Applications of a sulfonated-polymer wall-modified open-tubular fused-silica capillary in capillary zone electrophoretic separations

A fused-silica capillary that is wall-modified via chemically bonding a sulfonated polymer to the capillary wall has a uniform negative charge density on its surface and produces an electroosmotic flow (EOF) greater than 4 x 10(-4) cm2 V(-1) s(-1) The EOF is nearly independent of buffer pH over the pH range of 2 to 10 and is lower than the EOF obtained for the bare fused-silica capillary at the more basic pH but is higher at the more acidic buffer pH. Optimization of buffer pH can be based on analyte pKa values to improve the overall quality of the capillary zone electrophoresis (CZE) separation of complex mixtures of weak acid and base analytes. Because of the high EOF in an acidic buffer, the capillary is useful for the separation of weak organic bases which are in their cation forms in the acidic buffer. EOF for the sulfonic acid bonded phase capillary can be adjusted via buffer additives such as organic solvent, tetraalkylammonium salts, multivalent cations and alkylsulfonic acids. The advantages of utilizing buffer pH and the EOF buffer modifiers to enhance migration time, selectivity, and resolution in CZE separations with this capillary are illustrated using a series of test analyte mixtures of inorganic anions, carboxylic acids, alkylsulfonic acids, benzenesulfonic acids, sulfas, pyridines, anilines or small-chain peptides.     

Cationic diazacrown ether for the separation of positional isomers by capillary electrophoresis

The use of a positively charged diazacrown ether, N,N,N',N'-tetramethyl-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane dichloride (1), in capillary electrophoresis is reported, for the first time. The addition of the cationic diazacrown 1 to the running buffer permitted the successful separation of positional isomers of aromatic anions such as naphthalenedisulfonate, naphthalenedicarboxylate and phthalate. At a concentration of 1 of 2 mM, all the analyte anions were completely separated in less than 5 min. The ion association constants of the solute anions with diazacrown 1 were evaluated and the findings are discussed.     

Capillary-electrochromatographic separations with copolymeric reversed-stationary phase and ion-exchanger-packed columns

A macroporous, spherical, 7 μm, polystyrene–divinylbenzene (PS–DVB), reversed-phase adsorbent (PRP-1) was evaluated as a stationary phase for the capillary electrochromatographic (CEC) separation of neutral, acidic, and basic analytes of pharmaceutical interest. Electroosmotic flow (EOF) for a PRP-1 packed capillary is nearly constant over the pH 2 to 10 range and is higher than for a silica-based C₁₈ packed capillary on the acidic side. EOF increases with an increase in buffer acetonitrile concentration or as applied potential increases. As analyte hydrophobicity increases, analyte retention and migration time increases. Increasing buffer acetonitrile concentration reduces analyte partitioning with the PS–DVB stationary phase and analyte retention and migration time decreases. When exchange sites are present on the PS–DVB copolymer, EOF (EOF is reversed for the anion-exchanger) increases as the exchange capacity increases. An increased exchange capacity also reduces partitioning of the analyte with the PS–DVB matrix and analyte retention and migration time decrease. Because of excellent stability in an acid environment, the PRP-1 packed capillary can be used in strong acid buffer solution and weak acid and base analytes depending on pK_a values can be separated as neutral species and cations, respectively. CEC separations on a PRP-1 capillary of neutral steroids, weak base pharmaceuticals (separation as cations), purines and pyrimidines (as cations), fatty acids (as undissociated species), and sulfa derivatives (as cations) are described. Efficiency for the PRP-1 packed capillary for acetone or thiourea as the analyte is about 6·10⁴ plates m⁻¹.     

Semi-permanent surfactant coatings for inorganic anion analysis in capillary electrophoresis

Capillary electrophoretic separations of inorganic anions are performed using a capillary coated with a mixture of the cationic surfactant didodecyldimethylammonium bromide (DDAB) and the zwitterionic surfactant 1,2-dilauroyl-sn-phosphatidylcholine (DLPC). These double-chained surfactants form semi-permanent coatings on the capillary wall, which allows the excess surfactant to be removed from the buffer prior to separation. Interactions between surfactant aggregates in the buffer and analyte anions are thus eliminated. The electroosmotic flow (EOF) can be altered from fully reversed (100% DDAB) to near zero (100% DLPC) using different ratios of DDAB and DLPC. Controlling the EOF allows for improved resolution of the anions while maintaining a rapid, co-EOF separation, free from analyte-surfactant additive interactions.     

Use of 1-alkyl-3-methylimidazolium-based ionic liquids as background electrolytes in capillary electrophoresis for the analysis of inorganic anions

Separation of inorganic anions in CE is often a challenging task because the electrophoretic mobilities of inorganic anions are comparable to or even greater than the EOF mobility. In this study, we present the use of ionic liquids (ILs) as background electrolytes (BGEs) in CE of inorganic anions. The 1-alkyl-3-methylimidazolium-based ILs as BGEs dynamically coated the capillary wall and induced a reversed EOF. This allowed the anions to comigrate with the EOF and yielded a rapid separation. Increasing the alkyl chain length of the ILs and BGE concentration can significantly improve the separation resolution. With 40 mM 1-butyl-3-methylimidazolium tetrafluoroborate as BGE, good separations of five model anions (Br-, I-, NO2(-), NO3(-), and SCN-) were achieved in a range of buffer pH values. The separation efficiency was as high as 34 600-155 000, and the RSDs of the migration times were less than 0.8% (n = 5).     

Long-chained gemini surfactants for semipermanent wall coatings in capillary electrophoresis of proteins

In this paper, we presented the first example of using gemini surfactants as semipermanent coatings in CE for protein separation. These coatings are based on the self-assembly of a series of cationic gemini surfactants, alkanediyl-alpha,omega-bis(dimethylalkylammonium bromide) (m-s-m), on the capillary wall. The coatings can keep stable for a long time without surfactant in the buffer, e.g., after the surfactants were removed from the buffer, the reversed EOF only decreased by 3.6 and 3.9% for 18-2-18 and 16-2-16 coatings over 60 min under continuous electrophoretic conditions. The coating stability increased with the alkyl chain length m. The double long chains of geminis (m > or = 14) yielded a good coating stability; meanwhile, the spacer group acted as an EOF modifier. Thus, this bifunctional surfactant coating provided a new buffer-independent method for EOF control. For 18-s-18 series, the best coating stability and largest EOF were obtained at s = 10. Ranging s from 3 to 10 yielded a linear fine-tuning of EOF and thereby allowed the adjustment of the protein apparent mobility. Highly efficient separation (>500 000 plates/m) was achieved with all the 18-s-18 coatings. Excellent run-to-run and day-to-day reproducibility (RSD of migration time 相似文献   

New isoelectric buffers for capillary electrophoresis: N-carboxymethylated polyethyleneimine as a macromolecular isoelectric buffer

Isoelectric buffers are attractive for electrophoresis because of their low conductivity, and their compatibility with indirect photometric detection in capillary electrophoresis (CE) where they do not interfere with the detection by exhibiting competitive displacement of the UV-absorbing probe ion. N-carboxymethylated polyethyleneimine (CMPEI) was prepared by introducing a half molar equivalent of carboxylate groups onto a polyethyleneimine backbone. Its isoelectric point determined by conductometric titration and from the pH of its dilute aqueous solution is approx. 6.8, which allows isoelectric buffering at a lower pH compared to histidine (pI7.7). Although the isoelectric point is somewhat diffuse, as expected for a polymeric compound, it exhibits a buffering capacity at a pI point of about twice that of histidine. Studies of electroosmotic flow (EOF) profile at various pH values in fused silica capillaries showed that CMPEI adsorbs onto the fused silica wall and reverses the EOF at pH < 6.5. CMPEI was applied as a buffer in an electrolyte containing 0.5 mM of the anionic dye tartrazine used as the probe for indirect detection of anions. The separation system exhibited a stable baseline, no system peaks, separation efficiencies of up to 195,000 theoretical plates, and detection limits down to 0.2 microM or 2 amol of injected analyte.     

Suppression of electroosmotic flow and its application to determination of electrophoretic mobilities in a poly(vinylpyrrolidone)-coated capillary

A hydrophilic polymer, poly(vinylpyrrolidone) (PVP), was employed for suppressing the electroosmotic flow (EOF). A capillary was filled with aqueous PVP solution for coating the capillary wall with PVP; the PVP solution was then replaced by a migration buffer solution containing no PVP. Three types of PVP with different molecular weights were examined. The EOF was suppressed more effectively as the molecular weight of PVP increased. The EOF in the coated capillary was approximately 10-fold smaller than that of a bare capillary and was constant in the pH range of 6-8. The suppressed EOF was stable even when no PVP was added to the migration buffer. However, the EOF increased significantly when sodium dodecyl sulfate was added into the migration buffer. The method was applied for determining the electrophoretic mobilities of inorganic anions that have negative electrophoretic mobilities larger than the electroosmotic mobility of the bare capillary. A novel method for determining the electrophoretic mobilities was proposed based on the linear relationship between electric current and electrophoretic mobility. The electrophoretic mobility was proportional to the electric current. Therefore, the intercept of the regression equation represents the electrophoretic mobility at room temperature. The electrophoretic mobilities were in good agreement with the absolute electrophoretic mobilities.     

Fast and sensitive diagnosis of thalassemia by capillary electrophoresis

This paper demonstrates the diagnosis of -thalassemia by capillary electrophoresis in conjunction with laser-induced fluorescence using poly(ethylene oxide) (PEO) solutions in the presence of electroosmotic flow (EOF). During the electrophoretic separation, PEO solution entered a capillary from the anodic vial by EOF. The separation of a mixture of the polymerase chain reaction (PCR) products (330 and 334 base pairs) from a healthy person and a -thalassemia patient was accomplished within 15 min at 15 kV using 1.5% PEO containing 2 M urea at 30 °C. The electropherogram patterns instead of migration times were used to diagnose -thalassemia, with an accuracy of 100% for the analyses of 11 blood samples from suspected patients. After injecting a large volume of the mixture to the capillary filled with 800 mM Tris-borate buffer (pH 10.0), the DNA fragments stacked due to increases in viscosity and sieving when migrating into 1.5% PEO solution. As a result of improved sensitivity, only 15 PCR cycles were required when using 500 ng of DNA templates. The results shown in this study indicate the potential of this simple, rapid, and cost-effective method for the diagnosis of -thalassemia.Abbreviations CE Capillary electrophoresis - EOF Electroosmotic flow - EtBr Ethidium bromide - LIF Laser-induced fluorescence - PCR Polymerase chain reaction - PEO Poly(ethylene oxide) - TRIS Tris(hydroxymethyl)aminomethane - TB TRIS-borate     

Nucleoside monophosphates recognition using macrocyclic polyamine bonded phase in capillary electrochromatography

An open-tubular wall-coated macrocyclic polyamine capillary column (70 cm x 75 microm ID) with 50 cm effective length for the separation of nucleoside monophosphates is described. Some parameters with respect to concentration, pH, composition of the buffer, and voltage in order to optimize the separation were studied. The coated capillary showed reversed electroosmotic flow (EOF), allowing anions to be separated in the co-EOF mode. Baseline separations were achieved for the eight nucleotides in less than 26 min using a background electrolyte consisting of H(3)PO(4)-NaH(2)PO(4) (30 mM, pH 3.10), an applied voltage of -15 kV, and detection at 254 nm. The macrocyclic polyamine on the capillary wall introduced anion coordination for the interaction with the analytes, the strength of which could be moderated by the type and concentration of the competing ion used in the background electrolyte (BGE). With a low concentration of the competing ion (phosphate ion), the migration behavior followed that obtained in the electrophoretic system. Increasing the concentration of the competing ion resulted in a faster migration and more complete elution of the analyte. The method established was also employed for the analysis of nucleotides in mushrooms. Aqueous extracts of mushrooms from different species and various extraction methods were injected directly for the analysis. Uridine 5'-monophosphate, guanosine 5'-monophosphate, adenosine 5'-monophosphate, and cytidine 5'-monophosphate, were found in the sample tested.     

Protein separation by open tubular capillary electrochromatography employing a capillary coated with phenylalanine functionalized tentacle-type polymer under both cathodic and anodic electroosmotic flows

The use of a phenylalanine (Phe) functionalized tentacle-type polymer coated capillary column for protein separation by open tubular capillary electrochromatography (OTCEC) was demonstrated in this work. The tentacle-type stationary phase was prepared from silanized fused-silica capillaries of 50 microm I.D. by glycidyl methacrylate graft polymerization and subsequent Phe functionalization. Due to the amphoteric functional groups of the Phe bonded on the tentacle-type polymer stationary phase, protein separation in the prepared column can be performed under both cathodic and anodic electroosmotic flow (EOF) by varying the pH values of the mobile phase. Model proteins including ribonuclease A (RNase A), myoglobin, transferrin, insulin were baseline separated under cathodic EOF with a mobile phase of pH 8.8. Comparison between the separation result of the four proteins under conditions of OTCEC and capillary zone electrophoresis indicates that the migration behavior of the four proteins in the prepared column was the result of the interplay of chromatographic retention and electrophoretic migration. Besides, three basic proteins including RNase A, cytochrome c (Cyt-c) and lysozyme (Lys) were fully resolved under anodic EOF with an acidic running buffer (pH 2.5). The elution order was the same as the isoelectric point values of the proteins (RNase A相似文献   

Separation of atropisomers of anti-hepatitis drug dimethyl diphenyl bicarboxylate analogues by capillary electrophoresis with vancomycin as the chiral selector

Separation of atropisomers of analogues of the anti-hepatitis drug dimethyl diphenyl bicarboxylate (DDB) by capillary electrophoresis with vancomycin as the chiral selector is described. Among several tested chiral selectors, including various cyclodextrin derivatives as well as vancomycin, only the latter displayed the enantioselectivity to the studied atropisomers. However, relatively poor separation efficiency was obtained due to the adsorption of vancomycin on the capillary wall. This problem was overcome by modifying the capillary wall with a polycationic electrolyte named hexadimethrine bromide (HDB) to produce a positively charged coating, which minimized the adsorption of vancomycin on the capillary wall by electrostatic repulsion. Moreover, the positively charged coating could shorten the separation time by reversing the EOF because the reversed EOF migrated to the same direction as the negatively charged analyte. Effects of buffer pH, vancomycin and buffer concentrations and applied voltage on the separation were investigated and the optimal conditions were established as follows: 40 mM Tris-phosphate buffer (pH 6.0) containing 6.0 mM vancomycin and 0.001% HDB. Baseline separation of three racemic DDB analogues was obtained within 12 min under the optimal conditions.     

Enantiomeric separation by capillary electrophoresis with an electroosmotic flow-controlled capillary

Perfect control of electroosmotic flow (EOF) was achieved by dovetailing successive multiple ionic-polymer layer (SMIL) coated capillaries. The direction and magnitude of the EOF was perfectly controllable over the pH range 2-13. Zone diffusion was not observed, even if the inner wall of the dovetailed capillary was discontinuous, or if the sample zone passed through the connected part of the capillary because the RSDs of migration time, theoretical plates, symmetry factor and S/N of the marker were almost the same when seamless capillary and dovetailed capillary were compared. The dovetailed capillary was applied to cyclodextrin modified capillary zone electrophoresis. The control of the EOF enabled us to control both the resolution and the migration order of the enantiomers. The migration time was also controllable and, therefore, the best condition between separation and migration time could be determined by controlling the EOF. Partial filling affinity electrokinetic chromatography with a protein used as a chiral selector was also studied. The migration of the pseudostationary phase was controllable by EOF, and detection of the solute at 214 nm was possible. Therefore, the EOF-controlled dovetailed capillary has great potential to expand the application of the separation technique.     

Ion association with alkylammonium cations for separation of anions by capillary electrophoresis

A background electrolyte (BGE) containing a 100 mM concentration of an alkylammonium cation with ethyl, propyl or butyl groups provides an excellent medium for separation of anions by capillary electrophoresis (CE). Two major effects were noted. Use of one of a series of alkylammonium cations in the BGE at a selected pH provides a simple and effective way to vary and control electroosmotic flow (EOF) over a broad range. It is believed that the alkylammonium cations are coated onto the capillary surface through a reversible dynamic equilibrium. Secondly, alkylammonium cations modify the electrophoretic migration of sample anions and the electroosmotic migration of neutral organic analytes by association interaction. This selective interaction results in improved anion separations and permits the simultaneous separation of neutral analytes. The degree of association interaction varies with the bulk and hydrophobicity of the alkylammonium cations. Incorporation of an aliphatic amine salt of moderate molecular weight in the running electrolyte provides a valuable new way to vary the migration times of sample anions and to optimize their resolution. The interactions between alkylammonium cations and sample anions or neutral organics appear to take place entirely within the liquid phase and do not require a polymeric or micellar pseudo phase.     

Capillary electrochromatography with zwitterionic stationary phase on the lysine-bonded poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column

A polymer-based neutral monolithic capillary column was prepared by radical polymerization of glycidyl methacrylate and ethylene dimethacrylate in a 100 mum id fused-silica capillary, and the prepared monolithic column was subsequently modified based on a ring opening reaction of epoxide groups with 1 M lysine in solution (pH 8.0) at 75 degrees C for 10 h to produce a lysine chemically bonded stationary phases in capillary column. The ring opening reaction conditions were optimized so that the column could generate substantial EOF. Due to the zwitterionic functional groups of the lysine covalently bonded on the polymer monolithic rod, the prepared column can generate cathodic and anodic EOF by varying the pH values of running buffer during CEC separation. EOF reached the maximum of -2.0 x 10(-8) m2v(-1)s(-1) and 2.6 x 10(-8) m2v(-1)s(-1) with pH of the running buffer of 2.25 and 10, respectively. As a consequence, neutral compounds, ionic solutes such as phenols, aromatic acids, anilines, and basic pharmaceuticals were all successfully separated on the column by CEC. Hydrophobic interaction is responsible for separation of neutral analytes. In addition, the electrostatic and hydrophobic interaction and the electrophoretic migration play a significant role in separation of the ionic or ionizable analytes.     

pH effect on dynamic coating for capillary electrophoresis of DNA

A buffer consisting of tris(hydroxymethyl)aminomethane, 2-(N-moropholino)ethanesulfonic acid (Mes) and EDTA with constant ion strength was used to investigate the effect of buffer pH on the dynamic coating behavior of poly(N-isopropylacrylamide) (PNIPAM) for DNA separation. The atomic force microscopy (AFM) image illustrated that PNIPAM in lower-pH buffer was much more efficient in covering a silica wafer than that in higher-pH buffer. The coating performance of PNIPAM was also quantitatively analyzed by Fourier transform IR attenuated total reflectance spectroscopy and by measuring the electroosmotic flow (EOF). These results indicated that the stability of the dynamic coating was dependent on the pH of the sieving matrix and was improved by reducing the pH to the weak-acid range. The lower pH of the sieving buffer may induce the polymer more efficiently to adsorb on the capillary wall to suppress EOF and DNA–capillary wall interaction for DNA separation. The enhanced dynamic coating capacity of PNIPAM in lower-pH buffer may be attributed to the hydrogen bonds between the hydroxyl groups of the silica surface and the oxygen atom of the carbonyl groups of PNIPAM.     

Polymeric ionic liquid-coated capillary for capillary electrophoresis

A new application of the polymeric ionic liquid (PIL) in capillary electrophoresis is reported. Poly(1-vinyl-3-butylimidazolium bromide) was physically adsorbed on silica capillary as the simple and effective coating for capillary electrophoresis (CE) analysis, in which the PIL is not present in the background electrolyte. The electroosmotic flow (EOF) of the PIL-coated capillary as compared with that of the bare fused-silica capillary shows a different dependence on electrolyte pH values. The EOF is reversed over a wide pH range from 3.0 to 9.0 and shows good repeatability. It is also found that the coated capillary has a good tolerance to some organic solvents, 0.1 M NaOH and 0.1 M HCl. The PIL-coated capillary has been employed in different areas. Both the basic proteins and anionic analytes can be well separated by PIL-coated capillaries in a fast and easy way. The PIL-coated capillary is also able to separate organic acid additives in a grape juice. The results showed that this type of coating provides an alternative to the CE separation of anions and basic proteins.     

Adjusting the selectivity of inorganic anion separation by capillary electrophoresis

The separation of the principal inorganic anions (bromide, carbonate, chlorate, chloride, fluoride, nitrate, nitrite, sulfate, phosphate) has been achieved using a capillary electrophoresis system with indirect UV detection at 260 nm. Several types of cationic surfactants (quaternary ammonium, phosphonium or methonium) were tested as electroosmotic flow modifiers and added to a chromatebased buffer prepared from potassium dichromate. The influence of many physicochemical parameters such as nature and concentration of cationic surfactant, buffer pH, dichromate concentration buffer, voltage and temperature upon the migration time of an analyte anion, peak efficiency, asymmetry factor, and finally resolution has been investigated. A linear relationship between the corrected area and the anion concentration in the 2.5–50 ppm range was obtained, thus allowing the quantitative analysis of anions in mineral water. Finally, by increasing the hydrodynamic injection time, the separation of inorganic anions at a low concentration level of 50 ng/ml was achieved without any loss of resolution.     

Separation of cold medicine ingredients by capillary electrophoresis

This study demonstrates the separation of cold medicine ingredients (e.g., phenylpropanolamine, dextromethorphan, chlorpheniramine maleate, and paracetamol) by capillary zone electrophoresis and micellar electrokinetic chromatography. Factors affecting their separations were the buffer pH and the concentrations of buffer, surfactant and organic modifiers. Optimum results were obtained with a 10 mM sodium dihydrogen-phosphate-sodium tetraborate buffer containing 50 mM sodium dodecyl sulfate (SDS) and 5% methanol (MeOH), pH 9.0. The carrier electrolyte gave a baseline separation of phenylpropanolamine, dextromethorphan, chlorpheniramine maleate, and paracetamol with a resolution of 1.2, and the total migration time was 11.38 min.     

Polymeric ionic liquid as a background electrolyte modifier enhancing the separation of inorganic anions by capillary electrophoresis

A novel and easy method for the separation of inorganic anions by capillary electrophoresis using a polymeric ionic liquid (PIL), poly(1-vinyl-3-butylimidazolium bromide) as a background electrolyte modifier has been developed. The PIL has been proved to generate a reversed electroosmotic flow which reduces the analysis time and improves the separation significantly. Effects of the PIL concentration and buffer composition (pH and concentration) were evaluated on basis of the resolution and efficiency of the sample. Under optimum conditions, good separation of six model inorganic anions was achieved with high efficiency and excellent reproducibility within 3 min. The results obtained indicate that the combination of reversed EOF and the association between the analytes and the PIL on the capillary wall or BGE play a prominent role in the separation of anions. Therefore, the PIL presents a useful alternative for the BGE modifier in the study of inorganic anions by CE.