Influence of the kind of the Alcoholic Modifier on Chiral Separation of 4-Substituted-Pyrrolidin-2-Ones Using Amylose Based Chiral Stationary Phase

ABSTRACT

The enantioselective separation of 4-substituted-pyrrolidin-2-ones was investigated on a silica-based amylose tris (3, 5-dimetliylphenylcarbamate) (Chiralpak AD) using, normal phase methodology with a mobile phase consisting of H-hexane-alcohol (ethanol, 1-propanol or 2-propanol) in various percentage. Effects of variation of concentration of alcohol and the nature of aliphatic alcohols in the mobile phase were studied. In a general manner, for the ten compounds analysed, the retention times increased and the separations factors were improved on changing the alcohol modifier from 2-propanol to ethanol. The effects of substitution were analysed. Baseline separation was easily obtained in many cases with a maximum resolution factor of 11.     

Application of capillary zone electrophoresis with off-line solid-phase extraction to in vitro metabolism studies of antifungals

A simple and robust solid-phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itraconazole, fluconazole, and voriconazole) and their metabolites after incubation with human liver microsomes, as well as a simplified capillary zone electrophoresis (CZE) method for their rapid analysis, have been developed to determine the stability of these compounds in in vitro samples. Three different sample pretreatment procedures using SPE with reversed-phase sorbents (100 mg C8, 100 mg C18, and 30 mg Oasis-HLB) were studied. The highest and most reproducible recoveries were obtained using a 30 mg Oasis-HLB sorbent and methanol containing 2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates to a small volume. For the CZE separation, good separations without interfering peaks due to the in vitro matrix were obtained with a simple running electrolyte using a fused-silica capillary. The best separation for all components originated by each tested drug after incubation with human liver microsomes (unmetabolized parent drug and its metabolites) was obtained using a 0.05 M phosphate running buffer (pH 2.2) without additives. The effect of the injection volume was also investigated in order to obtain the best sensitivity. Performance levels in terms of precision, linearity, limits of detection, and robustness were determined.     

Poly(vinyl alcohol)-coated microfluidic devices for high-performance microchip electrophoresis

The channels of microfluidic glass chips have been coated with poly(vinyl alcohol) (PVA). Applied for microchip electrophoresis, the coated devices exhibited a suppressed electroosmotic flow and improved separation performance. The superior performance of PVA-coated channels could be demonstrated by electrophoretic separations of labeled amines and by video microscopy. While a distorted sample zone is injected using uncoated channels the application of PVA-coated channels results in an improved shape of the sample zone with less band broadening. Applying PVA-coated microchips for the separation of amines labeled with Alexa Fluor 350 even sub-second separations, utilizing a separation length of only 650 microm, could be obtained, while this was not possible using uncoated devices. By using PVA-coated devices rather than an uncoated chip a threefold increase in separation efficiencies could be observed. As the electroosmotic flow (EOF) was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Besides improved separation performance another important feature of the PVA-coated channels was the suppressed adsorption of fluorescent compounds in repetitive runs which results in an improved robustness and detection sensitivity. Applying PVA-coated channels, rinsing or etching steps could be omitted while this was necessary for a reliable operation of uncoated devices.     

Evaluation and comparison of a methylated teicoplanin aglycone to teicoplanin aglycone and natural teicoplanin chiral stationary phases

HPLC enantiomeric separations of a wide variety of racemic analytes was evaluated using chiral stationary phases (CSPs) based on the macrocyclic glycopeptides teicoplanin (T), teicoplanin aglycone (TAG), and methylated teicoplanin aglycone (Me-TAG) in two different mobile phase modes, i.e., the RP mode and the polar organic (PO) mode. Comparison of the enantiomeric separations using Chirobiotic T, Chirobiotic TAG, and the methylated form of TAG were conducted in order to gain a better understanding of the roles of the polar functional groups on the CSP. Substantial effects due to the cleavage of saccharides and/or methylation on chiral separations were observed in both separation modes. Improved separation efficiencies for many acidic analytes were obtained by methylating the H-bonding groups of TAG. These groups were believed to be a contributing factor to band broadening on TAG due to their negative effect on mass transfer between the stationary phase and mobile phase. Ionic/dipolar interactions between the carboxylate group of the analytes and the amine groups on T, TAG, or Me-TAG are important for chiral discrimination. Therefore, analytes possessing a carboxyl group are good candidates for successful separations on these CSPs. Hydrophobic interactions are important for enantiomeric separations in the RP mode where the H-bonding interactions between analytes and the chiral selectors are relatively weak. Me-TAG offers higher hydrophobicity, which can accentuate the interactions of analytes with hydrophobic moieties, but these interactions are not necessarily stereoselective. In the PO mobile phase, electrostatic/dipolar interactions between polar functional groups are the dominating interactions in chiral recognition. Another important factor is steric fit, which could be changed with every modification of the T structure. Therefore, substantial changes of enantioseparations were obtained within this studied group of CSPs. The PO mode was shown to be the most powerful mobile phase mode for enantiomeric separations on T-based stationary phases, mainly due to the improved efficiency. Methylation of the TAG proved to be a very useful tool for investigating the chiral recognition mechanism for this group of chiral selectors.     

磷酸钛离子交换薄层色谱分离无机离子(英文)

The chromatographic behavior of 30 inorganic cations has been studied on thin layers of titanium phosphate ion-exchanger using several aqueous,organic and mixed mobile phases.The separation of one ion from several other ions and also ternary and binary separations have been developed.Some important analytical separations are reported.The effect of pH of the mobile phase on retention factor(Rf)values of the cations in the presence of complex-forming anion along with the separation power of the ion-exchanger were studied.This ion-exchanger exhibits high sorption capacity and varying selectivity towards metal ions and makes it a suitable stationary phase in thin layer chromatography.     

CEC separation of peptides using a poly(hexyl acrylate-co-1,4-butanediol diacrylate-co-[2-(acryloyloxy)ethyl]trimethyl ammonium chloride) monolithic column

A polyacrylate-based monolithic column bearing cationic functionalities and designed for capillary electrochromatography (CEC) has been prepared via photopolymerization of a mixture of hexyl acrylate, butanediol diacrylate, 2-(acryloyloxy) ethyltrimethyl ammonium chloride (monomers), azobisisobutyronitrile (photoinitiator), acetonitrile, phosphate buffer, and ethanol (porogens). The polymerization process was initiated with UV light at 360 nm. The column performance was evaluated via the separations of alkylbenzenes, substituted anilines, basic drugs, peptides, and a protein digest. The separation of complex peptide mixtures was then studied since such separations constitute a promising application of capillary electrochromatography. In particular, the effects of mobile phase composition, including ionic strength of the buffer solution and the percentage of acetonitrile on the retention factor, the column efficiency, and the resolution were determined. The separations were affected by both interaction of the peptides with the stationary phase and their own electrophoretic mobility. Excellent separations with column efficiencies of up to 160 000 plates/m were achieved for both a mixture of ten well-defined peptides and a tryptic digest of cytochrome c. The fractions of eluent containing peptides of the digest separated in the monolithic column were collected and characterized using matrix-assisted laser desorption ionization mass spectrometry.     

应用四甲基氢氧化铵控制电渗流的毛细管电泳方法分离多沙唑嗪及其中间体的对映体

建立了毛细管电泳手性分离多沙唑嗪中间体对映体的方法,并同时分离了多沙唑嗪对映体。考察了不同种类季铵盐对电渗流及分离的影响,其中四甲基氢氧化铵(TMB)能有效控制电渗流并提高组分的分离度。实验还考察了其他因素,如pH值、分离电压和磷酸二氢钠浓度对分离的影响。所用的毛细管为40 cm(有效长度30 cm)×50 μm,缓冲液为12 mmol/L β-环糊精、30 mmol/L TMB、60 mmol/L 磷酸二氢钠(pH 2.2),分离电压为20 kV。在此条件下多沙唑嗪及其中间体的对映体均达到了基线分离。实验结果表明,一些用β-环糊精不能完全分离的对映体通过加入TMB控制电渗流能达到满意的分离效果。     

High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution

In this paper, we describe a method for size-based electrophoretic separation of sodium dodecyl sulfate (SDS)-protein complexes on a polymethyl methacrylate (PMMA) microchip, using a separation buffer solution containing SDS and linear polyacrylamide as a sieving matrix. We developed optimum conditions under which protein separations can be performed, using polyethylene glycol (PEG)-coated polymer microchips and electrokinetic sample injection. We studied the performance of protein separations on the PEG-coated PMMA microchip. The electrophoretic separation of proteins (21.5-116.0 kDa) was completed with separation lengths of 3 mm, achieved within 8 s on the PEG-coated microchip. This high-speed method may be applied to protein separations over a large range of molecular weight, making the PEG-coated microchip approach applicable to high-speed proteome analysis systems.     

High-performance membrane chromatography of serum and plasma membrane proteins.

Porous discs made of poly(glycidyl methacrylate) were used for high-performance membrane chromatography (HPMC) of proteins. In model experiments, separations of standard proteins by anion-exchange HPMC using a DEAE disc were carried out. The influences of sample distribution and disc diameter and thickness on separation performance were studied. The separation disc allowed a scaling-up from analytical (diameter 10 mm) to semi-preparative (diameter 50 mm) dimensions. In an application study, separations with anion-exchange and affinity HPMC were carried out using different complex samples such as rat serum and plasma membrane proteins. In all experiments the results on poly(glycidyl methacrylate) discs were comparable to those achieved on adequate high-performance liquid chromatographic (HPLC) columns. However, the separations on HPMC discs could be carried out faster than corresponding separations on HPLC columns. The pressure drop on the discs was low even at high flow-rates. The experiments show that the poly(glycidyl methacrylate) discs used are especially suitable for the isolation of proteins and other biopolymers which occur in a diluted state in complex mixtures.     

Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen as fluorescence inducing agent

The fast separation of oligonucleotide (oligos) sizing marker by CE using OliGreen and including effects due to the concentration of separation medium and urea denaturant is presented. OliGreen dye is found to be more sensitive than ethidium bromide (by a factor of about 6 based on S/N considerations) for the oligos' separations. Higher concentration of F127 in 1xTris-boricacid-EDTA (TBE) up to 30% w/v leads to better resolution of oligos separations. The addition of urea into the separation medium decreases the sensitivity. With an optimized running condition, the oligos sizing marker could be successfully separated with 1-base resolution within 1.3 min by using 30% w/v F127/1xTBE solution as the separation medium at an applied electric field of 800 V/cm in a 3 cm long capillary, the fastest capillary gel electrophoresis separation with high resolution reported to date for oligos in the similar size range.     

Direct separation of the stereoisomers of methoxytetrahydronaphthalene derivatives, new agonist and antagonist ligands for melatonin receptors, by liquid chromatography on cellulose chiral stationary phases

Analytical HPLC methods using derivatized cellulose chiral stationary phases were developed for the direct separation of the stereoisomers of disubstituted tetralin derivatives with two chiral centers, new agonist and antagonist ligands for melatonin receptors. The separations were made using normal-phase methodology with a mobile phase consisting of n-hexane-alcohol (methanol, ethanol, 1-propanol or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ). The effects of concentration of various aliphatic alcohols in the mobile phase were studied. A better separation was achieved on cellulose carbamate phase compared with the cellulose ester phase. The effects of structural features of the solutes on the discrimination between the stereoisomers were examined. Baseline separation (Rs>1.5) was easily obtained in many cases.     

毛细管电动色谱带电环糊精拆分N-FMOC氨基酸对映体

 将负电性磺丁基 -β -环糊精手性添加剂应用于毛细管电泳氨基酸对映体的拆分研究中 ,对 8种氨基酸对映体与 9-芴甲基氧基甲酰氯 (FMOC-Cl)生成的衍生物进行了分离 ,其中 5种得到了基线分离。考察了背景电解质 p H值及磺丁基 -β -环糊精的浓度对 N-FMOC氨基酸对映体拆分的影响。     

Optimization of a capillary electrophoresis method to determine the chiral purity of a drug

Capillary electrophoresis (CE) using hydroxypropyl-β-cyclodextrin (HP-β-CD) in the separation buffer was investigated to determine the overall chiral purity of a drug containing a single stereogenic center. The effects of primary factors —pH, buffer components, buffer concentration, cyclodextrin concentration and sample amount (concentration and injection volume) — on the resolution of the enantiomers were investigated. Secondary factors such as the HP-β -CD source, lot and degree of substitution that were expected to affect the robustness of the assay were investigated also. The linearity, quantitation limit for the trace enantiomer and the precision of the measurements were determined. This study shows that understanding and optimizing the assay conditions leads to a chiral CE separation that is comparable to that obtained by chiral HPLC. However, chiral CE separations achieved with buffer additives have the advantages of shorter run times, higher numbers of theoretical plates (greater resolution), smaller amounts of chiral additive (less cost) and greater ruggedness (separation virtually independent of column properties unlike HPLC).     

Fast turnaround bioanalysis in discovery and early clinical development

Summary The difect and indirect separation of enantiomers of secondary amino acids was studied by high-performance liquid chromatography. Direct separation was by using a macrocyclic glycopeptide, teicoplanin or ristocetin A, as chiral stationary phase. Indirect separation was via pre-column derivatization with (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester [(S)-NIFE] as a new chiral derivatizing agent. Both direct and indirect separations were performed by means of reversed-phase HPLC. Conditions for separations were optimized. The methods described offer good enantioselectivity for synthetic chiral imino acids. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Nonaqueous media for separation of nonionic organic compounds by capillary electrophoresis

For the separation of neutral compounds by micellar electrokinetic chromatography, separations are usually carried out in predominantly aqueous solution in order to preserve the charged micelle necessary for the separation. We now show that polycyclic aromatic hydrocarbon (PAH) compounds can be separated efficiently by capillary electrophoresis in pure methanol or in aqueous-organic mixtures containing a high percentage of methanol. Sodium tetradecyl sulfate was the preferred surfactant. The effects of pH, solvent composition, surfactant structure, and surfactant concentration on the separations were studied. Reproducible migration times and linear calibration plots were obtained.     

Ring-opening metathesis polymerization for the preparation of norbornene-based weak cation-exchange monolithic capillary columns

Functionalized monolithic columns were prepared via ring-opening metathesis polymerization (ROMP) within silanized fused silica capillaries with an internal diameter of 200 μm by in situ grafting. This procedure is conducted in two steps, the first of which is the formation of the basic monolithic structure by polymerization of norborn-2-ene (NBE) and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene (DMN-H6) in a porogenic system (toluene and 2-propanol) using RuCl₂(PCy₃)₂(CHPh) as ROMP initiator. In the second step the still active initiator sites located on the surface of the structure-forming microglobules were used as receptor groups for the attachment (“grafting”) of functional groups onto the monolithic backbone by flushing the monolith with 7-oxanorborn-2-ene-5,6-carboxylic anhydride (ONDCA). Functionalization conditions were first defined that did not damage the backbone of low polymer content (20%) monoliths allowing high-throughput chromatographic separations. Variation of the functionalization conditions was then shown to provide a means of controlling the degree of functionalization and resulting ion-exchange capacity. The maximum level of in situ ONDCA grafting was obtained by a 3 h polymerization in toluene at 40 °C. The weak cation-exchange monoliths obtained provided good separation of a standard peptide mixture comprising four synthetic peptides designed specifically for the evaluation of cation-exchange columns. An equivalent separation was also achieved using the lowest capacity column studied, indicative of a high degree of robustness of the functionalization procedure. As well as demonstrably bearing ionic functional groups enabling analyte separation in the cation-exchange mode, the columns exhibited additional hydrophobic characteristics which influenced the separation process. The functionalized monoliths thus represent useful tools for mixed-mode separations.     

Optimization of ion-exchange displacement separations. II. Comparison of displacement separations on various ion-exchange resins

A variety of stationary-phase materials are currently available for the chromatographic purification of biomolecules. However, the effect of various resin characteristics on the performance of displacement chromatography has not been studied in depth. In Part I, a novel iterative scheme was presented for the rapid optimization of displacement separations in ion-exchange systems. In this article, the optimization scheme is employed to identify the optimum operating conditions for displacement separations on various ion-exchange resin materials. In addition, the effect of different classes of separation problems (e.g., diverging, converging or parallel affinity lines) on the performance of displacement separations is also presented. The solid film linear driving force model is employed in concert with the Steric Mass Action isotherm to describe the chromatographic behavior in these systems. The results presented in this article provide insight into the effects of resin capacity and efficiency as well as the type of separation problem on the performance of various ion-exchange displacement systems.     

Optimisation and robustness analysis of a hydrophobic interaction chromatography step

Process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. The present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. Factorial experimental design is common practice in industry today for robustness analysis. The method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. In addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. The methodology consists of three consecutive steps. Firstly, screening experiments are performed using a factorial design. Secondly, a kinetic-dispersive model is calibrated using gradient elution and column load experiments. Finally, the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. The process studied in this work is the separation of polyclonal IgG from BSA using hydrophobic interaction chromatography.     

Enantioseparation of citalopram analogues with sulfated β‐cyclodextrin by capillary electrophoresis

Capillary electrophoresis methods were developed for the enantiomeric separation of 27 citalopram analogues. Sulfated β‐cyclodextrin was the most broadly selective and useful chiral selector. The separations of most of the citalopram analogue compounds reported in this work have not been reported previously. Excellent enantiomeric separations were obtained for 26 out of 27 compounds, and most of the separations were achieved within 10 min. The effects of chemical parameters such as chiral selector types, buffer types, chiral selector and buffer concentrations, buffer pH and organic modifiers on the separation were investigated. The influence of analyte structure on separation also was examined and discussed.     

Influence of buffer substances and urea on the beta-cyclodextrin-mediated chiral separation of dipeptides in CE

The influence of buffering substances and urea on the beta-CD-mediated chiral separations of the dipeptides Ala-Phe and Ala-Tyr was studied in the pH range of 2.5-3.8. Only minor effects of the buffer substances on the chiral separation selectivity alpha were observed at a beta-CD concentration of 15 mg/mL. In contrast, the selectivity improved at pH 2.5 but decreased at pH 3.8 upon the addition of 2 M urea. Complexation by beta-CD resulted in a shift of the pK(a) values toward higher values which was more pronounced for the DD-enantiomers of both dipeptides than for the LL-enantiomers. Addition of urea further increased the pK(a) shift. The consequence of this pK(a) shift is an increase of the fraction of the protonated, positively charged form of the peptides which explained the improved chiral separation at pH 2.5 and the reduced selectivity at pH 3.8. A pK(a) shift by the addition of urea was also observed for N-tert-butyloxycarbonyl phenylalanine (BOC-Phe) as a model compound that is strongly complexed by beta-CD. This effect was not stereospecific. Addition of urea resulted in a decrease of the apparent complexation constants between beta-CD and the BOC-Phe enantiomers to the same extent but this did not affect the separation selectivity alpha. For chiral separations that display strong pH dependence such as peptide enantioseparations close to the pK(a) values of the compounds, urea may not solely be regarded as a solubility enhancer for beta-CD but may also influence the separation.