Enantiomeric separation of amino acids derivatized with fluoresceine isothiocyanate isomer I by micellar electrokinetic chromatography using β- and γ-cyclodextrins as chiral selectors (original) (raw)
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Journal of Chromatography A, 2010
A validated two-dimensional HPLC system combining a microbore-monolithic ODS column and a narrowbore-enantioselective column has been established for a sensitive and simultaneous analysis of hydrophilic amino acid enantiomers (His, Asn, Ser, Gln, Arg, Asp, allo-Thr, Glu and Thr) and the nonchiral amino acid, Gly, in biological samples. To accomplish this goal, the amino acids were first tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to the respective fluorescent NBD derivatives which were separated in the first dimension by a micro-reversed-phase column. The automatically collected fractions of the target peaks were then transferred to the second dimension consisting of a Pirkle type enantioselective column generating separation factors higher than 1.13 for all the enantiomeric target analytes. The system was validated using standard amino acids and a rat plasma sample, and analytically satisfactory calibration and precision results were obtained. The present 2D-HPLC system enables the fully automated determination of hydrophilic amino acid enantiomers in mammalian samples. The d-isomers of all the investigated 9 amino acids were found in rat urine but at various enantiomeric ratios.
Journal of Microcolumn Separations, 1999
A number of amino acids and derivatives, bought or synthesized, were Ž . analyzed by capillary electrophoresis CE with optical active 18-crown-6-tetra-Ž . carboxylic acid 18C6H as chiral selector. Besides the primary interaction, i.e., 4 inclusion of the protonated primary amino function in the crown ether cavity, a second interaction mechanism between the substituents on the racemates and the carboxylic acid functions on the crown ether to enhance or suppress enantioselec-Ž . tivity. In certain cases, very high resolutions R ) 5 were noted, e.g., R 30 for s s serine benzyl ester.
Electrophoresis, 2000
A solution is prepared of 5 mm silica particles modified with (S)-N-3,5-dinitrobenzoyl-1naphthylglycine (particle 1) or (S)-N-3,5-dinitrophenylaminocarbonyl-valine (particle 2) suspended in liquid tetraethylorthosilicate, ethanol, and aqueous hydrochloric acid. This solution is injected under pressure into a 30 cm long, 75 mm inner diameter capillary column and heated for 1 h at 120 o C after which the modified particles are embedded in a monolithic column of sol gel. The packed column measures approximately 15 cm from the inlet to the window used to view the laser-induced fluorescence. Thirteen different amino acids and three nonprotein amino acids are derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) before injection onto the column for capillary electrochromatographic separation. The enantiomeric separation of the monolithic column packed with particle 1 results in a resolution ranging from 1.14 to 4.45, whereas that packed with particle 2 results in a resolution ranging from 0.79 to 1.17. On the basis of resolution and amount of chiral packing material the enantiomeric separation obtained by capillary electrochromatography is judged to be superior to that obtained previously with high performance liquid chromatography (HPLC).
Application of TLC, HPLC and GC methods to the study of amino acid and peptide enantiomers: a review
Biomedical Chromatography, 2013
ABSTRACTAmino acids are very important organic compounds in nature. The biological activity of amino acids depends mainly on their stereoisomeric configuration (d‐ or l‐). Thus, the stereochemical analysis of amino acids and peptides is an important aspect of their characterization. Owing to the increasing role of amino acid configuration in biomedical and pharmaceutical studies, numerous analytical methods have been described in the literature. Among a wide range of analytical techniques available for the steroselective separation of different amino acids, which were obtained from plants or biological samples, chromatographic methods such as thin‐layer chromatography, high‐performance chromatography and also gas chromatography are very useful. This review presents such systems developed for direct stereoisomeric separation and quantitative determination of amino acid and peptide enantiomers with emphasis on selected literature published during two last decades. Almost all aspects, ...
Analytical and Bioanalytical Chemistry, 2007
A feasibility study on the fast enantioselective two-dimensional HPLC separation of racemic amino acid derivatives is presented. The method involves the on-line coupling of a narrow-bore C18 RP column in the first dimension to a short enantioselective column based on nonporous 1.5 μm particles modified with quinidine carbamate as chiral selector in the second dimension. Conceptually, the system was designed to enable both time-controlled repeated transfer of fractions of the eluate and detectorcontrolled transfer of selected fractions from column 1 to column 2. To avoid volume overloading of the second chiral column, a narrow-bore reversed phase column was installed in the first dimension. Due to the fast (less than 1.5 minutes) enantiomer separation that occurs in the second dimension, the overall analysis time for the twodimensional separation of a mixture of nine racemic 3,5dinitrobenzoyl amino acids was optimized at 16 minutes.
Separation of amino acid enantiomers VIA chiral derivatization and non-chiral gas chromatography
Journal of Chromatography A, 2008
Two GC-MS methods for the enantioselective separation of the 20 proteinogenic amino acids are compared. Ethyl chloroformate and 2chloropropanol 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.
Analytical and Bioanalytical Chemistry, 2012
Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of D-Asp in the central nervous system. To achieve appropriate detection sensitivity fluorescent derivatization with 4fluoro-7-nitro-2,1,3-benzoxadiazole and laser-induced fluorescence detection was applied. Although, simultaneous baseline separation of the two enantiomer pairs could be achieved by using 3 mM 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin (HPA-β-CD), further improvement of the chemical selectivity was required because of the high excess of L-enantiomers in real samples to be analyzed. The system selectivity was fine-tuned by combination of 8 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin and 5 mM HPA-β-CD in order to increase the resolution between aspartate and glutamate enantiomers. The method was validated for biological application. The limits of detection for D-Asp and D-Glu were 17 and 9 nM, respectively, while the limit of quantification for both analytes was 50 nM. This is the lowest quantification limit reported so far for NBD-tagged D-Asp and D-Glu obtained by validated capillary electrophoresis laser-induced fluorescence method. The applicability of the method was demonstrated by analyzing brain samples of 1-day-old chickens. In all the studied brain areas, the D-enantiomer contributed 1-2 % of the total aspartate content, corresponding to 17-45 nmol/g wet tissue.
Journal of Chromatography A, 2010
The work compares two GC-MS methods for enantioselective separation of amino acids as suitable candidate for stereochemical analysis of chiral amino acids on board spacecrafts in space exploration missions of solar system body environments. Different derivatization reagents are used: a mixture of alkyl chloroformate-alcohol-pyridine to obtain the alkyl alkoxy carbonyl esters and a mixture of perfluorinated alcohols and anhydrides to form perfluoroacyl perfluoroalkyl esters. 20 proteinogenic amino acids were derivatized with the two procedures and submitted to GC-MS analysis on a Chirasil-l-Val stationary phase. The results were then compared in terms of the enantiomeric separation achieved and intensity of MS response. The combination of methyl chloroformate (MCF) and heptafluoro-1-butanol (HFB) allows separation of 14 enantiomeric pairs, five of which display a resolution (R s ≥ 1.2) supposed to be sufficient to quantify the enantiomeric excess. Three mixtures of trifluoroacetic (TFAA) and heptafluorobutyric (HFBA) anhydrides were combined with the corresponding perfluorinated alcohols -TFE (2,2,2-trifluoro-1-ethanol) and HFB (2,2,3,3,4,4,4-heptafluoro-1-butanol) -to give three different reagents (TFAA-TFE, TFAA-HFB, HFBA-HFB): the derivatives obtained show separation of the same number of proteinogenic amino acids (14 of 20) at a temperature lower than column bleeding limit (200 • C) and 8 of them give a separation with R s ≥ 1.2. Linearity study and limit of detection (X LOD ) computation show that both methods are suitable for quantitative determination of several amino acid diastereomers at trace level (X LOD ≈ 0.5 nmol as derivatized quantity). Both the procedures were coupled with automatic data handling to increase their suitability for space analysis: the simplified data treatment is especially helpful to handle the low quality data recovered from space experiments and labor and time are saved, as imposed by the space experiments requiring a rapid delivery of the results. To achieve this aim, a chemometric approach based on the computation of the Autocovariance Function (ACVF) was applied to extract information on the enantiomeric pairs present in the sample and the enantioseparation achieved on the chiral column.