Phospholipid-protein coatings for chiral capillary electrochromatography

A phospholipid-bovine serum albumin (BSA) coating was developed for chiral capillary electrochromatographic separation of d- and l-tryptophan. Temperature, liposome composition, and liposome-BSA mixing and extrusion were found to have critical effects on the chiral separation of d- and l-tryptophan in terms of resolution, separation efficiency, and migration times. A solution of 0.5mM phosphatidylcholine (PC)-1 mg/ml BSA performed better than a solution of 0.5mM PC/phosphatidylserine (PS) (80:20, mol%)-1 mg/ml BSA as capillary coating; baseline separation of the enantiomers with satisfactory resolution was then achieved. Temperature played a crucial role in the chiral separation, as demonstrated for phospholipid-coated capillaries immobilized with BSA and lysozyme. The d- and l-tryptophans showed a marked difference in separation efficiency on the PC-BSA-coated capillary; the theoretical plate number of l-tryptophan was above 500,000 m(-1), whereas that of d-tryptophan was only about 22,000 m(-1). Immobilized BSA (pI 4.7) showed better chiral separation selectivity for the enantiomers than did immobilized lysozyme (pI 10.5), alpha-chymotrypsin (pI 8.1-8.3), or avidin (pI 10.0-10.5); also resolution was better and analysis time was faster. Hydrophobic interactions played an important role in the BSA-immobilized phospholipid-coated capillaries. The importance of protein net charge and molar mass for its immobilization in phospholipid-coated capillaries is discussed.     

Recent advances in enantiomer separations by affinity capillary electrophoresis using proteins and peptides

Enantiomer separations by capillary electrophoresis (CE), using proteins as chiral selectors--affinity capillary electrophoresis (ACE) with free solutions and capillary electrochromatography (CEC)--with protein immobilized capillaries, are reviewed. The separation principle, recent advances in this field and some interesting topics are presented. In ACE, various enantiomer separations have been already reported using either plasma proteins or egg white ones. Miscellaneous proteins were also explored in the last few years. On the contrary, only a limited number of enantiomer separations have been successfully achieved in CEC. CEC is not yet mature enough to date, and further investigations, such as efficiency, durability and reproducibility of capillaries, will be necessary for the use of routine analyses. The study of enantioselective drug-protein binding is important in pharmaceutical developments. Some applications including high-performance CE/frontal analysis (HPCE/FA) are introduced in this paper.     

Molecularly imprinted polymer formats for capillary electrochromatography

The research aimed towards the adaptation of molecularly imprinted polymers (MIPs) to the capillary format and the use of these highly selective matrices for capillary electrochromatography (CEC) is reviewed in this article. The MIP is prepared by incorporation of a template molecule into a polymerization protocol. After polymerization and extraction of the template from the resulting polymer a highly selective material with recognition cavities complementary to the template in size, shape and chemical functionality is obtained. MIPs have been used as recognition elements in several different analytical techniques. In combination with CEC a novel separation system with a unique selectivity towards a predetermined target (the template) is achieved. The merge of molecular imprinting technology (MIT) and CEC have introduced several interesting polymer formats, due to the adaptation of the MIP to the miniaturized capillary format. The polymer formats can be classified according to their preparation protocols and appearance into three conceptually different categories, i.e. the monolith, the coating and the nanoparticles. The preparation protocols, characteristics and applications of these formats will be discussed.     

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.     

Hydroxyethylcellulose as an effective polymer network for DNA analysis in uncoated glass microchips: optimization and application to mutation detection via heteroduplex analysis

The nature of the sieving matrix for DNA fragment separation is of immense importance in capillary and microchip electrophoresis. The chemical nature of the surface of the capillary or microchannel wall is equally as important, particularly with DNA electrophoresis where a substantial electroosmotic flow (EOF) may be detrimental to the separation. Although DNA analysis has been carried out successfully in both coated and uncoated capillaries, analysis of unpurified polymerase chain reaction products has been carried out primarily with covalently coated surfaces, especially with microchip electrophoresis. In this report, double-stranded (ds) DNA fragment analysis using hydroxyethylcellulose (HEC) buffered in 1xTris-borate-EDTA is demonstrated both in uncoated capillaries and in microchips. EOF was suppressed 20% in the presence of 1.5% HEC, and the effectiveness of HEC as a polymer for dsDNA fragment analysis was dependent on the pH, with pH 8.6 being optimal. Using separation efficiency (number of theoretical plates) and resolution to gauge the effectiveness of a variety of polymers for the capillary separation of dsDNA fragments in the size range 60-587bp, HEC was found to be comparable in performance to polydimethylacrylamide (PDMA), and superior to linear polyacrylamide and polyethylene oxide for DNA analysis. With respect to longevity and robust performance, HEC could be used effectively in an uncoated capillary for more than 40 runs and for more than 90 runs (without replenishing the polymer) in an uncoated microchip. Application of the optimized HEC conditions is demonstrated through its ability to facilitate heteroduplex analysis.     

Enantioseparation of omeprazole and its metabolite 5-hydroxyomeprazole using open tubular capillary electrochromatography with immobilized avidin as chiral selector

The present paper demonstrates the enantiomeric separation of omeprazole and its metabolite 5-hydroxyomeprazole performed with open tubular capillary electrochromatography (OT-CEC). The protein avidin was used as the chiral selector. Avidin was immobilized by a Schiffs base type of reaction where the protein was via glutaraldehyde covalently bonded to the amino-modified wall of a fused-silica capillary, 50 microm i.d. Both racemates were baseline resolved. Resolution was 1.9 and 2.3, respectively, using ammonium acetate buffer, pH 5.8, 5% methanol, with UV-detection. These values of resolution using OT-CEC are higher than earlier published results regarding chiral separation of omeprazole and 5-hydroxyomeprazole on packed CEC. The number of theoretical plates also indicated good separation efficiency.     

The covalently bonded cellulose tris(3,5-dimethylphenylcarbamate) on a silica monolithic capillary column for enantioseparation in capillary electrochromatography

A chiral capillary monolithic column for capillary electrochromatography (CEC) was prepared by covalent bonding of cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) on the silica monolithic matrix within the confine of a 50-microm i.d. bare fused silica capillary. Several pairs of enantiomers including neutral and basic analytes were baseline resolved on the newly prepared chiral capillary monolithic column in CEC with aqueous mobile phases. Fast enantioseparation was achieved due to the favorable dynamic properties of silica monolith. The covalent bonding of CDMPC as the chiral stationary phase for CEC also enabled the use of THF in mobile phase for enantioseparation of prazquantel by overcoming the incompatibility of THF and the physically coated CDMPC on a column.     

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.     

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 μm 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 α-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 α values up to 5.     

Enantioseparations with cellulose tris(3-chloro-4-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Cellulose tris(3-chloro-4-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSPs) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the chiral selector loading onto silica, mobile phase composition and pH, as well as separation variables on separation of enantiomers was studied. It was found that CSPs based on cellulose tris(3-chloro-4-methylphenylcarbamate) can be used for preparation of very stable capillary columns useful for enantioseparations in nano-LC and CEC in combination with polar organic mobile phases.     

Enantioseparation of tetrahydropalmatine and Tröger's base by molecularly imprinted monolith in capillary electrochromatography

Two chiral compounds, Tr?ger's base and tetrahydropalmatine, were enantioseparated on the (5S, 11S)-(-)-Tr?ger's base and l-tetrahydropalmatine imprinted monolithic capillary columns with CEC, respectively. The monoliths were prepared by in situ thermal-initiated copolymerization of methacrylic acid (MAA) and ethylene dimethacrylate (EDMA). After optimizing the ratio of porogens (toluene and dodecanol), the obtained monolithic capillary columns show good flow-through property and enantioselectivity. The influences of CEC parameters such as pH of the buffer, organic solvent and salt concentration on the electroosmotic flow (EOF) and recognition selectivity were systematically investigated. Under the optimal conditions, baseline resolutions of two chiral compounds were achieved. In addition, the fast separation was obtained within 4 min by applying higher voltage and assisting pressure of 6 bar.     

Polyamidoamine dendrimer as a spacer for the immobilization of glucose oxidase in capillary enzyme microreactor

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups. In this study, different generations of PAMAM (G0-G4) were introduced onto the inner wall of fused-silica capillaries by microwave irradiation and a new type of glucose oxidase (GOx) capillary enzyme microreactor was developed based on enzyme immobilization in the prepared PAMAM-grafted fused-silica capillaries. The optimal enzymolysis conditions for β-d-glucose in the microreactor were evaluated by capillary zone electrophoresis. In addition, the enzymolysis efficiencies of different generations of PAMAM-GOx capillary enzyme microreactor were compared. The results indicate that enzymolysis efficiency increased with increasing generations of PAMAM. The experimental results provide the possibility for the development and application of an online immobilized capillary enzyme microreactor.     

Probing soft polymeric coatings of a capillary by atomic force microscopy

Atomic force microscopy (AFM) has been used to probe the surface of a capillary after coating with “soft” polymers, notably polyacrylamides. The aim was the investigation of the efficiency of coverage of the silica surface, so as to reduce or eliminate the electroosmotic flow (EOF), particularly noxious in the separation of macromolecules. The quality of such coating is strongly dependent on two variables: temperature and pH. In the first case, progressively higher temperatures produce open silica patches, where no polymer seems to be bound. The transition from coated to largely uncoated surfaces occurs at 50°C. Also the pH of the polymerizing solution strongly affects the coating efficiency. Since in all coating procedures the monomer solution is not buffered, addition of accelerator (TEMED, N,N,N′N′-tetramethylethylendiamine) induces polymer growth at pH 10–11. These pH values generate hydrolysis of the siloxane bridge anchoring the bifunctional agent (Bind Silane, onto which the polymer chain should grow) to the wall. Thus, coating and de-coating occur simultaneously. Low temperatures during polymer growth (typically 10°C) and buffered solutions (pH 7, titrated after TEMED addition) ensure a most efficient and thorough coating, with virtual elimination of EOF: well coated capillaries exhibit residual EOF values, at pH 10, of the order of 10⁻⁷ cm² V⁻¹ s⁻¹ vs. a standard value for uncoated capillaries of the order of 10⁻⁴ cm² V⁻¹ s⁻¹. The AFM data have been fully confirmed by direct measurement of EOF in coated and uncoated capillaries under an electric field.     

Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors

Proteins display interesting chiral discrimination properties owing to multiple possibilities of intermolecular interactions with chiral compounds. This review deals with proteins which have been used as immobilized chiral selectors for the enantioseparation of drugs in liquid chromatography and capillary electrophoresis. The main procedures allowing the immobilization of proteins onto matrices, such as silica and zirconia particles, membranes and capillaries are first presented. Then the factors affecting the enantioseparation of drugs in liquid chromatography, using various protein-based chiral stationary phases (CSPs), are reviewed and discussed. Last, chiral separations already achieved using immobilized protein selectors in affinity capillary electrochromatography (ACEC) are presented and compared in terms of efficiency, stability and reproducibility.     

Enantioseparation of tetrahydropalmatine and Tröger's base by molecularly imprinted monolith in capillary electrochromatography

Two chiral compounds, Tröger's base and tetrahydropalmatine, were enantioseparated on the (5S, 11S)-(-)-Tröger's base and l-tetrahydropalmatine imprinted monolithic capillary columns with CEC, respectively. The monoliths were prepared by in situ thermal-initiated copolymerization of methacrylic acid (MAA) and ethylene dimethacrylate (EDMA). After optimizing the ratio of porogens (toluene and dodecanol), the obtained monolithic capillary columns show good flow-through property and enantioselectivity. The influences of CEC parameters such as pH of the buffer, organic solvent and salt concentration on the electroosmotic flow (EOF) and recognition selectivity were systematically investigated. Under the optimal conditions, baseline resolutions of two chiral compounds were achieved. In addition, the fast separation was obtained within 4 min by applying higher voltage and assisting pressure of 6 bar.     

DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.

Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.     

无胶筛分毛细管电泳分离DNA片段的现状与展望

毛细管电泳已DNA片段分离分析的重要手段。本简述了毛细管电泳中采用无胶筛分介质分离DNA片段的机理研究,介绍了筛分介质近年的研究发展状况,依据分离介质的化学组成,分单聚物、共聚物和混聚物等3个部分进行了评述,并对其发展前景进行了展望。     

Capillary electrophoresis of DNA in uncrosslinked polymer solutions: evidence for a new mechanism of DNA separation

The electrophoretic separation of DNA molecules is usually performed in thin slabs of agarose or polyacrylamide gel. However, DNA separations can be achieved more rapidly and efficiently within a microbore fused silica capillary filled with an uncrosslinked polymer solution. An early assumption was that the mechanism of DNA separation in polymer solution(SINGLEBOND)capillary electrophoresis (PS(SINGLEBOND)CE) is the same as that postulated to occur in slab gel electrophoresis, i.e., that entangled polymer chains form a network of "pores" through which the DNA migrates. However, we have demonstrated that large DNA restriction fragments (2.0(SINGLEBOND)23.1 kbp) can be separated by CE in extremely dilute polymer solutions, which contain as little as 6 parts per million [0.0006% (w/w)] of uncrosslinked hydroxyethyl cellulose (HEC) polymers. In such extremely dilute HEC solutions, far below the measured polymer entanglement threshold concentration, pore-based models of DNA electrophoresis do not apply. We propose a transient entanglement coupling mechanism for the electrophoretic separation of DNA in uncrosslinked polymer solutions, which is based on physical polymer/DNA interactions. (c) 1996 John Wiley & Sons, Inc.     

Separation of DNA fragments and single strand conformation polymorphism analysis in bare capillaries using poly(acrylamide–dimethylacrylamide) as a separation medium

A short chain poly(acrylamide–dimethylacrylamide) (PADMA) was synthesized in aqueous phase using isopropanol as a chain transfer agent, and was characterized according to the chemical composition and molecular mass. This polymer can form a stable dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA–capillary wall interaction. The sieving medium has low viscosity and capillary filling with this medium and medium replacement were conveniently carried out by commercial capillary electrophoresis instruments. The effects of components and concentration of copolymers on the separation of DNA fragments were investigated. Highly efficient separation of DNA fragments, successful single strand conformation polymorphism (SSCP) analysis and good reproducibility of the migration time were obtained in bare capillaries using these copolymers as sieving media. Our preliminary results demonstrate that PADMA will become an alternative matrix for DNA separation by capillary electrophoresis.     

Simultaneous enantioselective separation of azelastine and three of its metabolites for the investigation of the enantiomeric metabolism in rats. I. Liquid chromatography-ionspray tandem mass spectrometry and electrokinetic capillary chromatography

Enantioselective separation methods and the enantioselective determination of the anti-allergic drug azelastine and of three of its main phase I metabolites in a biological matrix underwent chromatographic and electrophoretic investigations. An enantioselective assay of a coupling of HPLC using a beta-cyclodextrin chiral stationary phase to ionspray tandem mass spectrometry is presented. Additionally, this assay is compared to another enantioselective assay using electrokinetic capillary chromatography with beta-cyclodextrin and carboxymethyl-beta-cyclodextrin in polyacrylamide-coated capillaries. For capillary electrophoresis (CE) the importance of polyacrylamide coating for the validation of this separation method is highlighted. Extracted rat plasma samples of enantioselective metabolism studies were measured by both validated assays. Differences in the pharmacokinetics and pharmacodynamics were evaluated for the main substance azelastine and its main metabolite demethylazelastine. So, a first hint about the enantioselectivity of biotransformation of azelastine in rats was seen after oral application of either enantiomer or the racemate to rats.