Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self‐assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3‐cyanopropyldimethylchlorosilane (CPDCS) or n‐octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m^?2, respectively, compared to 17 ± 1 mJ m^?2 for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2‐dilauroyl‐sn‐glycero‐3‐phosphocholine or 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine to CPDCS‐ or ODCS‐modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3–1.9 × 10^?4 cm² V^?1s^?1) compared to CPDCS‐ and ODCS‐modified or bare capillaries (3.6 ± 0.2 × 10^?4 cm² V^?1s^?1, 4.8 ± 0.4 × 10^?4 cm² V^?1s^?1, and 6.0 ± 0.2 × 10^?4 cm² V^?1s^?1, respectively), with increased stability compared to phospholipid bilayer coatings. HPB‐coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.     

Coupled fused silica capillaries for rapid capillary zone electrophoresis of proteins

A novel two-dimensional electrophoretic system for the control of electroosmosis in capillary zone electrophoresis has been developed and evaluated for rapid separations of proteins. The system comprises uncoated and polyether-coated fused silica capillaries coupled in series. An equation relating the average electroosmotic flow velocity in the coupled capillaries to the intrinsic electroosmotic velocities of the connected segments and their corresponding lengths has been derived and verified experimentally. This approach has the advantage of enabling the electroosmotic flow to be tuned independently of the applied voltage. As a consequence, rapid protein analysis at relatively low field strength was achieved without sacrificing the high separation efficiencies obtained with surface-modified capillaries.     

Application of open tubular electrochromatography for acidic and basic analytes

Summary The separation of charged analytes by electrochromatography has been examined on porous-layer open tubular capillaries prepared using the sol gel method. An electroosmotic flow of about 2.10×10⁴ cm²V¹s¹ was obtained using between 10 and 30% acetonitrile in the mobile phase. Acidic diuretic drug compounds were successfully separated at high pH as were theN-alkylanilines in their basic and neutral forms. The limitation of open tubular columns was observed on separating some basic pharmaceutical drugs. These components showed severe peak tailing and were not resolved on a 20 μm i.d. porous silica layer open tubular column. Strongly acidic components could not be detected on these columns due to their higher counter electromobilities. The successful separation of neutral aryl alkyl ketones with an efficiency of 101,533 plates m¹ for butyrophenone was an indication of the improved phase ratio on this type of open tubular columns.     

Development of the resolution theory for electrophoretic exclusion

Electrophoretic exclusion, a technique that differentiates species in bulk solution near a channel entrance, has been demonstrated on benchtop and microdevice designs. In these systems, separation occurs when the electrophoretic velocity of one species is greater than the opposing hydrodynamic flow, while the velocity of the other species is less than that flow. Although exclusion has been demonstrated in multiple systems for a range of analytes, a theoretical assessment of resolution has not been addressed. To compare the results of these calculations to traditional techniques, the performance is expressed in terms of smallest difference in electrophoretic mobilities that can be completely separated (R = 1.5). The calculations indicate that closest resolvable species (Δμ_min) differ by approximately 10^?13 m²/Vs and peak capacity (n_c) is 1000. Published experimental data were compared to these calculated results.     

Mobilization of electroosmotic flow markers in capillary zone electrophoresis

UV-absorbing neutral substances are commonly used as markers of mean electroosmotic flow in capillary electrophoresis for their zero electrophoretic mobility in an electric field. However, some of these markers can interact with background electrolyte components and migrate at a different velocity than the electroosmotic flow. Thus, we tested 11 markers primarily varying in their degree of methylation and type of central atom in combination with five background electrolyte cations differing in their ionic radii and surface charge density, measuring the relative electrophoretic mobility using thiourea as a reference marker. Our results from this set of experiments showed some general trends in the mobilization of the markers based on the effects of marker structure and type of background electrolyte cation on the relative electrophoretic mobility. As an example, the effects of an inadequate choice of marker on analyte identification were illustrated in the electrophoretic separation of glucosinolates. Therefore, our findings may help electrophoretists appropriately select electroosmotic flow markers for various electrophoretic systems.     

Direct determination of bromide ions in seawater by capillary zone electrophoresis using polyethyleneimine-coated capillaries

A rapid and simple capillary electrophoretic method was developed for the direct determination of bromide ion in seawater. We have found an effective method, based on the use of polyethyleneimine-coated capillaries and the addition of sodium chloride to the background electrolyte. The use of coated capillaries with a cationic polymer changes the direction of the electroosmotic flow in the capillary, which favors the migration speed of the bromide ion and enables the use of low salt concentrations in the separation electrolyte. Bromide ion in seawater can be determined within 2 min using this system and 20 mmol L^-1 NaCl-containing separation electrolyte. The detection limit for the bromide ion was 0.45 g ml^-1. The method was applied to the determination of bromide ion in seawater samples collected from the Bosphorus and the Black Sea. Bromide contents in samples from 0 to 72 m depths varied between 33.2 and 72.8 mg L^-1 with a mean 3.0% RSD.     

Modification of silica surfaces for CZE by adsorption of non-ionic hydrophilic polymers or use of radial electric fields

Polyvinylalcohols (PVA) and hydroxyethylcelluloses (HEC) have been used as additives in cyclodextrin-modified capillary zone electrophoretic chiral separations of aromatic amines such as tocainide and its analogs in unpretreated 50 μm i.d. fused silica capillaries. The additives were used at low concentrations (<0.05%) in common buffers, together with the γ-cyclodextrin as chiral selector. They reduce the electroosmotic flow, i.e. increase the migration times of the analytes in these chiral separations, and, moreover, considerably improve both peak symmetry and the widths of the peaks relative to migration time. In terms of the chromatographic theory of efficiency, more than 200000 theoretical plates can be achieved with unpretreated fused silica capillaries. This enhancement of efficiency arises because adsorptive “dynamic” coating with the hydroxylic modifier molecules suppresses adsorption of the analyte molecules by the capillary walls. The influence of field strength and buffer composition on the separation efficiency attainable with and without modifier in the buffers has also been investigated. Alternative experiments on the influence of analyte adsorption on efficiency have been performed by superimposing radial electric fields on the capillary to modify the ζ potentials. Although the EOF could be freely adjusted, it was not possible to obtain an improvement in efficiency comparable with that furnished by coating the adsorptive surface with PVA or HEC.     

Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations

A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10⁻⁹ m² V⁻¹ s⁻¹) when compared with unmodified fused silica (5.9 ± 0.1 10⁻⁸ m² V⁻¹ s⁻¹). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1–1.8% coefficient-of-variation (CV) within a day) and 2–3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use.     

Fast separation of enantiomers by capillary electrophoresis using a combination of two capillaries with different internal diameters

The combination of capillaries with different internal diameters was used to accelerate the separation of enantiomers in capillary electrophoresis. Separation of R ,S‐1,1′‐binaphthalene‐2,2′‐diyl hydrogen phosphate using isopropyl derivative of cyclofructan 6 was studied as a model system. The best separation conditions included 500 mM sodium borate pH 9.5 with 60 mM concentration of the chiral selector. Separation lasted approx. 1.5 min using the combination of 50 and 100 μm id capillaries of 9.7 cm and 22.9 cm, respectively. It allowed approx. 12‐fold acceleration in comparison to the traditional long‐end separation mainly due to the higher electroosmotic flow generated in the connected capillaries.     

Capillary electrokinetic chromatography with polyethyleneimine as replaceable cationic pseudostationary phase. Influence of methanol and acetonitrile on separation selectivity.

The effect of methanol and acetonitrile, respectively, on the separation of neutral compounds (benzyl alcohol, phenols) is investigated in electrokinetic chromatographic (EKC) systems consisting of polyethyleneimine (PEI) as charged, polymeric, replaceable pseudostationary phase. The separation systems consist of a buffer solution (2-morpholinoethanesulfonic acid, pH 7.0, 20 mM) containing 0.3-0.9% (w/v) PEI as additive and a varying percentage of methanol (0-50%, v/v) or acetonitrile (0-30%, v/v). EKC is carried out in fused-silica capillaries [47.0 cm (effective length 40.3 cm) x 100 microns I.D.]. They are dynamically coated with PEI, resulting in an electroosmotic flow directed towards the anode. The neutral analytes are migrating with the electroosmotic flow, and are retarded by the electrically driven counterflow of PEI. Separation of the analytes follows in the sequence benzyl alcohol, phenol, resorcinol, pyrogallol, reflecting the increasing hydrogen bond acidity and polarity (polarizibility) of the solutes. However, addition of methanol or acetonitrile causes a drastic loss of resolution, whereby the relative retention of the separands (related to benzyl alcohol) indicates a decrease of retardation upon addition of the organic solvents.     

Covalent anionic copolymer coatings with tunable electroosmotic flow for optimization of capillary electrophoretic separations

We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10⁻⁹ m² V⁻¹ s⁻¹ interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2–10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.     

Sedimentation behavior of high-temperature concentrated colloidal suspension based on potassium cryolite

The paper describes the sedimentation behavior of concentrated high-temperature oxide-fluoride slurries with alumina particles volume fraction range 0.24?≤?φ?≤?0.32 at ～700 °C. The behavior is of interest due to perspectives of the non-Newtonian fluids usage in the future aluminium reduction technology. To characterize sedimentation behavior several techniques were used: density analysis, X-Ray diffraction, microphotography and electron scanning microscopy. Sample with ?63?µm dispersed phase as well as smelter-grade alumina which contains particles in a range of 10–150?µm has been examined. It has been found that particle settling occurs with the initial velocity 0.97·10^?5 m·s^?1 at φ?=?0.24 and gradually reduces reaching zero at φ close to 0.32 which was considered as a maximum packing fraction. MS^?1 sedimentation mode has been identified, the type of sedimentation was found to be changed from type 1 to type 3 in the sample with φ?=?0.24. The alumina partial density distribution in suspension as a function of time was determined. Alumina average motion velocity during sedimentation was calculated to be 154·10^?3?kg·m^?2·s^?1 at φ?=?0.24 in the top layer of the sample within the first 150?s of sedimentation. It had been reducing drastically before it reached zero after 300?s. An alumina flow was higher in the top layers. Some of the rheological properties (such as relaxation time, which increased from 7.2·10^?3 to 17.7·10^?3?s after the increase of φ from 0.24 to 0.28) has been determined.     

Electrophoretic separation of linear and supercoiled DNA in uncoated capillaries.

We report electrophoretic separation of supercoiled plasmids (2-16 kilo base pairs) and linear double-stranded DNA (0.6-23 kilo base pairs) in uncoated capillaries filled with dilute hydroxyethylcellulose. Because electroosmotic flow reverses the order of elution, long plasmids spend less time in the capillary and their bandwidths are narrower than observed in coated capillaries. However, resolution is similar to that obtained in coated capillaries, because it is governed by the distribution of unresolved topoisomers. In the presence of electroosmotic flow migration of supercoiled plasmids does not follow the elastic rod model that has been observed in coated capillaries.     

Microchannel protein separation by electric field gradient focusing

A microchannel device is presented which separates and focuses charged proteins based on electric field gradient focusing. Separation is achieved by setting a constant electroosmotic flow velocity against step changes in electrophoretic velocity. Where these two velocities are balanced for a given analyte, the analyte focuses at that point because it is driven to it from all points within the channel. We demonstrate the separation and focusing of a binary mixture of bovine serum albumin and phycoerythrin. The device is constructed of intersecting microchannels in poly(dimethylsiloxane)(PDMS) inlaid with hollow dialysis fibers. The device uses no exotic chemicals such as antibodies or synthetic ampholytes, but operates instead by purely physical means involving the independent manipulation of electrophoretic and electroosmotic velocities. One important difference between this apparatus and most other devices designed for field-gradient focusing is the injection of current at discrete intersections in the channel rather than continuously along the length of a membrane-bound separation channel.     

Impact of organic solvents on the resolution of synthetic peptides by capillary electrophoresis

The effect of variations in the concentrations of different organic solvents, including acetonitrile, methanol, ethanol, propanol and isopropanol, with aqueous buffer electrolytes of defined composition and pH on the electroosmotic flow velocity, v(EOF), of uncoated fused silica capillaries and on the electrophoretic mobility, mu(e), of synthetic peptides in high-performance capillary electrophoresis (HPCE) has been systematically investigated. In these experiments, the volume fractions of the organic solvent in the aqueous buffer electrolyte were changed from psi = 0.0 to 0.80. The addition of these organic solvents to the aqueous buffer electrolyte reduced the electroosmotic flow (EOF) of the system, but to significantly different extents. For the protic solvents as the alkyl chain of the alcohol increased, at the same volume fraction the greater was the influence on the electroosmotic flow. However, for the aprotic solvent, acetonitrile, the EOF did not change substantially as the volume fraction was varied. The electrophoretic mobility of synthetic peptides under the different buffer electrolyte conditions showed similar trends, confirming that the content and type of the organic modifier can be rationally employed to subtly manipulate the separation selectivity of synthetic peptides. These results, therefore, provide fundamental insight into the experimental options that can be used to maximise resolution of synthetic peptides in HPCE with aqueous buffer-organic solvent mixtures as well as a basis to select optimal binary or ternary buffer electrolyte compositions for the analysis of peptides when hyphenated techniques, such as HPCE-electrospray ionisation mass spectrometry (ESI-MS), are contemplated for the analysis of peptide samples of low abundance as can often be experienced in proteomic investigations.     

Bis(4‐nitraminofurazanyl‐3‐azoxy)azofurazan and Derivatives: 1,2,5‐Oxadiazole Structures and High‐Performance Energetic Materials

Bis(4‐nitraminofurazanyl‐3‐azoxy)azofurazan ( 1 ) and ten of its energetic salts were prepared and fully characterized. Computational analysis based on isochemical shielding surface and trigger bond dissociation enthalpy provide a better understanding of the thermal stabilities for nitramine‐furazans. These energetic compounds exhibit good densities, high heats of formation, and excellent detonation velocity and pressure. Some representative compounds, for example, 1 (v_D: 9541 m s^?1; P: 40.5 GPa), and 4 (v_D: 9256 m s^?1; P: 38.0 GPa) exhibit excellent detonation performances, which are comparable with current high explosives such as RDX (v_D: 8724 m s^?1; P: 35.2 GPa) and HMX (v_D: 9059 m s^?1; P: 39.2 GPa).     

Nanosized cation exchanger for the electrophoretic separation and preconcentration of catecholamines and amino acids

The first example of application of nanosized polystyrene-based cation exchanger (NSCE) with sulfo groups as a dynamic coating of capillary walls was demonstrated. The conditions of dynamic coating formation were optimized and ensured the long-term stability of the coating. Capillary-to-capillary and day-to-day repeatabilities were 4% and 3%, correspondingly. The NSCE coating stability at various pH and influence of pH on the EOF mobility were investigated. The developed NSCE-modified coated capillaries provided improved resolution (R_s = 0.9–3.2 for catecholamines and R_s = 1.7–2.8 for amino acids) and efficiencies (330–520 ×10³ t.p./m) of basic analytes, which are 1.5 times higher compared to untreated capillary. The optimized conditions were as follows: 50 mM phosphate buffer solution at pH 2.2 with 5 μM NSCE. The effect of the NSCE concentration in BGE on the electrophoretic mobilities of the analytes was investigated. The various online concentration techniques were tested in order to decrease the LODs. The simultaneous application of NSCE capillaries and field-amplified sample stacking provided the lowest LODs of catecholamines and amino acids and allowed to determine these analytes in human urine.     

Capillary Electrophoretic Separation of Tricyclic Antidepressants Using 1‐Alkyl‐3‐Methylimidazolium‐Based Ionic Liquids as Background Electrolyte

A capillary electrophoretic (CE) method coupled with the use of 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (1E‐3MI‐TFB) ionic liquid as background electrolyte (BGE) has been developed for the simultaneous separation of nine tricyclic antidepressants, viz. amitriptyline (Ami), clomipramine (Clo), desipramine (Des), fluphenazine (Flu), imipramine (Imi), nortriptyline (Nor), promazine (Pro), thioridazine (Thi) and trimipramine (Tri). Resolution of TCAs with similar molecular structures and pK_a values was accomplished by minute manipulation of the electrophoretic velocities of TCAs via reversed electroosmotic flow (EOF) generated by adsorption of 1E‐3MI cations onto the capillary wall. The optimal separation was obtained with a 50 mM 1E‐3MI‐TFB as the sole BGE at pH 3. Symmetric peaks with efficiencies up to 2.4 × 10⁵ plates/m were achieved. RSD values on migration times and peak areas were in the ranges of 0.63–0.95% and 3.41–6.34% (n = 4), respectively. The role of different alkyl groups on the imidazolium cations was also investigated.     

The effect of electroosmotic and hydrodynamic flow profile superposition on band broadening in capillary electrophoresis

The effect of the superposition of electroosmotic flow and pressureinduced hydrodynamic counterflow on efficiency has been investigated for different capillary electrophoretic systems. Results are shown for 50 and 75 μm internal diameter capillaries at several voltage and counterpressure levels. Hydrodynamic counterflows were successfully applied in electrokinetic chromatography in order to delay the entry of a UV-active pseudostationary phase, tetraphenyl porphyrintetrasulfonate, into the detection zone allowing the separation of neutral nitroaromatics. The separations are based on the weak charge-transfer interactions between the porphyrin and the analytes.     

超支化聚合物化学键合毛细管电泳柱的制备及其对蛋白质的分离行为

分别合成了以三羟甲基丙烷和季戊四醇为核的超支化聚(胺-酯),并对其进行了红外测定、羟值测定、粘度测定等表征。采用化学键合方法将其涂于毛细管内壁,并测定涂层柱的电渗流以及对碱性蛋白质的分离能力,结果表明,涂层柱能有效地抑制碱性蛋白质在毛细管内壁上的吸附,大大降低电渗流;以三羟甲基丙烷为核的超支化聚(胺-酯)涂层柱的塔板数达105/m,而以季戊四醇为核的超支化聚(胺-酯)涂层柱的分离柱效更高,塔板数达107/m。实验结果表明这两类涂层柱都具有较好的分离效果和稳定性。