Ligand-exchange chromatography amino acids

Achiral Columns Together with Chiral Mobile Phases. Ligand-exchange chromatography for chiral separation has been introduced (59), and has been appHed to the resolution of several a-amino acids. Prior derivatization is sometimes necessary. Preparative resolutions are possible, but the method is sensitive to small variations in the mobile phase and sometimes gives poor reproducibiUty. 

Galli, B. et al., Enantiomeric separation of DNS-amino acids and DBS-amino acids by ligand exchange chromatography with (5) - and (R)-phenylalaninamide modified silica gel, J. Chromatogr. A, 666,11, 1994. 

Chiral ligand-exchange chromatography (CLEC) ° separates enantiomers by the formation of diastereomeric metal complexes. In a first instance the technique was mainly used for the separation of amino acids. Impressive results of the first separations gave rise to intensive investigation in the field and numerous publications appeared in the literature, which have been reviewed. 

Based on preliminary results from Helfferich130, further developments by Davankov and co-workers5 131 133 turned the principle of chelation into a powerful chiral chromatographic method by the introduction of chiral-complex-forming synlhetie resins. The technique is based on the reversible chelate complex formation of the chiral selector and the selectand (analyte) molecules with transient metal cations. The technical term is chiral ligand exchange chromatography (CLEC) reliable and complete LC separation of enantiomers of free a-amino acids and other classes of chiral compounds was made as early as 1968 131. 

Figure 19. Resolution of analytes by chiral ligand exchange chromatography (CLEC). A hydroxy acids (reprinted with permission from ref 138) B dansyl amino acids (reprinted with permission from ref 139),...

C Tao, TB Huang. Resolution of DL-a-amino acids on a L-hydroxyproline chiral phase by ligand-exchange chromatography. Chin Chem Lett 6 383-384, 1995. 

A modification of the pyridoxal—amino acid reaction (mentioned above) has been made for automatic analysis of amino acids by ligand-exchange chromatography [95]. This technique involves separation of the amino acids prior to fluorimetric reaction and determination. As the amino acids are eluted from the column, they are mixed with the pyridoxal-zinc(II) reagent to produce a highly fluorescent zinc chelate. Amounts of as low as 1 nmole of amino acid may be detected. The first reaction involved is the formation of the pyridoxyl-amino acid (Schiff base) as in Fig.4.46. The zinc then forms a chelate which probably has the structure shown in Fig. 4.48. 

V. A. Davankov and A. A. Kurganov, The role of achiral sorbent matrix in chiral recognition of amino acid enantiomers in ligand-exchange chromatography, Chromatographia, 77 696 (1983). 

Chiral ligand-exchange chromatography resolves enantiomers on the basis of their ability to complex with transition metal ions, such as copper, zinc, and cadmium, as illustrated by the separation of amino acid racemates using copper102 (Fig. 2.21). The principle of exchange is similar to that... 

Further examples of separation techniques that exploit the asymmetric distribution of amino acid residues at the surface of folded proteins include metal ion affinity chromatography (HP-IMAC), ligand exchange chromatography (HP-LEC), immunoaffinity chromatography (HP-IAC), hydrophilic chromatography (HP-HILIC), and the various modes of biospecific (HP-BAC), and biomimetic (HP-BMC) chromatography. For example, the... 

Ligand exchange chromatography is a very powerful method for separating enantiomers. However, it is limited to enantiomeric compounds that are able to undergo metal complexes with the chiral stationary phase such as amino acids, amino acid derivatives, and amino alcohols. 

For a quite long period of time, chiral ligand-exchange chromatography (CLEC) has been the standard method for the enantioseparation of free amino acids. Meanwhile, other methods became available for these target molecules, such as teicoplanin or chiral crown-ether-based CSPs. However, for the enantioseparation of aliphatic a-hydroxy carboxylic acids, it is still one of the most efficient methods. 

Chiral ligand-exchange chromatography is based on the formation of diastereomeric ternary complexes that involve a transition metal ion (M), usually copper II a single enantiomer of a chiral molecule (L), usually an amino acid and the eitantiomers of the racemic solute R and S). The diastereomeric mixed chelate complexes formed in this system are represented by the formulas L-M-R and L-M-S. When these complexes have different stabilities, the less stable complex is eluted first, and the enantiomeric solutes are separated. 

The most important technique for enantiomeric separation in TLC is chiral ligand-exchange chromatography (LEC). LEC is based on the copper(II) complex formation of a chiral selector and the respective optical antipodes. Differences in the retention of the enantiomers are caused by dissimilar stabilities of their diastereomeric metal complexes. The requirement of sufficient stability of the ternary complex involves five-membered ring formation, and compounds such as a-amino and a-hydroxy-acids are the most suitable. 

In ligand-exchange chromatography (LEC), the separation of analytes is due to the exchange of ligands from the mobile phase with other ligands coordinated to metal ions immobilized on a stationary phase. LEC has been used successfully for the resolution of free amino acids, amino acid derivatives, and for enantiomeric resolution of racemic mixtures [3]. 

Blackwell and Carr investigated the ligand exchange chromatography of free amino acids on copper-loaded zirconia. It was shown that the use of Lewis base buffers in this system improved the operating efficiency. Acetate, sulfate, fluoride, and phosphate are the effective competing ions. 

Blackwell, J.A. Carr, P.W. Ligand-exchange chromatography of free amino-acids and proteins on porous micro- 39. particulate zirconium-oxide. J. Liq. Chromatogr. 1992,15, 1487-1506. 

V. I., Dostavalov, I. N., Myasoedov, N. F. Ligand exchange chromatography for analysis and preparative separation of tritium-labeled amino acids,/. Radioanal. Nud. Ch., 1988,121, 469-478. 

A cross-linked polystyrene resin with fixed ligands of the type (R)-N, N -dibenzyl-l,2-propanediamlne In the form of a copper (II) complex displays high enantloselectlvlty In ligand-exchange chromatography of amino acids (11). A microbore column (100 mm X 1 mm i.d.) packed with particles of dp 5-10 ym generated up to 3500 theoretical plates, enabling a complete resolution of a mixture of three racemic amino acids Into six components under isocratlc conditions. 

Chemically modified layers CHIRalplate Enantiomer separation based on ligand exchange chromatography Chiral amino acids, a-hydroxy-carboxyhc acids and other compounds which can form chelate complexes with Cu(II) ions... 

Chiral ligand exchange chromatography utilizes immobilized tremsition metal complexes that selectively bind one enantiomer of the analyte, which is usually an amino acid. 

Siegel, A. and Degens, E.T., 1966. Concentration of dissolved amino acids from saline waters by ligand exchange chromatography. Science, 151 1098—1101. 

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