Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC.

This paper deals with the chiral separation of triiodothyronine (T3) and thyroxine (T4) by HPLC and micro-HPLC. The separation of T3 and T4 is of great pharmaceutical and clinical interest, since the enantiomers exhibit different pharmacological activities. The HPLC measurements were performed on a chiral stationary ligand-exchange phase using l-4-hydroxyproline bonded via 3-glycidoxypropyltrimethoxysilane to silica gel as a selector. Also a chiral teicoplanin (Chirobiotic) phase was used. In micro-HPLC the chiral separation behaviour of l-4-hydroxyproline, and of the macrocyclic antibiotics teicoplanin and teicoplanin aglycone was investigated for the enantioseparation of T3 and T4. l-4-Hydroxyproline was bonded to 3 microm and the glycopeptide antibiotics were bonded to 3.5 microm silica gel and separations were accomplished by microbore HPLC columns (10 cmx1 mm I.D.). With both techniques and all chiral selectors investigated T3 and T4 were baseline resolved. micro-HPLC was found to be superior to analytical HPLC with respect to low consumption of packing material, mobile phase and analyte.

Enantioseparation by ligand-exchange using particle-loaded monoliths: capillary-LC versus capillary electrochromatography.

Particle-loaded monoliths containing a polymethacrylamide backbone were prepared by suspending a silica-based chiral phase in the mixture of the monomers followed by in-situ polymerization in the capillary. As chiral selector l-4-hydroxyproline chemically bonded to 3 microm silica particles was used following the separation principle of ligand-exchange. Electrolytes containing Cu(II) ions were used. Amino acid enantiomers were separated by capillary-LC and CEC, whereby the latter showed the better resolution properties. For the chiral separation of alpha-hydroxy acids the EOF was reversed by copolymerizing diallyldimethylammonium chloride instead of vinylsulfonic acid as charge providing agent. Short columns of 6 cm were found to be sufficient in the case of CEC for baseline separations of amino acids with alpha values up to 5.

Chiral separation of T3 enantiomers using stereoselective antibodies as a selector in micro-HPLC.

This work deals with the application of stereoselective antibodies against L-T3 as a tailor-made chiral selector in micro-HPLC. The separations were performed in microbore columns using commercially available anti-L-T3 antibodies chemically bonded to 5 microm silica gel. The enantiomers of T3 were baseline separated under mild continuous isocratic elution conditions using 10 mM phosphate buffer, pH 7.4. The D-enantiomer eluted with the void volume, while the L-enantiomer was retained by the antibody phase and eluted second. An indirect competitive and non-competitive enzyme linked immunosorbent assay (ELISA) was used for testing the stereoselectivity of anti-L-T3 antibodies.

Chiral separation of sympathomimetics and beta-blockers by ligand-exchange CE using Cu(II) complexes of L-tartaric acid and L-threonine as chiral selectors.

This paper deals with the use of Cu(II) complexes of L-tartaric acid or L-threonine as selectors for the chiral separation of drugs containing amino alcohol structure by ligand-exchange CE. Using Cu(II) ions as a complexing agent, a series of sympathomimetics and beta-blockers were resolved. It was found that the resolution strongly depends on selector concentration and pH. The optimum pH for complexation was 12.

Chiral resolution of tryptophan derivatives by CE using canine serum albumin and bovine serum albumin as chiral selectors.

This work deals with the application of BSA and canine serum albumin (CSA) for enantioseparation of tryptophan derivatives with CE. The aim of this work was the investigation of the influence of different functional groups of tryptophan derivatives on enantioseparation. CSA as a chiral selector was tested to compare its selector properties with those of BSA. The enantiomers of the tryptophan derivatives were separated by adding BSA or CSA to the BGE. The influence of pH, temperature, BSA and CSA concentration and organic modifiers was investigated. It was found that the stereoselectivity for the different tryptophan derivatives is dependent on the albumin species. It turned out that the different functional groups of the derivatives showed a significant influence on stereoselectivity.

Chiral separation of halogenated amino acids by ligand-exchange capillary electrophoresis.

The chiral separation of halogenated amino acids by ligand-exchange CE is described. Halogenated amino acids attracted increasing interest in recent years because of their physiological activities. Different chiral selectors, as there are L-4-hydroxyproline, L-histidine, and N-alkyl derivatives of L-4-hydroxyproline in form of their copper(II) complexes, are compared for their chiral recognition ability for halogenated amino acids. The influence of various parameters, such as selector concentration, pH, organic modifier, and field strength, on the resolution was investigated. All halogenated amino acids investigated were baseline-separated under optimized conditions.

New particle-loaded monoliths for chiral capillary electrochromatographic separation.

Fritless particle-loaded monoliths for chiral capillary electrochromatographic (CEC) separation were prepared. Silica particles containing a chiral selector are suspended in a monomer solution, which is drawn into the capillary followed by in situ polymerization. Thereby the silica-based particles containing the chiral selector are embedded in a nonchiral continuous bed. This kind of chiral stationary phase is inexpensive, easy, and reproducible to prepare and circumvents the preparation of frits. As a model, teicoplanin aglycone as chiral selector bonded to 3 microm silica particles was used. The applicability of this approach is demonstrated by means of the chiral separation of aliphatic and aromatic amino acids and dipeptides. As a further application, the chiral selector ristocetin A bonded to 3 microm silica particles was used for the enantiomeric separation of chiral alpha-hydroxy acids. Since alpha-hydroxy acids migrate toward the anode, a cationic charge-providing agent was copolymerized with the matrix. This served to reverse the direction of the electroosmatic flow (EOF).