Chiral separations using polymeric surfactants and polyelectrolyte multilayers in open-tubular capillary electrochromatography

In this study, fused-silica capillaries are modified using a polyelectrolyte multilayer (PEM) coating procedure in open-tubular capillary electrochromatography. The PEM coating was constructed in situ with alternating rinses of positively and negatively charged polymers. The quaternary ammonium salt poly (diallyldimethylammonium chloride) was used as the cationic polymer, and the polymeric surfactant poly (sodium N-undecanoyl-l-leucylvalinate) was used as the anionic polymer. Previous studies have shown that the PEM-coated capillaries used for achiral separations have excellent reproducibilities and high stabilities against extreme pH values. In the current study, this PEM coating approach was applied to chiral separations of 1,1'-binaphthyl-2,2'-dihydrogenphosphate (BNP), 1,1'-bi-2-naphthol, secobarbital, pentobarbital, and temazepam. However, the PEM coating procedure used in the achiral studies needed to be significantly modified in order to achieve chiral separations. Optimal conditions were established by varying the additives (sodium chloride, 1-ethyl-3-methyl-1H-imidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium tetrafluoroborate) in the polymer deposition solutions, the salt concentration, the column temperature, and the bilayer number. Reproducibilities were evaluated by use of the relative standard deviation (RSD) values of the electroosmotic flow (EOF) and the first peak ((R)-(+)-BNP). In all cases, the run-to-run and capillary-to-capillary RSD values of EOF were less than 0.5%, and the run-to-run RSD values of the (R)-(+)-BNP peak were less than 1%. In addition, more than 230 runs were performed on a single PEM-coated capillary.     

Application of cyclam-capped beta-cyclodextrin-bonded silica particles as a chiral stationary phase in capillary electrochromatography for enantiomeric separations

Two novel types of substituted cyclam-capped beta-cyclodextrin (beta-CD)-bonded silica particles have been prepared and used as chiral stationary phases in capillary electrochromatography (CEC). The two stationary phases have a chiral selector with three recognition sites: beta-CD, cyclam, and the latter's sidearm. They exhibit excellent enantioselectivities in CEC for a wide range of compounds as a result of the cooperative functioning of the anchored beta-CD and cyclam. After inclusion of the metal ion (Ni2+) from the running buffer into the substituted cyclams and their sidearm ligands, the bonded stationary phases become positively charged and can provide extra electrostatic interactions with ionizable solutes and enhance the dipolar interactions with some polar neutral solutes. This enhances the host-guest interaction with some solutes and improves chiral recognition and enantioselectivity. These new types of stationary phases exhibit great potential for fast chiral separations in CEC.     

Chiral separations using a polypeptide and polymeric dipeptide surfactant polyelectrolyte multilayer coating in open-tubular capillary electrochromatography

A polyelectrolyte multilayer (PEM) coating consisting of the polypeptide, poly(l-lysine) hydrobromide, poly(l-lysine) and the polymeric dipeptide surfactant, poly(sodium undecanoyl-l-leucyl-alaninate), poly(l-SULA), is investigated as a new medium for the separation of chiral analytes in open-tubular capillary electrochromatography (OT-CEC). In this approach, a stable PEM is constructed in situ by alternative rinses of the cationic polymer poly(l-lysine) and the anionic polymer poly(l-SULA). In previous studies, the PEM coating has been constructed by use of the cationic polyelectrolyte poly (diallydimethylammonium chloride), PDADMAC. In this study, we investigate the use of a biopolymer as the cationic polyelectrolyte. The results reported here indicate an increase in selectivity and resolution when poly(l-lysine) is used as the cationic polymer in place of PDADMAC. To evaluate the chromatographic performance of the PEM coating as a chiral stationary phase, the separation of the beta-blockers, labetalol and sotalol, and the binaphthyl derivatives, 1,1'-bi-2-naphthyl-2,2'-dihydrogen phosphate, 1,1'-bi-2-naphthol, and 1,1-binaphthyl-2,2'-diamine, are investigated. In addition, the effect of varying the amino acid order of the polymeric dipeptide surfactant on resolution is investigated. The number of bilayers also significantly influences the separation efficiency and resolution of enantiomers. The run-to-run and capillary-to-capillary reproducibilities are evaluated by calculating the relative standard deviations (RSDs) of the electroosmotic flow. These RSD values were found to be less than 1%. The coating is also stable and allows more than 290 runs to be performed in the same capillary. In addition, coupling of this chiral OT-CEC column with mass spectrometry is investigated.     

Amino acid order in polymeric dipeptide surfactants: effect on physical properties and enantioselectivity

The effect of amino acid order on chiral selectivity in polymeric dipeptide surfactants, as well as the physical properties of the surfactants, is investigated. An understanding of enantioselectivity of such dipeptide surfactants is crucial to the design of more efficient polymeric surfactants and has implications in other areas of research such as enantioselective interactions of amino acid based compounds (i.e., enzymes, hemoglobin, antibodies, etc.). It should be noted that such polymeric surfactants are not easily crystallized. Therefore, in a manner similar to the study of proteins, fluorescence spectroscopy is a powerful tool used to study the structure-function relationship of these polymeric surfactants. The microenvironments inside the core of 18 polymeric surfactants were characterized using the environmentally sensitive probes pyrene and 6-propionyl-2-(dimethylamino)naphthalene (Prodan). The surfactants examined in this study include all possible dipeptide combinations of the L-form of alanine, valine, and leucine and the achiral amino acid glycine (except glycine-glycine) as well as the single amino acid surfactants of alanine, valine, and leucine. The results of the fluorescent probe studies led to a proposed structure of the polymeric dipeptide surfactants in solution. The implications of the proposed structure for chiral selectivity were tested with two model atropisomers, (+/-)1,1'-bi-2-naphthol and (+/-)1,1'-bi-2-naphthyl-2,2'-diyl hydrogen phosphate, using capillary electrokinetic chromatography.     

Chiral monolithic columns for enantioselective capillary electrochromatography prepared by copolymerization of a monomer with quinidine functionality. 2. Effect of chromatographic conditions on the chiral separations

The effect of chromatographic conditions on the performance of chiral monolithic poly(O-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroqui nidine-co-ethylene dimethacrylate-co-2-hydroxyethyl methacrylate) columns in the capillary electrochromatography of enantiomers has been studied. The flow velocity was found to be proportional to the pore size of the monolith and both the pH and the composition of the mobile phase. The length of both open and monolithic segments of the capillary column was found to exert a substantial effect on the run times. The use of monoliths as short as 8.5 cm and the "short-end" injection technique enabled the separations to be achieved in approximately 5 min despite the high retentitivity of the quinidine selector. Very high column efficiencies of close to 250000 plates/m and good selectivities were achieved for the separations of numerous enantiomers using the chiral monolithic capillaries with the optimized chromatographic conditions.     

Evaluation of a vancomycin chiral stationary phase in capillary electrochromatography using polar organic and reversed-phase modes

A vancomycin chiral stationary phase (CSP) was fully evaluated in capillary electrochromatography (CEC) in reversed-phase and polar organic modes for a number of racemic pharmaceutical compounds. High efficiency and resolution values were obtained for a number of compound classes including thalidomide in both the polar organic mode (190000 plates meter(-1) and Rs = 13.8) and reversed-phase mode (125000 plates meter(-1) and Rs = 13.0). Experimental parameters, including organic modifier, organic solvent ratio, ionic strength, pH, temperature, and voltage, were examined in both the aqueous and nonaqueous modes to deduce their effect on the resultant EOF, retention times, resolution, and efficiency of chiral separations. All results were consistent with and found to be a combination of what is known from existing literature on CEC theory and experience obtained with macrocyclic antibiotic CSPs in LC. Column stability was excellent, and each column packed was found to offer repeatable separations even when switching from the aqueous to the nonaqueous mode.     

Quenched phosphorescence as a detection method in capillary electrophoretic chiral separations. Monitoring the stereoselective biodegradation of camphorquinone by yeast

Quenched phosphorescence detection of camphorquinone in cyclodextrin-based electrokinetic chromatography provides very favorable detection limits, i.e., 7 x 10(-)(7) M, 3 orders of magnitude lower than conventional UV absorption detection at 200 nm. The detection is based on the dynamic quenching by the analyte of the strong phosphorescence emission of brominated naphthalenesulfonate, under deoxygenated buffer solution conditions. This approach has been used to detect (1S)-(+)- and (1R)-(-)-camphorquinone after enantiomeric separation by CE. Although the use of the negatively charged carboxymethyl beta-cyclodextrin (CM-beta-CD) alone was not successful, the addition of a second, neutral cyclodextrin, alpha-CD, provided an adequate enantiomeric separation of camphorquinone. Using 25 mM borate buffer (pH 8.5) with 10 mM CM-beta-CD and 20 mM alpha-CD (applied voltage 20 kV, ambient temperature), the enantiomeric separation was performed in approximately 14 min. The chiral method was applied to monitor the stereoselectivity of the biotransformation of a racemic mixture of camphorquinone by yeast. It was found that the enantiomeric ratio calculated from the peak areas in the electropherogram (RSD = 5%) after 24 h of incubation decreased from 0.92 for the control solution (culture medium without yeast) to 0.24 for the culture medium; a similar ratio of 0.25 was observed for cell extract solutions. Therefore, racemic camphorquinone is enantioselectively degraded by yeast, the biodegradation of (1S)-(+)-camphorquinone being faster than that of the (1R)-(-)-enantiomer.     

A new territory and its pioneer: opening up a dominant research stream for a translational research area

Scientometrics - An academic pioneer makes many trials to find relations and results that were unknown before. The verified relations and repeatable results the pioneer finds guide followers to...     

Combination of chiral capillary electrochromatography with electrospray ionization mass spectrometry: method development and assay of warfarin enantiomers in human plasma

The hyphenation of chiral capillary electrochromatography (CEC) with electrospray ionization mass spectrometry (ESI-MS) is very challenging but promising due to the fact that it combines sensitivity with high specificity and selectivity. In this work, CEC capillaries packed with (3R,4S)-Whelk-O1 chiral stationary phase were used for simultaneous enantioseparation of (+/-)-warfarin and its internal standard, (+/-)-coumachlor. Furthermore, both the chiral CEC separation and MS detection parameters were examined in detail. First, the influence of different column fabrication was investigated. Second, enantioseparation was optimized by varying CEC parameters, including acetonitrile concentration, buffer pH, and ionic strength. Under the optimum chiral CEC conditions, ESI-MS parameters such as sheath liquid pH and composition, sheath liquid flow rate, drying gas flow rate, drying gas temperature, nebulizer pressure, and fragmentor voltage were investigated to achieve maximum MS signals of the separated enantiomers. Finally, using solid-phase extraction as sample preparation method, (+/-)-warfarin spiked in 100-microL human plasma samples were analyzed. The calibration curves showed good linearity for both (R)-warfarin (R = 0.9979) and (S)-warfarin (R = 0.9978) enantiomers. The experimental limit of detection was approximately 25 ng/mL for both enantiomers. Even though the data are still preliminary, we can state with confidence that chiral CEC-ESI-MS has the potential to establish itself as a very powerful technique for the determination of enantiomeric ratios in human body fluid.     

丙烯酸酯水性涂料用高分子乳化剂的合成及性能研究

以甲基丙烯酸、苯乙烯、丙烯酸羟乙酯和丙烯酸-2-乙基己酯为主要原料,通过溶液共聚法合成了用于丙烯酸酯水性涂料的高分子表面活性剂.使用FT-IR对合成产物的结构进行了表征,测试了共聚物的表面张力、临界胶束浓度(CMC)、表观黏度,初步探讨了甲基丙烯酸用量对产物表面活性的影响.结果表明,共聚物的表观黏度(异丙醇溶剂,浓度85%)为100～530 mPa·s,具有较低的临界胶束浓度(CMC)(4～9 g/L),最低表面张力降至31.5mN/m(25℃).以此产物为乳化剂,皆能制得体系稳定的丙烯酸酯乳液,其乳胶粒粒径分布均匀.     

Polymeric sulfated amino acid surfactants: a class of versatile chiral selectors for micellar electrokinetic chromatography (MEKC) and MEKC-MS

In this work, three amino acid-derived (l-leucinol, l-isoleucinol, l-valinol) sulfated chiral surfactants are synthesized and polymerized. These chiral sulfated surfactants are thoroughly characterized to determine critical micelle concentration, aggregation number, polarity, optical rotation, and partial specific volume. For the first time the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy. The polysodium N-undecenoyl-l-leucine sulfate shows distinct tubular structure, while polysodium N-undecenoyl-l-valine sulfate also shows tubular morphology but without any distinct order of the tubes. On the other hand, polysodium N-undecenoyl-l-isoleucine sulfate (poly-l-SUCILS) displays random distribution of coiled/curved filaments with heavy association of tightly and loosely bound water. All three polymeric sulfated surfactants are compared for enantioseparation of a broad range of structurally diverse racemic compounds at very acidic, neutral, and basic pH conditions in micellar electrokinetic chromatography (MEKC). A small combinatorial library of 10 structurally related phenylethylamines (PEAs) is investigated for chiral separation under acidic and moderately acidic to neutral pH conditions using an experimental design. In contrast to neutral pH conditions, at acidic pH, significantly enhanced chiral resolution is obtained for class I and class II PEAs due to the compact structure of polymeric sulfated surfactants. It is observed that the presence of a hydroxy group on the benzene ring of PEAs resulted in deterioration of enantioseparation. A sensitive MEKC-mass spectrometry (MS) method is developed for one of the PEAs (e.g., (+/-)-pseudoephedrine) in human urine. Very low limit of detection (LOD) is obtained at pH 2.0 (LOD 325 ng/mL), which is approximately 16 times better compared to pH 8.0 (LOD 5.2 microg/mL). Another broad range of chiral analytes (beta-blockers, phenoxypropionic acid, benzoin derivatives, PTH-amino acids, benzodiazepinones) studied also provided improved chiral separation at low pH compared to high-pH conditions. Among the three polymeric sulfated surfactants, poly-l-SUCILS with two chiral centers on the polymer head group provided overall higher enantioresolution for the investigated acidic, basic, and neutral compounds. This work clearly demonstrates for the first time the superiority of chiral separation and sensitive MS detection at low pH over conventional high-pH chiral separation and detection employing anionic chiral polymeric surfactants in MEKC and MEKC-MS.     

Enantiomer separations in capillary electrophoresis in the case of equal binding constants of the enantiomers with a chiral selector: commentary on the feasibility of the concept

It is generally acknowledged that enantiomer separations in capillary electrophoresis are based on differences in the affinities of the analyte enantiomers toward the chiral selector expressed as equilibrium constants of the resulting temporary diastereomeric associates. However, as can be derived from theoretical considerations, a separation of enantiomers by CE is in principle also possible solely based on differences in the mobilities of the temporary diastereomeric complexes per se, when equal binding constants between analyte enantiomers and the chiral selector are assumed and observed.     

Living cells of Staphylococcus aureus immobilized onto the capillary surface in electrochromatography: a tool for screening of biofilms

Microorganisms attach to nonliving surfaces in many natural, industrial, and medical environments, enveloped within extracellular polymeric substances. The result is a biofilm. Biofilms are reported to exist in 65-80% of bacterial infections refractory to host defenses and antibiotics therapy and are regarded as a central problem in present-day medical microbiology. Understanding of the parameters governing the interaction of antimicrobials with biofilms is thus of great interest in any attempt to increase biocide efficacy. In this work, study was made of the feasibility of using open tubular capillary electrochromatography (CEC) in bacterial biofilm studies with living cells. Staphylococcus aureus was selected as model bacterium. First, S. aureus was shown, under various conditions, to form a biofilm on the inner wall of a fused-silica capillary coated with poly(L-lysine). Optimal conditions for biofilm formation, such as bacterial concentration, growing time, and the stability of the ensemble, were preliminarily defined with conventional 96-microtiter well plates. Continuous flushing of the capillary with fresh cells meant that no growth medium was needed. The presence of biofilm in the capillary was confirmed by atomic force microscopy. Interactions between S. aureus biofilms and different antibiomicrobial agents were studied by capillary electrochromatography. The effect of five antibiotics (penicillin G, oxacillin, fusidic acid, rifampicin, vancomycin) on biofilms was examined in terms of retention factors and reduced mobilities of the antibiotics. The antibiotic susceptibility profile for S. aureus is similar as the result of minimal inhibitory concentrations registered on the 96-microtiter well plates for both planktonic and biofilm cells. The results show, for the first time, that bacterial biofilms can be studied by CEC. The technique allows highly efficient and easy characterization of interactions between S. aureus biofilms and potentially active antimicrobial compounds under different conditions. Reagent and cell consumption are minimal.     

Examination of structural changes of polymeric amino acid-based surfactants on enantioselectivity: effect of amino acid order, steric factors, and number and position of chiral centers

In this study, a large number of polymeric chiral surfactants were examined and their performances in terms of enantiomeric resolution compared for a variety of chiral analytes. The surfactants investigated in this study include all possible dipeptide combinations of the L-form of alanine, valine, leucine, and the achiral amino acid glycine (except glycine-glycine). Also included in this study were the single amino acid surfactants of alanine, valine, and leucine as well as the single chiral center dipeptide surfactant poly(sodium undecyl-L-leucine-beta-alanine) (poly L-SULbetaA). Several different aspects of polymeric dipeptide surfactants, as they pertain to chiral separations, are examined. Some of the factors investigated in this report include the effect of position and number of chiral centers, amino acid order, and steric effects.     

Use of a mixed-mode packing and voltage tuning for peptide mixture separation in pressurized capillary electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer detector.

A mixed-mode (reversed-phase/anion-exchange) stationary phase has been used as the capillary column packing for investigation of the separation of peptide mixtures in pressurized capillary electrochromatography (pCEC). This stationary phase contains both octadecylsilanes and dialkylamines. The amine groups of the stationary phase determine the charge density on the surface of the packing and can produce a strong and constant electroosmotic flow (EOF) at low pH. A comparison was made in terms of the capability of separating tryptic digests between the mixed-mode phase and C18 reversed phase. In addition, the constant EOF enabled the tuning of the retention and the selectivity of the separation by adjusting the mobile phase pH from 2 to 5. Furthermore, the magnitude and the polarity of the electric voltage were demonstrated to greatly influence the elution profiles of the peptides in pCEC. An ion trap storage/reflectron time-of-flight mass spectrometer was used as an on-line detector in these experiments due to its ability to provide rapid and accurate mass detection of the sample components eluting from the separation column.     

Synthesis of a new family of Schiff base nonionic surfactants and evaluation of their corrosion inhibition effect on X-65 type tubing steel in deep oil wells formation water

Some new Schiff base nonionic surfactants were synthesized and characterized using FTIR and ¹H NMR spectroscopy. The corrosion inhibition effect of the synthesized surfactants on X-65 type tubing steel in deep oil wells formation water has been studied by polarization, electrochemical impedance spectroscopy (EIS) and weight loss measurements. Polarization data indicate that the selected surfactants act as mixed type (cathodic/anodic) inhibitors. EIS results show that the change in impedance parameters with the concentration of the surfactants studied is indicative of the adsorption of surfactant molecules on carbon steel surface. The adsorption of the inhibitors was well described by the Langmuir adsorption isotherm. Effect of temperature on the efficiency of the corrosion inhibition process was studied and the values of activation energy, enthalpy of activation and entropy of activation were calculated and discussed. The corrosion inhibition efficiency of the surfactants is correlated with surface tension measurements data. The protective film formed on the carbon steel surface was studied by scanning electron microscopy (SEM) and energy dispersive analysis of X-rays (EDX) techniques.     

Exploring the possibilities of cryogenic cooling in liquid chromatography for biological applications: a proof of principle

The possibilities to use cryogenic cooling to trap components in liquid chromatography was investigated. In a first step, van 't Hoff plots were measured with a reversed-phase column using the temperature control unit of a conventional high performance liquid chromatography (HPLC) system to gain insight in the retention behavior of proteins at low temperatures. It was estimated that retention factors in the range of k = 10(4) could be achieved at T = -20 °C for lysozyme, indicating that temperature is a usable parameter to trap components in LC. In a next step, trapping experiments were carried out on a nano-LC system, equipped with a UV-detector, using a commercial reversed-phase column. An in-house built setup, allowing cooling of a segment of the column down to temperatures below T = -20 °C, was used to trap components. Experiments were conducted under isocratic and gradient conditions with methanol as organic solvent. It is demonstrated that, by thermally trapping and elution of components, an enhanced S/N ratio and decreased peak widths can be obtained. At the same time, a significant increase in pressure drop occurs during the cooling process. Limitations and benefits of the technique are further discussed.     

Accuracy in the determination of isoelectric points of some proteins and a peptide by capillary isoelectric focusing: utility of synthetic peptides as isoelectric point markers

To evaluate the accuracy ofisoelectric point determination by capillary isoelectric focusing, the pI values of nine proteins and a peptide, the pI values of which had been determined by other methods and ranging pI 3.55-9.60, were determined by capillary isoelectric focusing by cofocusing of recently developed peptide pI markers ranging 3.38-10.17, and the consistency of the pI values was examined. Isoelectric focusing was carried out in neutral polymer-coated capillaries, and the pH gradient was mobilized by pressure toward the cathode, to detect samples with absorption at 280 nm at a fixed detection point. Carrier ampholytes from two different suppliers and in different pH ranges were used. The sharp peaks of the highly pure peptide pI markers greatly facilitated the unambiguous identification of the peaks. When a carrier ampholyte ranging over the acidic side was used, the detection of acidic pI samples was anomalously delayed. This could be partly mitigated by reducing the viscosity of the anode solution in comparison with the pH gradient formed in the capillary. Since the detection times vs the pH relationships were not linear in most cases, the use of a linear calibration line over an entire pH gradient would be erroneous. Instead, the pI values of samples were calculated by assuming a linear relation for pH against detection time between two flanking marker peptides. Close agreement between the pI values, determined by capillary isoelectric focusing, and the reference values of the samples was observed within an average difference range of 0.04-0.08 pH unit with a sample consumption of 10-100 ng within 30-60 min. Some carrier ampholytes were preferentially more effective at either the acidic or the basic side of the pH gradient. For confirmation of the completion of focusing, the use of two different focusing times is recommended.     

Synthesis and characterization of a new inorganic cation-exchanger-Zr(IV) tungstomolybdate: analytical applications for metal content determination in real sample and synthetic mixture

An amorphous sample of inorganic cation-exchanger Zr(IV) tungstomolybdate was prepared by mixing varying ratios of 0.1M aqueous solution of sodium tungstate and 0.1M aqueous solution of sodium molybdate into 0.1M aqueous solution of zirconium oxychloride at pH 1. This cation-exchanger was found to have a good ion-exchange capacity (2.40 mequiv.g(-1) for Na(+)), high thermal and chemical stability. A tentative structural formula was proposed on the basis of chemical composition, FTIR and thermogravimetric analysis. Distribution coefficients (K(d)) values of metal ions in various solvent systems were determined. Some important and analytically difficult quantitative binary separations viz. Ni(II)-Pb(II), Ni(II)-Zn(II), Ni(II)-Cd(II), Mg(II)-Al(III), etc. were achieved. The practical applicability of the cation-exchanger was demonstrated in the separation of Cu(II)-Zn(II) from a synthetic mixture as well as from real samples of pharmaceutical formulation and brass alloy.