Ligands analytical chromatography

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),...

Although the majority of reports of macrocycles in analytical chromatography have involved ligand association with the stationary phase, their use as mobile phase constituents has also been investigated. Lamb and Drake [11] showed that addition of water-soluble crown ethers to the mobile phase altered the retention of alkali metal cations on an underivatized reversed phase column. Nakagawa et al. [63-66] also used crown ether-containing mobile phases in the separation of protonated amines, amino acids and peptides, and [1-lactam antibiotics. 

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]. 

The basic mechanism of separation of carbohydrates is by ligand exchange chromatography but is quite similar to ion-exclusion chromatography described earlier in this chapter for weak organic and inorganic acids. The column contains fully sulfonated polystyrene polymer beads cross-linked with polydivinylbenzene. The polymers are fully hydrated and contain occluded water within the gel polymer matrix, just as in ion-exclusion polymer beads. Analytes partition between the occluded water within the bead matrix and the mobile phase. Water is most often used as the mobile phase and the detection method is most often refractive index. 

Another form of ion chromatography is ligand exchange chromatography where a cation exchange resin is used to separate analytes that can form coordination complexes with the metal attached to the resin. 

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

Muckenschnabel, 1., Falchetto, R., Mayr, L.M., and Filipuzzi, 1. (2004) SpeedScreen label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands. Analytical Biochemistry, 324 (2), 241-249. 

Chiral Chromatography. Chiral chromatography is used for the analysis of enantiomers, most useful for separations of pharmaceuticals and biochemical compounds (see Biopolymers, analytical techniques). There are several types of chiral stationary phases those that use attractive interactions, metal ligands, inclusion complexes, and protein complexes. The separation of optical isomers has important ramifications, especially in biochemistry and pharmaceutical chemistry, where one form of a compound may be bioactive and the other inactive, inhibitory, or toxic. 

---