Enantioseparation by chromatographic and electromigration techniques using ligand-exchange as chiral separation principle.

This article gives a short overview of the application of the principle of chiral ligand-exchange in HPLC, CE, and CEC. Since its introduction by Davankov, more than thousand articles have appeared in this field. Citing all these papers would extend the scope of this review-it would fill several books. Therefore only some milestones are mentioned in this article and it will focus on our own activities in this field. Some new developments are mentioned, and selected biochemical and biomedical application are presented.

Chiral separation of the ß2-sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies.

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5-dimethylphenylcarbamate)-coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid-liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors.

Enantioseparation of amino acids and alpha-hydroxy acids on ligand-exchange continuous beds by capillary electrochromatography.

A new chiral stationary phase based on continuous bed (CB) technology using L-prolinamide as a chiral selector was prepared. Its ability for enantioseparation of amino acids and alpha-hydroxy acids by ligand-exchange CEC was compared with that of a CB containing L-4-hydroxyproline as chiral selector. Preparation was done by a one-step in situ copolyermerization procedure using methacrylamide as monomer, vinylsulfonic acid as charge providing agent and piperazine diacrylamide as crosslinker. L-Prolinamide showed marked enantioselective properties for the separation of amino acids and alpha-hydroxy acids. Furthermore, we show the possibility of simultaneous enantioseparation of amino acids and alpha-hydroxy acids using the CB with L-4-hydroxyproline as chiral selector.

Enantioseparation of amino acids, alpha-hydroxy acids, and dipeptides by ligand-exchange CEC using silica-based chiral stationary phases.

This work deals with the application of silica-based ligand-exchange chiral stationary phases (CSPs) for the enantioseparation of underivatized amino acids, alpha-hydroxy acids, and dipeptides with packed CEC. Two different possibilities of preparing silica-based CSPs are presented. One phase contains L-4-hydroxyproline chemically bonded via a spacer to 3 mum silica material. The other approach makes use of N-decyl-L-4-hydroxyproline dynamically coated on a reversed-phase packed capillary. Dynamical coating of reversed-phase material represents a simple alternative to prepare CSP. A comparison of the chemically bonded phase with the dynamically coated CSP by means of resolution of complex-forming analytes is presented. The chemically bonded phase was found to be superior to the dynamically coated phase in terms of resolution of amino acids and dipeptides. However, the dynamically coated CSP was found to be especially suitable for the separation of alpha-hydroxy acids. Both techniques are applicable for enantiomer purity tests.

Chiral separation by capillary electromigration techniques.

This review gives an overview of chiral separation principles and their applications in capillary electromigration techniques. The basic chiral separation principles are explained and the mechanisms discussed. Recent developments and new techniques in CZE and capillary electrochromatography (CEC) are highlighted. New chiral selectors among cyclodextrins, crown ethers, carbohydrates, macrocyclic antibiotics, proteins, chiral ion-pairing reagents, chiral surfactants and chiral metal ion complexes and their chiral recognition ability are discussed. Recent advances in chip technology for chiral separation and new approaches regarding improvement of detection sensitivity are presented. Due to the tremendous number of publications dealing with applications, in this review only recent applications are summarized.

Chiral separation of amino acids and glycyl dipeptides by chiral ligand-exchange capillary electrophoresis comparing Cu(II), Co(II), Ni(II) and Zn(II) complexes of three different sugar acids.

This paper deals with comparative studies on the use of copper(II), cobalt(II), nickel(II) and zinc(II) complexes of d-gluconic acid, d-saccharic acid and l-threonic acid as chiral selectors for the enantioseparation of aromatic amino acids and glycyl dipeptides using the principle of ligand-exchange capillary electrophoresis. Although copper(II) is the most frequently used central ion in ligand-exchange capillary electrophoresis, in the case of d-gluconic acid cobalt(II) was shown to be an alternative for the enantioseparation of amino acids. Glycyl dipeptides, however, were resolved only with copper(II) complexes. Zn(II) as a central ion was not effective in all cases and with Ni(II) only some partial separations were achieved.

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.

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.

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.

Advances in chiral separation using capillary electromigration techniques.

This review gives an overview of recent developments in CZE, EKC, and CEC covering the literature since the year 2004. Since there appeared a special issue on applications, this review focuses on the progress in electromigration techniques and new methodological developments. New techniques, new chiral selectors as well as new chiral stationary phases for CEC are discussed.

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.

Fast chiral separation by ligand-exchange HPLC using a dynamically coated monolithic column.

The preparation and application of dynamically coated ligand-exchange chromatography phases for enantioseparation is described. The phases were prepared by pumping a solution of N-decyl-L-4-hydroxyproline, N-hexadecyl-L-4-hydroxyproline, or N-2-hydroxydodecyl-L-4-hydroxyproline through a commercially available monolithic RP-18 column. These coatings are stable against desorption for months at ambient temperature when aqueous mobile phases are used. The columns were applied to the chiral separation of amino acids, glycyl dipeptides and diastereomeric dipeptides, and tripeptides. The chiral selector can be removed or changed easily by washing the column with ACN or methanol. Ultrafast separations in the range of seconds were achieved using high flow rates.

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.

Enantioseparation of glycyl-dipeptides by CEC using particle-loaded monoliths prepared by ring-opening metathesis polymerization (ROMP).

Novel particle-loaded monolithic capillary electrochromatography (CEC) phases for chiral separations were prepared via ring-opening metathesis polymerization (ROMP) within the confines of fused silica columns with 200 microm i.d. using norborn-2-ene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8,exo,endo-dimethanonaphthalene (DMN-H6) as monomers, 2-propanol and toluene as porogens, RuCl2(PCy3)2(CHPh) as initiator and silica-based particles containing the chiral selector. By suspending silica particles bearing the chiral selector in the polymerization mixture, particle-based monoliths are easily prepared. This approach has several advantages compared to particle-based separation media: (i) the concept of particle-based monoliths is broadly applicable, as any silica-based chiral phase can be used; (ii) they are inexpensive to prepare; and (iii) the manufacturing process is very simple, no sophisticated packing procedures or the preparation of end frits are required. To show the usefulness of this concept for chiral CEC, the chiral separation performance of particle-loaded CEC monoliths bearing teicoplanin aglycone, chemically bonded to 3 microm silica gel, was investigated for a set of glycyl-dipeptides. Particle-loaded ROMP CEC monoliths showed good separation performance for glycyl-dipeptides.

Chiral separation principles in chromatographic and electromigration techniques.

Almost half of the drugs in use today are chiral. It is well established that the pharmacological activity is mostly restricted to one of the enantiomers (eutomer). There can be qualitative and quantitative differences in the activity of the enantiomers. In many cases, the inactive enantiomer (distomer) shows unwanted side effects or even toxic effects. Even if the side effects are not that drastic, the distomer has to be metabolized and this represents an unnecessary burden for the organism. Therefore, the development of methods for the separation of enantiomers, both on analytical and preparative scale, has become increasingly important. Chromatographic techniques such as thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), and above all high-performance liquid chromatography (HPLC) have been used for enantiomer separation for about two decades. More recently, electromigration techniques, such as capillary electrophoresis and capillary electrochromatography, have been shown to be powerful alternatives to chromatographic methods. This review gives a short overview of different chiral separation principles and their application. Several new developments are discussed.

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.

Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography.

This review summarizes recent developments in chiral separation in capillary zone electrophoresis (CZE), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC) covering literature published since the year 2000. New chiral selectors and innovative approaches for CE and CEC are introduced. Recent progress in column technology for CEC is highlighted and the development of new chiral stationary phases is discussed. This review is not dedicated to list applications but will focus on new developments.

Enantioseparation of dipeptides and tripeptides by micro-HPLC comparing teicoplanin and teicoplanin aglycone as chiral selectors.

Chiral separation of glycyl- and diastereomeric dipeptides and tripeptides was performed by micro-HPLC using macrocyclic antibiotics as chiral selectors. Teicoplanin was compared with teicoplanin aglycone (TAG) regarding selectivity, efficiency and separation time. The stationary phases are based on teicoplanin and TAG chemically bonded to 3.5 mum silica gel. The material was packed into 10 cm x 1 mm stainless steel microcolumns. Different mobile phases were checked using the reversed phase mode. Both teicoplanin and TAG were found to show good chiral separation ability for dipeptides. Glycyl-dipeptides were baseline resolved and most of the diastereomeric dipeptides and tripeptides were separated into their four isomers. In this study, teicoplanin was found to be advantageous compared to TAG regarding separation time, although TAG showed the higher resolution power. Baseline resolution for some glycyl-dipeptides was obtained within 3 min, diastereomeric dipeptides were resolved in 7 min. This method was also shown to be applicable for enantiomer purity control.

Chiral separation of natural and unnatural amino acid derivatives by micro-HPLC on a Ristocetin A stationary phase.

This paper deals with the chiral separation of Fmoc- and Z-derivatives of natural and unnatural sulfur containing amino acids by micro-HPLC. The separations were carried out in microbore columns packed with a new material based on Ristocetin A bonded to 3.5 microm silica gel. The columns were run in the normal phase, polar organic mode as well as in the reversed phase mode, whereby best results were obtained with the reversed-phase mode using mixtures of triethylamine acetate (TEAA) buffer and methanol as mobile phases.

Determination of lipoic acid in dietary supplement preparations by capillary electrophoresis.

The present study deals with the development of a method for the quantitative determination of lipoic acid in a dietary supplement preparation. A rapid capillary electrophoretic method is developed using UV detection at 208 nm. Although lipoic acid is only weakly UV-absorbing, at this wavelength it could be detected with sufficient sensitivity with an LOD and LOQ of 0.8 and 2.5 microg/ml, respectively. Analysis time was less than 9 min. The compound was extracted from tablets with a recovery of 98.3% and a precision of 2.8% RSD.