deals with the application of BSA and canine serum albumin (CSA) for enantioseparation of tryptophan derivatives with CE. The aim of this work was the investigation of the influence of different functional groups of tryptophan derivatives on enantioseparation. CSA as a chiral selector was tested to compare its selector properties with those of BSA. The enantiomers of the tryptophan derivatives were separated by adding BSA or CSA to the BGE. The influence of pH, temperature, BSA and CSA concentration and organic modifiers was investigated. It was found that the stereoselectivity for the different tryptophan derivatives is dependent on the albumin species. It turned out that the different functional groups of the derivatives showed a significant influence on stereoselectivity.     

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

A 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.     

Separation of enantiomers of norephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE and NMR studies

In this study, the enantiomer migration order (EMO) of norephedrine (NEP) in the presence of various CDs was investigated by CE. NMR and CE techniques were used to analyze the mechanism of the chiral recognition between NEP enantiomers and four CDs, i.e., native α-CD, β-CD, heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD), and heptakis(2,3-di-O-methyl-6-O-sulfo)-β-CD (HDMS-β-CD). EMO was reversed in the presence of α-CD and β-CD, although only minor differences in the structures of the complexes formed between NEP and these CDs could be derived from rotating frame nuclear Overhauser experiments (ROESY). The complexes between the enantiomers of NEP and the sulfated CDs, HDMS-β-CD, and HDAS-β-CD, were substantially different. However, EMO of NEP was identical in the presence of these CDs. HDAS-β-CD proved to be the most suitable chiral selector for the CE enantioseparation of NEP.     

Ternary mixed-ligand copper(II) complexes with chiral aminophosphonic acids and amino acids

Ternary mixed-ligand complexes of divalent copper, where one ligand is an -aminoalkylphosphonic acid (APA) and the second ligand is L-phenylalanine, L-tyrosine, or L-dihydroxyphenylalanine, are studied by potentiometric titration in aqueous medium at 25°C and =0.1. A maximum in the formation of the ternary complexes is exhibited at pH 7–8. The log K_st for the complexes formed and the statistical parameters characterizing the ternary complexes logK and logX are determined. The effect of substituents on the -carbon atom of the -APA and of hydroxy groups on the phenyl substituent of the -amino acids on the nature of the distribution of the complexes formed is studied.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 11, pp. 2482–2486, November 1989.The authors thank V. P. Solov'ev for helpful discussions.     

NMR and HPLC study of chiral selectors with naphthyl unit

Doubling of resonances in NMR spectra of chiral selectors with naphthyl group attached to the tertiary amide nitrogen atom has been noticed what revealed the presence of two isomers. To test the enantiorecognition ability of these chiral selectors they are covalently bonded to the HPLC silica gel. Those kind of chiral stationary phases were compared with analogous commercial leucine chiral stationary phase. They exhibit the better enantioseparation results which indicate that the existence of cis/trans isomers does not have the negative influence on their enantioselective ability.

Development of a chiral non-aqueous capillary electrophoretic system using the partial filling technique with UV and mass spectrometric detection

A chiral non-aqueous CE system with UV and mass spectrometric detection has been developed. The enantioseparation was promoted by diastereomeric complex (ion-pair) formation between the amines (e.g. salbutamol, atenolol) and the chiral selector, (-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid [(-)-DIKGA]. Different solvent mixtures were studied, as well as different concentrations of (-)-DIKGA and ammonium acetate in the background electrolyte. A partial filling technique was developed with a selector plug composed of (-)-DIKGA and ammonium acetate in a solvent mixture of methanol and 2-propanol. The separated enantiomers of pronethalol were detected by a Q-TOF MS system equipped with a sheath-flow electrospray ionization interface.     

Diversity-oriented synthesis of amino acids using chiral enolates

We report a facile diversity oriented synthesis of α- and β-amino acids, by utilizing the pluripotent α-methylene group in a chiral bicyclic lactam as our key point of transformation.     

Electrochemical detection of amino acids

Conclusion A method for the separation and the quantification of amino acids by high performance liquid chromatography and electrochemical detection using an isocratic mobile phase is possible and satisfactory.

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Elektrochemischer Nachweis von Aminosäuren