Preparation and characterization of a polystyrene/bovine serum albumin nanoparticle‐coated capillary for chiral separation using open‐tubular capillary electrochromatography

Polystyrene (PS) nanoparticles coated by BSA, hereafter denoted as PS/BSA, were prepared and chemically immobilized for the first time onto a capillary inner wall for open‐tubular CEC (OTCEC). EOF and scanning electron micrography were used to characterize the prepared nanoparticle‐coated capillaries. To investigate the performance of the prepared columns in OTCEC, chiral separation of d ,l ‐tryptophan (dl ‐Trp) was performed in monolayer BSA‐modified capillary and PS/BSA nanoparticle‐coated columns. The results indicated that the nanoparticle‐modified column afforded a higher resolution compared with the monolayer type. Rapid enantioseparation of dl ‐Trp (within 3 min) was achieved with the PS/BSA‐immobilized column using an electroosmotic pump‐assisted CEC. Enantiomer separations of other compounds like dl ‐tyrosine and warfarin were also achieved with the column. Besides, run‐to‐run and column‐to‐column repeatabilities of the PS/BSA‐coated column in the chiral separation were systematically introduced.     

Contribution of ionically immobilized bovine serum albumin to the retention of enantiomers.

The retention of the enantiomers of mandelic acid and N-benzoylalanine was studied on columns prepared by immobilizing bovine serum albumin (BSA) on an anion exchanger. The amount of BSA fixed on the column is easy to adjust and measure. The adsorption isotherms were determined. For each enantiomer, the isotherm is well accounted for by a bi-Langmuir equation. One term of the isotherm (which is the same for both enantiomers) corresponds to non-selective interactions and the other term to the chiral selective interactions. The column saturation capacity of this second term is 8% larger for the less strongly retained enantiomer. This saturation capacity corresponds approximately to one enantiomer molecule adsorbed for five BSA molecules immobilized. This result is in agreement with the assumption of the hydrophobic cavity of BSA being the chiral selective site.     

金纳米粒子修饰的手性毛细管电色谱固定相的制备及性能表征

制备了粒径为15 nm的金纳米粒子(GNPs)并将其修饰到氨基衍生化的硅胶整体柱内,通过化学键合法将牛血清白蛋白(BSA)固载到GNPs的表面作为手性固定相。通过透射电子显微镜、扫描电子显微镜等方法进行表征,结果表明,GNPs分散性良好,并被成功地修饰到毛细管柱内,含量高达17.18%。优化了BSA手性柱的制备条件,最终确定了体积分数为10%的3-氨丙基三乙氧基硅烷(APTES)和15 g/L BSA为最佳反应条件。在毛细管电色谱分离模式下,对缓冲液pH值、电压等分离条件进行了考察,最终选择了10 mmol/L pH 7.4的磷酸缓冲液和15 kV运行电压作为最佳分离条件。手性柱对3种手性化合物(色氨酸、阿替洛尔和麻黄碱)有拆分效果,对色氨酸能实现基线分离。与物理吸附法相比,化学键合法制备的手性柱拆分效果好,分析物无需柱前衍生化,且色谱柱稳定性良好。该文的制备方法也为其他类型手性选择剂的引入提供了良好的思路。     

温度对蛋白和β-环糊精手性固定相拆分对映体的影响

 采用三聚氯氰为活化剂分别合成了牛血清白蛋白 (BSA)、人血清白蛋白 (HSA)和 β 环糊精手性固定相 ,研究了温度在色氨酸 ,华法令 ,酮基布洛芬和丹酰化苏氨酸手性拆分中的影响。结果表明 ,在蛋白手性固定相上对映体间的熵变对色氨酸 ,华法令和酮基布洛芬的拆分有很大的影响 ,而丹酰化苏氨酸对映体在 β 环糊精手性固定相上的拆分为典型的焓控过程 ,与蛋白柱有着不同的热力学特性。由于键合方式不同 ,色氨酸在我们合成的BSA手性固定相上的最佳分离温度为 35℃左右 ,而不是文献报道的以戊二醛为活化剂的 2 4℃。     

Comparative Enantiomer-Resolving Ability of Coated and Covalently Immobilized Versions of Two Polysaccharide-Based Chiral Selectors in High-Performance Liquid Chromatography

The separation of enantiomers of over 175 randomly selected chiral acidic, basic, and neutral compounds was studied on 4 polysaccharide-based chiral columns made by coating or covalent attachment of cellulose 3,5-dichlorophenylcarbamate or amylose 3,5-dimethylphenylcarbamate on the surface of silica. Triscarbamate derivatives of cellulose or amylose were used for the preparation of coated-type columns, while in the case of covalently immobilized chiral stationary phases, the respective polysaccharides were not completely carbamoylated but only close to triscarbamates. It was found that this minimal difference in the chemical composition of the polysaccharide derivatives resulted in significantly different enantiomer-resolving ability for certain groups of chiral compounds while only marginally different for other chiral analytes. This potential difference between coated- and covalently immobilized versions of the “same” chiral selector must be considered in method development with these columns, as well as in method transfer between them.     

流动相的pH值和离子强度对牛血清蛋白高效液相色谱手性柱分离性能的影响

利用羰基咪唑-柱上衍生法制得牛血清蛋白(BSA)生物手性柱,并研究它在高效液相色谱中对3种对映异构体(色氨酸、匹多莫德及4-苯基-1,3-恶唑烷-2-硫酮(L-苯))的手性分离性能.实验结果表明:随着pH值从5.0上升到7.0,由于L-色氨酸与BSA有固定的作用位点,使得其保留值随着pH值的增加而大幅增大.而D-色氨酸与BSA无固定作用位点,其保留值随pH变化基本不变,分离度由1.15上升到8.91,增加了6.8倍;酸性样品匹多莫德与BSA主要是静电作用,与色氨酸相反,其对映体的保留值随着pH的增加逐渐减小,分离度逐渐下降,pH 7.0时为单峰,pH 5.0时Rs=1.27;中性样品L-苯的分离度随着pH值的增加有小幅增大.随着离子强度的减小,3对对映体的保留都增强,分离度增大,增加的幅度依次为色氨酸＞匹多莫德＞L-苯.     

Monolithic silica columns with chemically bonded tert-butylcarbamoylquinine chiral anion-exchanger selector as a stationary phase for enantiomer separations

An enantioselective silica rod type chiral stationary phase (CSP) is presented as a novel combination of the well-known enantiomer separation properties of immobilized tert-butyl-carbamoylquinine chiral anion-exchanger selector with the unique properties of monolithic silica material. The chromatographic behavior of the tert-butyl-carbamoylquinine silica rod was studied and compared with a similar prepared particulate material. Good selectivities were achieved for a spectrum of chiral test components like N-derivatized amino acids (DNB- Ac-, DNZ-, Bz-, Z-amino acids) and for Suprofen. The influence of mobile phase parameters, as well as the effect of serially coupling up to six 10 cm monolithic silica columns was studied and put in context to conventional columns of particulate 5 microm type CSP. Using that 60 cm long monolithic column it was possible to improve the enantiomer separation of Suprofen and achieve a baseline separation in less than 10 min of total separation time.     

A novel lipase-based stationary phase in liquid chromatography

In this paper, a new chiral stationary phase (CSP) based on Candida antarctica lipase B (CALB) bounding to the surface of macroporous silica gel was developed and its stereoselectivity in enantioseparation and asymmetrical hydrolysis was evaluated. Three CALB-based HPLC columns with different amounts of enzyme immobilized were prepared by employing the immobilization method, namely “in batch”. In this technique two chromatographic supports epoxy silica and aminopropyl silica were considered. This novel CSP was proven capable of hydrolyzing chiral esters asymmetrically as bioreactor and separating several aromatic alcohols and diniconazole enantiomers.     

Immobilized penicillin G acylase as reactor and chiral selector in liquid chromatography

In this paper, the use of penicillin G acylase (PGA) as a biocatalyst and as a chiral selector is described. Penicillin G-acylase is an interesting enzyme used in the manufacture of semisynthetic antibiotics and, in particular, in the production of 6-APA by hydrolysis of penicillin G. Five PGA-based HPLC columns have been prepared by using two different silica supports by employing two immobilization methods, namely "in situ" and "in batch". The effects of the immobilization techniques and of different silica pore size on the catalytic properties of the enzyme as well as the applicability of the PGA-bonded stationary phases as chiral selectors for a number of chiral drugs have been investigated. The HPLC columns based on immobilized PGA combine the hydrolytic activity and the chiral recognition properties of PGA, therefore they have been used for the development of a combined reaction-separation system for chiral and achiral substrates.     

Recent trends in chiral separations on immobilized polysaccharides CSPs

Polysaccharide CSPs are recognized widely in chiral chromatography but the introduction of immobilized phases (Chiralpak IA, Chiralpak IB and Chiralpak IC columns) is a remarkable achievement. The immobilized CSPs can be used with organic, normal and reversed phase modes; even with prohibited solvents too (tetrahydrofuran, chlorofom, dichloromethane, acetone, 1,4-dioxane, ethylacetate, and certain other ethers). Their susceptibilities to work with a wide range of solvents have increased the range of applications including chiral recognition mechanisms. Besides, these are also useful for monitoring the progress of stereo-specific reactions; normally need prohibited solvents. The present review describes the various aspects of commercial available immobilized chiral columns. Attempts have been made to discuss immobilized polysaccharides CSPs, immobilized vs coated CSPs, comparison of immobilized CSPs, method development, optimization, chiral recognition mechanism and applications. The chiral recognition capabilities of commercial columns were in the order of Chiralpak IA > Chiralpak IB > Chiralpak IC columns; but complimentary to each other. Of course, these CSPs are not fully developed and need more advancements and applications. Definitely, the future of immobilized CSPs is quite better. Hopefully, in the coming years they will be the choice of the chromatographers for chiral separations in liquid chromatography.     

On-chip chiral separation based on bovine serum albumin-conjugated carbon nanotubes as stationary phase in a microchannel

A novel method of chiral separation based on protein-stationary phase immobilized in a poly(methyl methacrylate) microfluidic chip was developed. BSA conjugated with the shortened carboxylic single-walled carbon nanotubes (SWNTs) was employed as the chiral selector. Successful separation of tryptophan enantiomers was achieved in less than 70 s with a resolution factor of 1.35 utilizing a separation length of 32 mm. This is the first example of chiral separation based on SWNTs-BSA conjugates as stationary phase immobilized in microchip channel. The stability of the stationary phase in the channel was examined by microchip electrophoresis with laser-induced fluorescence detection. Factors that influenced the chiral separation resolution were examined. Under the optimized conditions, the proposed modified chip revealed adequate repeatability concerning run-to-run. These results show that the use of SWNTs-BSA conjugates within microfluidic channels hold great promise for a variety of analytical schemes.     

Analysis of spherical polyelectrolyte brushes by small angle X-ray scattering

Spherical polyelectrolyte brushes （SPBs） with PS core and poly（acrylic acid） （PAA） brushes were prepared and analyzed by SAXS in this article. A radial electron density profile of SPB was brought up, which fits well with the SAXS result and shows a core-shell structure. The effect of pH on SPB form was represented by SAXS and it proves that the chains of SPB will stretch in response to increased pH owning to the increased electrostatic repulsion. SPBs immobilized with magnetic nanoparticles or bovine serum albumin （BSA） were prepared and analyzed by SAXS as well. SAXS could characterize the changes of electron density inside brushes of SPBs due to the immobilization of magnetic nanoparticles or BSA. This provides significant supports for further application of immobilized metal nanoparticles or proteins.     

High-performance affinity monolith chromatography for chiral separation and determination of enzyme kinetic constants

A new kind of immobilized human serum albumin (HSA) column was developed by using the sub-micron skeletal polymer monolith based on poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-EDMA)] as the support of high-performance affinity chromatography. Using the epoxide functional groups presented in GMA, the HSA immobilization procedure was performed by two different means. The affinity columns were successfully adopted for the chiral separation of d,l-amino acids (AAs). Then this method was shown to be applicable to the quantitative analysis of d-tryptophan, with a linear range between 12.0 μM and 979.0 μM, and a correlation coefficient above 0.99. Furthermore, it was used for the analysis of urine sample. This assay is demonstrated to be facile and relatively rapid. So it allows us to measure the enzyme catalytic activity in the incubation of d,l-AAs with d-AA oxidase and to study the kinetics of the enzyme reaction. It implied that the affinity monolithic columns can be a useful tool for studying DAAO enzyme reaction and investigating the potential enzyme mechanism requirement among chiral conversion.     

Binding of dl-tryptophan to BSA adsorbed in multilayers by polymer chains grafted onto a porous hollow-fiber membrane in a permeation mode

An anion-exchange-group-containing porous membrane in the form of a hollow fiber was prepared to immobilize bovine serum albumin (BSA) as a chiral selector. First, an epoxy-group-containing polymer chain was grafted onto the pore surface of the polyethylene porous hollow-fiber membrane pre-irradiated with an electron beam. Second, the epoxy group was converted to diethylamino and 2-hydroxyethylamino groups. Third, a BSA solution was forced to permeate through the pores of the membrane. As a result, 190 mg BSA per gram of membrane, which amounted to a degree of multilayer binding of about four, were immobilized. Subsequently, a racemic solution of tryptophan (0.02 mM) was forced to permeate through the BSA-multilayered porous membrane at a flow rate ranging from 10 to 80 ml/h. A two-stage stepwise concentration change of tryptophan in the effluent was observed due to independent chiral recognition of d- and l-tryptophan by BSA adsorbed in multilayers within the graft chains.     

Characterization of the Binding Performance of Silica-Based Immunoaffinity Adsorbents Prepared via Online Coupling Reactions

Antibodies against a conjugate of daidzein and bovine serum albumin (BSA) were immobilized onto dihydrazide-activated silica beads via online coupling reactions. The binding performances of the obtained immunoaffinity adsorbents to the corresponding hapten and carrier protein were all optimized and characterized. It was found that online coupling reactions offered a convenient and practical way to generate the requisite immunoaffinity columns. The optimum binding buffers for BSA and daidzein were found to be pH 5.0, 0.01 mol L^?1 phosphate-buffered saline (PBS) and 0.05% Tween-20 in pure water, indicating different interactions between the immobilized antibodies with different parts of the antigen. Under the optimum operating conditions, the silica-based immunoaffinity adsorbents showed specific and selective binding properties to the target protein; while for small molecules, a mixed adsorption mechanism caused by both specific and non-specific interactions was observed. For comparison, offline coupled silica-antibody immunoaffinity adsorbents and Sepharose 4B support immobilized with the same antibody were also prepared and tested. The experimental results of this study may provide useful information for further development of other high-performance immunoaffinity chromatographic methods for different purposes.     

Effects of organic modifiers on the chiral recognition by different types of silica-immobilized bovine serum albumin

We prepared three columns containing bovine serum albumin immobilized on silica by different means and the effects of organic modifiers in the eluent on chiral separation were studied using N-substituted amino acids. Adsorption on silica, covalent immobilization to diol-silica with carbonyldiimidazole (CSP-II) and covalent immobilization to amino-silica with glutaraldehyde were studied. CSP-II had the highest stereoselectivity and was the most affected by organic modifiers in the eluent. The hydrophobicity of amino acid moiety affected the chiral recognition of N-benzoylamino acids and the aromaticity of the N-substituted group was important.     

Synthesis and chromatographic properties of a chiral stationary phase derived from bovine serum albumin immobilized on magnesia-zirconia using phosphonate spacers

A novel bovine serum albumin (BSA)-modified magnesia-zirconia stationary phase was prepared using the sodium salt of cis-(3-methyloxiranyl)phosphonic acid (fosfomycin) as spacer and glutaraldehyde as coupler. Baseline separation of six derivatized amino acids (DNB-Leu, Dansyl-Val, etc.) was achieved on this column using ammonium acetate buffer-isopropanol mobile phase at a flow rate of 1.0 mL/min. The effects of mobile phase composition, eluent pH value, column temperature, and flow rate on the retention and separation of chiral compounds were also investigated. The BSA chiral stationary phase (BSA-CSP) was relatively stable under experimental conditions. The coupling reaction in this method was mild, reliable, and reproducible; thus it was also suitable for the immobilization of various biopolymers with amino groups in the preparation of chromatography stationary phases.     

Enantioselectivity of monolithic silica stationary phases immobilized with different concentrations cellulose tris (3,5-dimethylphenylcarbamate), analyzed with different mobile phases in capillary electrochromatography

The 3,5-dimethylphenylcarbamate derivatives of cellulose bearing 3-(triethoxysilyl)propyl residues were immobilized in a capillary format onto a monolithic silica support by intermolecular polycondensation of the triethoxysilyl groups. The resulting columns were used for chiral separations using capillary electrochromatography. The effects of the synthesizing solvent, the selector coating procedure, the chiral selector concentration onto the silica monolith and the mobile phase pH value, on the separation of enantiomers were studied. The column-to-column reproducibility and stability also were evaluated. A test set of 14 chiral substances, including acidic, neutral, bifunctional and basic compounds, was used to investigate the effects of the factors mentioned above. Twelve pairs of enantiomers showed enantioselectivity at some of the different conditions tested. The column-to-column repeatability was satisfactory, and the prepared columns were stable under the adopted analysis conditions.     

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.     

Reversal of the enantiomeric elution order of some aromatic amino acids using reversed-phase chromatographic supports coated with the teicoplanin chiral selector

In this paper, two chiral stationary phases were prepared by coating the surface of both C8 and C18 high-performance liquid chromatography (HPLC) supports with the teicoplanin chiral selector. The hydrophobic C11 acyl side chain, attached to the d-glucosamine group of teicoplanin, served as anchor moiety for the immobilization of the chiral selector on the apolar support material. The retention and enantioselectivity of these coated stationary phases were studied using some aromatic amino acids as probe solutes and an aqueous solution as mobile phase. It was found that the enantiomer elution order on the modified C8 and C18 stationary phases was reversed (l > d) relatively to that classically observed with a teicoplanin covalently immobilized on a silica support (d > l). Such a dynamic coating on the reversed-phase supports was found to be of interest since the apparent enantioselectivity was not significantly changed by the use during an extended period of time or following a long-term storage of the columns.