Amino acid enantiomers, chiral separation

Owing to the different biological activity of D- and L-enantiomers of seleno-amino acids, the chiral separation of optical isomers has been undertaken in sele-nized yeast and in yeast-based commercial supplements. Both, chiral stationary phase (crown ether) and chiral derivatization prior to reversed-phase HPLC were used [16, 77, 78],... 

As separation materials, the obvious advantage offered by MIPs is a relatively straightforward and predetermined selectivity. Based on molecular imprinting, some difficult separations, particularly enantiomer separations, have been solved. Amino acid enantiomers, chiral dipeptide, and racemic naproxen are examples. In particular, molecular imprinting is probably currently the only choice where no suitable biomolecule is available. When coupling with appropriate transducers such as electro-, photo- or magnetochemical transducers, MIPs may be applied as the monitors in several sys-... 

Berthod et al. also tried copper complexation with teicoplanin and TAG CSPs [19]. Similar results were obtained. Amino acid enantiomers perfectly separated by both teicoplanin and TAG CSP could no longer be separated as soon as copper was present in the mobile phase. The copper-teicoplanin complex is also formed with the primary amine group on the peptidic teicoplanin basket (Figs. 1 and 3). However, unlike the vancomycin-copper complex which was very stable [18], the teicoplanin-copper complex was found to be reversible. Indeed, amino acid enan-tioselectivity was mostly restored after washing the chiral column with several column volumes of copper-free clean mobile phase [19]. 

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

S Einarsson, B Josefsson, P Moller, D Sanchez. Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-l-(9-fluorenyl)ethyl chlo-roformate and reversed phase liquid chromatography. Anal Chem 59, 1191, 1987. 

Ilisz, I., Berkecz, R., and Peter, A., HPLC separation of amino acid enantiomers and small peptides on macrocyclic antibiotic-based chiral stationary phases a review, J. Sep. ScL, 29, 1305, 2006. 

Peyrin, E. et al., Dansyl amino acid enantiomer separation on a teicoplanin chiral stationary phase effect of eluent pH, J. Chromatogr. A, 923, 37, 2001. 

Amino acid enantiomers can be separated on a chiral stationary phase after derivatization with chloroformates (Abe et al., 1996). The derivatization procedure is quite simple and rapid, but the derivatizing reagent must be synthesized, which complicates the assay. Another method for the analysis of amino acid enantiomers uses N,0-pentafluoropropionyl isopropyl derivatives and a chiral column with NPD detection (Hashimoto et al., 1992). 

Analysis using a CMPA is usually resolved on a nonchiral column. A transient diastereomeric complex is formed between the enantiomer and the chiral component in the mobile phase, similar to the complexes formed with chiral stationary phases. A review by Liu and Liu (2002) cites several papers where addition of CPMAs has been used in analyzing amphetamine-related compounds. Some CPMAs include amino acid enantiomers, metal ions, proteins, and cyclodextrins. Advantages of this method of analysis include the use of less expensive columns and more flexibility in the optimization of chiral separation (Misl anova and Hutta, 2003). 

OPA in combination with chiral thiols is one method used to determine amino acid enantiomers. A highly fluorescent diastereomeric isoindole is formed and can be separated on a reverse-phase column. Some of these chiral thiols include N-acetyl-L-cysteine (NAC), N-tert-butyloxy-carbonyl- L-cysteine (Boc-L-Cys), N-isobutyryl- L-cysteine (IBLC), and N-isobutyryl- D -cysteine (IBDC). Replacing OPA-IBLC with OPA-IBDC causes a reversal in the elution order of the derivatives of D- and L-amino acids on an ODS column (Hamase et al., 2002). Nimura and colleagues (2003) developed a novel, optically active thiol compound, N-(tert-butylthiocarbamoyl)- L-cysteine ethyl ester (BTCC). This reagent was applied to the measurement of D-Asp with a detection limit of approximately 1 pmol, even in the presence of large quantities of L-ASP. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

Frank H, G. J. Nicholson and E. Bayer, Rapid gas chromatographic separation of amino acid enantiomers with a novel chiral stationary phase , J. Chromatogr. Sci. 15 174-176(1977). 

Analytical Properties Separation of amino acid enantiomers good chiral recognition of A/-acetyl amino acid methyl esters depends on hydrogen bond interactions hexane with isopropanol modifier has been used as the liquid phase usually prepared on LiChrosorb (10 pm)... 

Chiral separation of FITC-labeled amino acid enantiomers was performed on a glass chip using fluorescent detection. Analysis time ranged from 75 s for the most basic amino acids to 160 s for the most acidic ones. y-CD was used as the chiral selector [627]. Chiral separation of amino acids in extraterrestrial samples or meteorites were also performed [610,628],... 

R. Wernicke, Separation of underivatized amino acid enantiomers by means of a chiral solvent-generated phase, J. Chromatogr., 318 117(1985). 

As a further test of the etched open tubular approach for the analysis of optical isomers, another column was fabricated based on the selector naphthylethylamine that had been attached to porous silica by the silanization/hydrosilation method for use in HPLC [70]. As in the HPLC experiments, this column was best suited for the resolution of the optical isomers of dinitrobenzoyl methyl esters of amino acids. The best separation (a = 1.14) was obtained for the alanine derivative. In addition, the peak symmetry and efficiency for the naphthylethylamine column was significantly better than that obtained on the cyclodextrin column. However, as shown in HPLC experiments, changes in the amino acid moiety (replacing alanine with valine, etc.) often results in a loss of chiral resolution. In the case of optical isomers, the separation mechanism in HPLC and CEC modes is identical since only interaction between the solute and the bonded phase can result in resolution of the enantiomers. 

The FITC labeling method was also applied to chiral separations of amino acids on a microchip to determine the enantiomeric ratios of amino acids found on a meteorite [27], Since biotic amino acids are normally single enantiomers, chiral separations of amino acids are not truly clinical in nature, but illustrate the potential for chiral separations of small molecules of clinical interest. Ma-thies and co-workers used this technique to search for evidence of life in extraterrestrial environments. Enantiomeric forms of Val, Ala, Glu, and Asp could be discriminated by addition of a-, (3-, or y-cyclodextrin (CD) to the run buffer. Improved resolution with faster separations was found with respect to conventional CE. This method has been modified, by addition of SDS to the buffer, to perform cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) [28]. Increasing the SDS concentration decreased the magnitude of elec-troosmotic flow (EOF), increasing the effective migration distance, and therefore the resolution on the microchips. 

The resorcarene 23a, on the other hand, was obtained by O-alkylation of the corresponding octol. Its incorporation in a dimethylpolysiloxane backbone led to a stationary phase by which proteinogenic amino acids could be separated into their enantiomers by GC of their 7V(0,S)-trifluoroacetylmethylesters with separation factors aLD = 1.025-1.102.50 The question remains in this case (and in similar cases), whether the chiral amide functions have to be attached to the resorcarene skeleton, or if a direct attachment to the polymer backbone via suitable spacers would lead to similar results. The chiral resorcarene octaamides 23b prepared by... 

Type IV includes chiral phases that usually interact with the enantiomeric analytes through the formation of metal complexes. There are usually used to separate amino acid enantiomers. These types of phases are also called ligand exchange phases. The transient diastereomeric complexes are ternary metal complexes between a transitional metal (usually Cu +), an amino acid enantiomeric analyte, and another compound immobilized on the CSP which is able to undergo complexation with the transitional metal (see also the ligand exchange section. Section 22.5). The two enantiomers are separated based on the difference in the stability constant of the two diastereomeric species. The mobile phases used to separate such enantiomeric analytes are usually aqueous solutions of copper (II) salts such as copper sulfate or copper acetate. To modulate the retention, several parameters—such as the pH of the mobile phase, the concentration of the copper ion, or the addition of an organic modifier such as acetonitrile or methanol in the mobile phase—can be varied. 

Although enantiomer separation of amino adds has always been a domain of chiral diamide phases such as Chirasil-val (1] or XE-60-L-val- S)-a-phenylethylamide [2], the development of hydrophobic cyclodextrm derivatives has affected even this field of applications of enantioselective gas chromatography. Octakis(3-O-butyryl-2,6-di-0-pentyI)-7-cyclodextrin [Lipodex E , (32) (see footnote on p. 115)] is highly selective for amino acid enantiomers. Since hydrogen bonding is not necessary for a determination of the enantiomers, some unusual amino adds can be separated that could not be resolved before, including p-amino adds, a-alkylated, and N-alkyl-ated amino adds. 

The three general approaches to enantiomer separation entail a chiral stationary phase, a chiral mobile phase, or a chiral reagent. Tandem columns, with reversed and chiral stationary phases, were used to separate 18 D-L pairs of PTC-amino acids in 150 min. OPA-amino acid enantiomers have been separated on both ion-exchange and reversed-phase columns using a sodium acetate buffer with a L-proline-cupric acetate additive. Chiral reagents, such as Marphey s reagent and OPA/IBLC (A-isobutiril-L cysteine), were successfully used for racemization analysis within 80 min. 

T. Ueda, F. Kitamura, R. Mitchell, T. Metcalf, T. Kuwana, and A. Nakamoto, Chiral separation of naph-thalene-2,3-dicarboxaldehyde-labeled amino-acid enantiomers by cyclodextrin-modified micellar electro-kinetic chromatography with laser-induced fluorescence detection, Chem. 63 2919 (1991). 

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