Capillary electrophoresis in a fused‐silica capillary with surface roughness gradient

The electro‐osmotic flow, a significant factor in capillary electrophoretic separations, is very sensitive to small changes in structure and surface roughness of the inner surface of fused silica capillary. Besides a number of negative effects, the electro‐osmotic flow can also have a positive effect on the separation. An example could be fused silica capillaries with homogenous surface roughness along their entire separation length as produced by etching with supercritical water. Different strains of methicillin‐resistant and methicillin‐susceptible Staphylococcus aureus were separated on that type of capillaries. In the present study, fused‐silica capillaries with a gradient of surface roughness were prepared and their basic behavior was studied in capillary zone electrophoresis with UV‐visible detection. First the influence of the electro‐osmotic flow on the peak shape of a marker of electro‐osmotic flow, thiourea, has been discussed. An antifungal agent, hydrophobic amphotericin B, and a protein marker, albumin, have been used as model analytes. A significant narrowing of the detected zones of the examined analytes was achieved in supercritical‐water‐treated capillaries as compared to the electrophoretic separation in smooth capillaries. Minimum detectable amounts of 5 ng/mL amphotericin B and 5 μg/mL albumin were reached with this method.     

Comparison of three modifications of fused‐silica capillaries and untreated capillaries for protein profiling of maize extracts by capillary electrophoresis

In this work, capillary electrophoresis was applied to protein profiling of fractionated extracts of maize. A comparative study on the application of uncoated fused‐silica capillaries and capillaries modified with hydroxypropylmethylcellulose, ω‐iodoalkylammonium salt and a commercially available neutral capillary covalently coated with polyacrylamide is presented. The coating stability, background electrolyte composition, and separation efficiency were investigated. It was found that for zeins separation, the most stable and efficient was the capillary coated with polyacrylamide. Finally, the usefulness of these methods was studied for the differentiation of zein fraction in transgenic and nontransgenic maize. Zeins extracted from maize standards containing 0 and 5% m/m genetic modification were successfully separated, but slight differences were observed in terms of the zein content. Albumin and globulin fractions were analyzed with the use of unmodified fused‐silica capillary with borate buffer pH 9 and the capillary coated with polyacrylamide with phosphate buffer pH 3. In the albumin fraction, additional peaks were found in genetically modified samples.     

Gas chromatographic retention in uncoated fused silica capillaries

Understanding of retention in uncoated fused‐silica capillaries is of interest due to increased attention on precolumn backflushing in capillary GC. Uncoated capillaries offer several advantages as precolumns compared to coated precolumns. In order to examine the possibility of predicting elution temperatures of alkanes from uncoated capillaries a priori, several sizes of deactivated but uncoated fused‐silica capillaries were evaluated under various operating conditions. Retention was found to depend on dimensionless ramp rate (°C/t_M), sample loading (capacity), flow mode, and column dimensions (probably related to surface area).     

Investigation of the separability of thaumatin by capillary electrophoresis

The electrophoretic behaviour of the highly basic protein thaumatin was explored in strongly acid (pH 2) and mildly acid (pH 4.5) separation systems using both bare and coated fused silica capillaries. The separation selectivity for thaumatin I, thaumatin II, and for other sample constituents was insufficient for their baseline separation at pH 2 in an uncoated capillary because the separation efficiency was markedly lower than is common in the electrophoretic separations of proteins. A separation selectivity higher by up to one order of magnitude has been reached at pH 4.5. A pronounced asymmetry of zones, which impaired resolution at this pH, was effectively suppressed by coating of the capillary wall with a polymer. In fact, adsorption on the capillary coating always plays a contributory role whenever a good separation of thaumatin constituents is attained. This indicates that electrochromatographic separation systems based on capillaries coated with the layer of either cationic or hydrophilic uncharged polymer hold promise for the development of methods for thaumatin analysis.     

Surface initiated polymerization of a cationic monomer on inner surfaces of silica capillaries: Analyte separation by capillary electrophoresis versus polyelectrolyte behavior

[2‐(Methacryloyl)oxyethyl]trimethylammonium chloride was successfully polymerized by surface‐initiated atom transfer radical polymerization method on the inner surface of fused‐silica capillaries resulting in a covalently bound poly([2‐(methacryloyl)oxyethyl]trimethylammonium chloride) coating. The coated capillaries provided in capillary electrophoresis an excellent run‐to‐run repeatability, capillary‐to‐capillary and day‐to‐day reproducibility. The capillaries worked reliably over 1 month with EOF repeatability below 0.5%. The positively charged coated capillaries were successfully applied to the capillary electrophoretic separation of three standard proteins and five β‐blockers with the separation efficiencies ranging from 132 000 to 303 000 plates/m, and from 82 000 to 189 000 plates/m, respectively. In addition, challenging high‐ and low‐density lipoprotein particles could be separated. The hydrodynamic sizes of free polymer chains in buffers used in the capillary electrophoretic experiments were measured for the characterization of the coatings.     

CE coupled to MALDI with novel covalently coated capillaries

CE offers the advantage of flexibility and method development options. It excels in the area of separation of ions, chiral, polar and biological compounds (especially proteins and peptides). Masking the active sites on the inner surface of a bare fused silica capillary wall is often necessary for CE separations of basic compounds, proteins and peptides. The use of capillary surface coating is one of the approaches to prevent the adsorption phenomena and improve the repeatability of migration times and peak areas of these analytes. In this study, new capillary coatings consisting of (i) derivatized polystyrene nanoparticles and (ii) derivatized fullerenes were investigated for the analysis of peptides and protein digest by CE. The coated capillaries showed excellent run‐to‐run and batch‐to‐batch reproducibility (RSD of migration time ≤0.5% for run‐to‐run and ≤9.5% for batch‐to‐batch experiments). Furthermore, the capillaries offer high stability from pH 2.0 to 10.0. The actual potential of the coated capillaries was tested by combining CE with MALDI‐MS for analysing complex samples, such as peptides, whereas the overall performance of the CE‐MALDI‐MS system was investigated by analysing a five‐protein digest mixture. Subsequently, the peak list (peptide mass fingerprint) generated from the mass spectra of each fraction was entered into the Swiss‐Prot database in order to search for matching tryptic fragments using the MASCOT software. The sequence coverage of analysed proteins was between 36 and 68%. The established technology benefits from the synergism of high separation efficiency and the structure selective identification via MS.     

Capillary electrophoresis using core‐based hyperbranched polyethyleneimine (CHPEI) static‐coated capillaries

With unique 3‐D architecture, the application of core‐based hyperbranched polyethyleneimine (CHPEI), as a capillary coating in capillary electrophoresis, is demonstrated by manipulation of the electroosmotic mobility (EOF). CHPEI coatings (CHPEI5, M_w ≈? 5000 and CHPEI25, M_w ≈? 25 000) were physically adsorbed onto the inner surface of bare fused‐silica capillary (BFS) via electrostatic interaction of the oppositely charged molecules by rinsing the capillaries with different CHPEI aqueous solutions. The EOF values of the coated capillaries were measured over the pH range of 4.0–9.0. At higher pH (pH >6) the coated capillary surface possesses excess negative charges, which causes the reversal of the EOF. The magnitudes of the EOF obtained from the coated capillaries were three‐fold lower than that of BFS capillary. Desirable reproducibility of the EOF with % RSD (n = 5) ? 2 was obtained. Effect of ionic strength, stability of the coating (% RSD = 0.3) and the dependence of the EOF on pH (% RSD = 0.5) were also investigated. The CHPEI‐coated capillaries were successfully utilized to separate phenolic compounds, B vitamins, as well as basic drugs and related compounds with reasonable analysis time (<20 min) and acceptable migration‐time repeatability (<0.7% RSD for intra‐capillary and <2% RSD for inter‐capillary).     

Rapid determination of globin chains in red blood cells by capillary electrophoresis using INSTCoated fused‐silica capillary

A laboratory‐made INSTCoated fused‐silica capillary has been newly used for CE separation of four mixtures of proteins in sodium phosphate BGEs at pH 3.0 and 2.5, respectively. The obtained separation efficiencies range from 145 000 theoretical plates per meter for myoglobin to 1 216 000 m^?1 for lysozyme. A total of 49–89% of the number of theoretical plates was obtained in a commercial polyvinyl alcohol coated capillary compared to the INSTCoated capillary under the same experimental conditions, 0–86% was obtained in a laboratory polyacrylamide‐coated capillary, and only 0–6% was obtained in an uncoated fused‐silica capillary. The RSD values for the intraday repeatability for an INSTCoated capillary were 0.1–1.0% (migration time) and 0.3–2.4% (peak area); RSD values for the interday repeatability in the same capillary are 0.6–1.4% (migration time) and 2.4–5.5% (peak area); RSD values for interday repeatability between different capillaries equaled 1.7–2.1% (migration time) and 2.8–10.9% (peak area). The INSTCoated capillary has been further used for rapid determination of globin chains isolated from red blood cells. A separation of α and β chains prepared from adult blood has been completed in 3 min with a peak resolution of 1.3, and the separation of α and ^Gγ chains prepared from newborn blood took 3 min with a peak resolution of 3.6.     

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye–DNA complexes may prevent from their wide applications in CE‐LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris‐glycine buffer and Tris‐glycine‐acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused‐silica capillary‐based CE‐LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium‐4‐pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4×10^?22 mol (150 molecules) in mass. We further demonstrate that the non‐gel sieving CE‐LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip‐based CE DNA analysis.     

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.     

Analysis of calcitonin and its analogues by capillary zone electrophoresis and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry

Capillary zone electrophoretic (CZE) separations and mass spectrometric analysis of salmon calcitonin and related analogues were performed to generate electrophoresis and mass fingerprints for quality control of the recombinant polypeptide pharmaceutical salmon calcitonin. The calcitonins and their corresponding tryptic digests were successfully separated by CZE at low pH in fused silica capillaries dynamically modified with poly-cationic polymers. The poly-cationic modified inner surface of the fused silica capillaries generated a strong anionic electroosmotic flow (EOF). Analytes of negative, neutral, and positive charge were all swept through the capillary toward the positive electrode. Compared to Polybrene-coated capillaries, capillaries coated with PEI showed a markedly slower but much more stable electroosmotic flow. The migration order of the analytes was predicted by comparing approximate values of the charge to (molecular mass)2/3 ratios. The predicted migration order was confirmed by off-line analysis of CZE fractions with matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Etched chemically modified capillaries: novel separation media for electrophoretic analyses

The fabrication, properties, and applications of etched chemically modified capillaries for electrophoretic analysis are reviewed. It is shown that the etching process creates a surface that is fundamentally different than a bare fused silica capillary. The new surface matrix produces unique electroosmotic flow properties and is more compatible with basic and biological compounds. After chemical modification of the surface, the bonded organic moiety (stationary phase) contributes to the control of migration of solutes in the capillary. Both electrophoretic and chromatographic processes take place in the etched chemically modified capillaries leading to a variety of experimental variables (pH, buffer type, presence and amount of organic modifier, and temperature) that can be used to optimize separations. A number of examples of separations on these capillaries are presented as well as data on column ruggedness and reproducibility.     

A cationic β‐cyclodextrin as a dynamic coating for the separation of proteins in capillary electrophoresis

A cationic cyclodextrin was used as dynamic coating for the capillary electrophoresis of a model mixture of proteins (i.e., ubiquitin, α‐lactoglobulin, cytochrome‐c, and myoglobin) as positively charged species in a fused silica capillary. An interesting feature of the coating is that by simple adjustment of the concentration of cyclodextrin added into the background electrolyte, a neutral or positively charged surface, which was beneficial in preventing protein adsorption at the inner capillary wall surface, was obtained. This is the first demonstration of a dynamic coating that yielded a neutral surface for protein separations in capillary electrophoresis. Based on electro‐osmotic flow measurements, addition of 0.05 to 0.10 mg/mL quaternary β‐cyclodextrin in a low pH electrolyte resulted in a neutral or positive surface (undetectable to very slow anodic electro‐osmotic flow). The coating approach afforded the electrophoretic separation of the mixture of proteins at positive polarity with good repeatability and separation performance.     

Electrophoretic separation of aniline derivatives using fused silica capillaries coated with acid treated single-walled carbon nanotubes

This paper reports on a new strategy for coating fused silica capillaries based on the ionic adsorption of acid treated single-walled carbon nanotubes (SWCNTs) on a poly(diallydimethylammonium chloride)-modified fused silica surface. The coated capillaries were used to demonstrate their performance for baseline separation of a mixture of seven nitrogen-containing aromatic compounds compared to capillary zone electrophoresis. This combined layer formed a coating material that could be useful for improvement of the selectivity of the solutes in an electrical field. We reasoned that the interaction of the solutes and the modified capillary wall occurred mainly via ionic interactions with the charged moieties of CNTs. The single-walled CNT modified capillaries were very stable and could be used for over 200 repeated analyses without compromising its analytical performance.     

Behaviour of fused silica capillaries subjected to gamma radiation. Part 2: Physical aspects

Five commercial varieties of uncoated fused silica capillary tubing used in high resolution gas chromatography were subjected to cobalt-60 gamma radiation. The advantages and superior performance of fused silica open tubular columns coated with OV-1 and subsequently immobilized or crosslinked in situ by radiation is reported in a previous paper in this Journal; however, in this investigation a loss in flexibility of irradiated capillaries was also noted. Quantitative measurements on flexibility were performed on specimens of irradiated fused silica capillaries, indicating that changes do indeed occur upon irradiation. Scanning electron micrographs also illustrate slight deterioration of the outer protective polyimide coating. Most surprisingly, it was also established that a variation does exist in the flexibility of the commercially available raw material studied.     

Hydride-Based Separation Materials for High Performance Liquid Chromatography and Open Tubular Capillary Electrochromatography

 Silica hydride is a recent development in chromatographic support materials for high performance liquid chromatography (HPLC) where hydride groups replace 95% of the silanols on the surface. This conversion changes many of the fundamental properties of the material as well as the bonded stationary phases that are the result of further chemical modification of the hydride surface. Some unique chromatographic properties of hydride-based phases are described as well as some general application areas where these bonded materials may be used in preference to or have advantages not available from typical stationary phases. The fabrication, properties and applications of etched chemically modified capillaries for electrophoretic analysis are also reviewed. It is shown that the etching process creates a surface that is fundamentally different than a bare fused silica capillary. The new surface matrix produces unique electroosmotic flow properties and is more compatible with basic and biological compounds. After chemical modification of the surface, the bonded organic moiety (stationary phase) contributes to the control of migration of solutes in the capillary. Both electrophoretic and chromatographic processes take place in the etched chemically modified capillaries leading to a variety of experimental variables that can be used to optimize separations. A number of examples of separations on these capillaries are described.     

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.     

Separation of α‐lactalbumin grafted‐ and non‐grafted maghemite core/silica shell nanoparticles by capillary zone electrophoresis

The use of nanoparticles (NPs) in immunodiagnostics is a challenging task for many reasons, including the need for miniaturization. In view of the development of an assay dedicated to an original, miniaturized and fully automated immunodiagnostics which aims to mimic in vivo interactions, magnetic zwitterionic bifunctional amino/polyethyleneoxide maghemite core/silica shell NPs functionalized with allergenic α‐lactalbumin were characterized by CE. Proper analytical performances were obtained through semi‐permanent capillary coating with didodecyldimethylammonium bromide (DDAB) or permanent capillary wall modification by hydroxypropylcellulose. The influence of experimental conditions (e.g. buffer component nature, pH, ionic strength, and electric field strength) on sample stability, electrophoretic mobility, and dispersion was investigated using either DDAB‐ or hydroxypropylcellulose‐coated capillaries. Adsorption to the capillary wall and aggregation phenomena were evaluated according to the CE conditions. The proper choice of experimental conditions, i.e. separation under −10 kV in a 25 mM ionic strength MES/NaOH (pH 6.0) with a DDAB‐coated capillary, allowed the separation of the grafted and the non‐grafted NPs.     

A fully automated linear polyacrylamide coating and regeneration method for capillary electrophoresis of proteins

Surface modification of the inner capillary wall in CE of proteins is frequently required to alter EOF and to prevent protein adsorption. Manual protocols for such coating techniques are cumbersome. In this paper, an automated covalent linear polyacrylamide coating and regeneration process is described to support long‐term stability of fused‐silica capillaries for protein analysis. The stability of the resulting capillary coatings was evaluated by a large number of separations using a three‐protein test mixture in pH 6 and 3 buffer systems. The results were compared to that obtained with the use of bare fused‐silica capillaries. If necessary, the fully automated capillary coating process was easily applied to regenerate the capillary to extend its useful life‐time.     

Measurement of the differences in electrophoretic mobilities of individual molecules of E. coli beta-galactosidase provides insight into structural differences which underlie enzyme microheterogeneity

The electrophoretic mobility and catalytic activity of individual molecules of Escherichia coli beta-galactosidase were measured using CE-LIF detection. Both the mobility and activity were reproducible for each molecule but differed between individual molecules. Assays were performed using uncoated capillaries and capillaries coated with different polymers, using enzymes from different sources and by three different experimental protocols. In all cases the observed ranges in electrophoretic mobilities were similar. The observed range in the electrophoretic mobility may be explained by structural microheterogeneity resulting in a gain or loss of up to 1.6 suppressed charge units. There was no observed relationship between the observed activities and electrophoretic mobilities. If the finding that individual beta-galactosidase molecules have heterogeneous electrophoretic mobility can be extended to other proteins, this may limit the resolution possible for capillary zone electrophoresis protein separations.