Simultaneous chirality separation of amino acids and their derivative by natamycin based on mobility measurements

Direct chiral separation of amino acids derivatized with 2-(9-anthryl)ethyl chloroformate by capillary electrophoresis using cyclodextrins as chiral selectors Effect of organic modifiers on resolution and enantiomeric elution order

Enantioselective recognition and chiral separation of amino acids hold significant importance in chemistry, materials science, and life science. Here, we report a water-soluble chiral fluorescent probe that enables visual chiral recognition and separation by incorporating a morpholinium quaternary cation into the 1,1’-bi-2-naphthol frameworks. Upon binding with free amino acid enantiomers, the probe achieves rapid chiral discrimination within 100 s, accompanied by distinct changes in luminescence color or intensity. The underlying mechanism of this chiral recognition involves imine formation and electrostatic interactions, accompanied by aggregation-induced emission. These processes collectively promote selective aggregation and precipitation between the probe and specific enantiomers of amino acids. Furthermore, the enantiomers can be efficiently separated from D-/L- amino acid mixtures through a simple filtration process. Comparative analyses using a fluorescence visualization and chiral high performance liquid chromatography further validate the probe’s efficacy in achieving efficient chiral separation. This study provides a practical approach for the precise detection and separation of amino acid enantiomers.

Chiral analysis is critical to many research fields due to different biological functions of enantiomers in living systems. Although the use of ion mobility spectrometry (IMS) has become an alternative technology in the area of chiral measurements, there is still a lack of a general chiral selector for IMS-based chiral recognition, especially for small chiral molecules. Here, a new method using oligosaccharides as the chiral selector has been developed to discriminate chiral amino acids (AAs) by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). We analyzed 21 chiral amino acids, including small molecules (e.g., alanine and cysteine). Our data showed that the use of nonreducing tetrasaccharides was effective for the separation of chiral AAs, which differentiated 21 chiral AAs without using metal ions. By further incorporating a copper ion, the separation resolution could be improved to 1.64 on average, which accounts for an additional 52% improvement on top of the already achieved separation in metal-free analysis. These results indicate that the use of tetrasaccharides is an effective strategy for the separation of AA enantiomers by TIMS. The method developed in this study may open up a new strategy for effective IMS-based chiral analysis.

Chiral separation of amino acids and peptides by capillary electrophoresis

Investigation of hydroxypropyl-β-cyclodextrin-based synergistic system with chiral nematic mesoporous silica as chiral stationary phase for enantiomeric separation in microchip electrophoresis

In situ instrumentation that can detect amino acids at parts-per-billion concentration levels and distinguish an enantiomeric excess of either d- or l-amino acids is vital for future robotic life-detection missions to promising targets in our solar system. In this article, a novel chiral amino acid analysis method is described, which reduces the risk of organic contamination and spurious signals from by-products by avoiding organic solvents and organic additives. Online solid-phase extraction, chiral liquid chromatography, and mass spectrometry were used for automated analysis of amino acids from solid and aqueous environmental samples. Carbonated water (pH ∼3, ∼5 wt % CO2 achieved at 6 MPa) was used as the extraction solvent for solid samples at 150°C and as the mobile phase at ambient temperature for chiral chromatographic separation. Of 18 enantiomeric amino acids, 5 enantiomeric pairs were separated with a chromatographic resolution >1.5 and 12 pairs with a resolution >0.7. The median lower limit of detection of amino acids was 2.5 μg/L, with the lowest experimentally verified as low as 0.25 μg/L. Samples from a geyser site (Great Fountain Geyser) and a geothermal spring site (Lemon Spring) in Yellowstone National Park were analyzed to demonstrate the viability of the method for future in situ missions to Ocean Worlds.

Previous work has demonstrated that copper complexation strategies can be used with tandem MS (MS/MS) and, more recently, ion mobility-mass spectrometry (IM-MS) to differentiate chiral isomers based upon enantiomeric-specific binding. In this study, we investigate the separation of chiral amino acids (AAs) forming trinuclear complexes that can be directly resolved by IM-MS analyses. Twenty standard AAs of both d- and l-chirality were investigated. Specific AAs including d/l-histidine, d/l-proline, d/l-glutamine, d/l-tyrosine, and d/l-tryptophan were evaluated as "chiral selectors" that, when combined with copper, were found to promote selective complexation with specific AA enantiomers. Significant enantiomer differentiation was observed in the IM spectra for hydrophobic AAs acids with peak-to-peak resolutions ranging from 0.63 to 1.15. Among the chiral selectors investigated, histidine provided the best enantioselectivity, followed by tryptophan, suggesting the aromatic structure plays an important role in forming chiral-specific ion complexes. Unlike MS/MS methods where chiral selectors with l-stereochemistry enhance the differentiation, the chirality of the selector was found to have no significant effect on observed IM separation with both d- and l-selectors providing similar resolutions but with inverted IM arrival time ordering. To investigate the structural differences between resolvable chiral complexes, a combination of MS/MS, collision cross-section (CCS) measurements, and molecular mechanics techniques was used. Candidate trinuclear structures of the stoichiometry [(Cu2+)3(d/lIle)3(lHis)2 - 5H]+ were constructed with guidance from empirical MS/MS results. Of the 48 theoretical structures generated, one enantiomeric cluster pair yielded close correlation (<1%) with experimental CCS measurements, suggesting the most enantioselective ion complexes observed in this work are bridged by three coppers.

Recent advances in chiral separation of amino acids using capillary electromigration techniques

Chiral distinction of phenyl-substituted ethanediol enantiomers by measuring the ion mobility of their ternary complexes

(+)-(18-Crown-6)-tetracarboxylic acid (18C6H4) is used as a chiral selector for various amino acids, where the l-isomer is usually eluted prior to the d-isomer in HPLC using 18C6H4-linked column. To clarify the structural scaffold of (+)-18C6H4 responsible for chiral separation of amino acids, we have previously investigated the interaction mode between (+)-18C6H4 and amino acids using X-ray analysis. However, no conclusive results could be obtained to explain the reverse elution order in the case of serine and to establish a general separation rule of chiral amino acid by (+)-18C6H4 in HPLC. Thus, to clarify the exceptional result obtained with serine and to set a general separation rule, interaction between (+)-18C6H4 and α-amino-n-butyric acid, valine, and alanine, as methyl substitutes of the methyl groups for the hydroxy groups of serine and threonine, and the simplest chiral amino acid, respectively, was investigated both in solution and solid states. Consequently, it was found that an asymmetric bowl-like conformation of (+)-18C6H4 is necessary for chiral separation. This conformation is constructed by chiral-specific interaction between the Cα–H groups of the amino acid and the polar oxygen atoms of (+)-18C6H4. It was also found that the exceptional reverse elution observed with serine is due to additional interaction between the polar groups of the amino acid side chain and (+)-18C6H4.

3-[(3-cholamidopropyl)-dimethylammoniol-1-propane sulfonate (CHAPS) can be used as an effective chiral selector for the separation of dansyl-amino acids by capillary electrophoresis (CE). While CHAPS can serve as an chiral selector, better enantiomeric separation can be performed by using CHAPS not as the sole chiral selector but as one of a [CHAPS-SDS-cyclodextrin] three-component system. In this CHAPS-SDS-CD system, enantiomeric separations of the amino acids can be readily accomplished by judiciously adjusting the pH of the solution, concentrations of CHAPS and SDS, and the concentration and type of CD. All amino acids can be baseline resolved in less than 15 minutes with resolution as high as 2.01 at pH 6.5 with 50 mM of CHAPS and 75 mM of SDS. The resolution is also dependent on the size of the CD. Substantial increase in the resolution can be readily achieved by replacing β-CD with γ-CD. For example, theR s for Leu was increased by four-folds (from 1.65 to 6.29) while the elution time still remains as short as 20 min when β-CD was replaced by γ-CD.

A chiral ligand-exchange phase for capillary electrochromatography based on continuous bed technology was developed. The chiral stationary phase is prepared by a one-step in situ copolymerization procedure using methacrylamide, piperazine diacrylamide, vinylsulfonic acid and N-(2-hydroxy-3-allyloxypropyl)-L-4-hydroxyproline. These chiral continuous beds are inexpensive and easy to prepare. They also have several advantages over silica-based packed capillaries. Since the bed is covalently attached to the capillary wall, no frit is required. The applicability of this new approach to the chiral separation of underivatized amino acids is demonstrated.

Ultrafast simultaneous chiral analysis of native amino acid enantiomers using supercritical fluid chromatography/tandem mass spectrometry

Chiral separation of amino acids by copper(II) complexes of tetradentate diaminodiamido-type ligands added to the eluent in reversed-phase high-performance liquid chromatography: a ligand exchange mechanism

Chiral separation of amino acids by capillary electrophoresis with octyl-beta-thioglucopyranoside as chiral selector.