PVA-coated Capillary Research Articles

Electrokinetic preconcentration of magnetite core – carboxylic shell nanoparticles by capillary electrophoresis

Online electrokinetic preconcentration of magnetite core/carboxylic shell nanoparticles (MNPs) was studied by capillary electrophoresis using reversed and suppressed electroosmotic flow (EOF). 50mM sodium borate pH 9.5 was used as a background electrolyte. CTAB additive was used to reverse EOF and commercial polyvinylalcohol (PVA)-coated capillaries were used for EOF suppressed studies. Analyses in PVA-coated capillaries were more reproducible and therefore, the setup was further optimized in terms of water plug injection time, sample injection time, and voltage. Within the optimal conditions, the MNPs dispersed in water are electrokinetically loaded into BGE consisting of 50mM sodium borate pH 9.5 using −10kV for 120s. In comparison with the hydrodynamic injection of 5s by 50mbar, the electrokinetic injection allows 860-fold preconcentration of MNPs.

One single, fast and robust capillary electrophoresis method for the direct quantification of intact adenovirus particles in upstream and downstream processing samples

During development of adenovirus-based vaccines, samples have to be analyzed in order to either monitor the production process or control the quality and safety of the product. An important quality attribute is the total concentration of intact adenoviruses, which currently is determined by quantitative polymerase chain reaction (qPCR) or anion exchange-HPLC. Capillary Electrophoresis (CE) was evaluated as alternative to the current methods with the aim to have one single method that allows reliable and fast quantification of adenovirus particles throughout the full process. Intact adenoviruses samples from downstream processing and upstream processing were analyzed directly by CE with UV-detection at 214nm. Only the samples with high amounts of DNA required a simple sample pretreatment by benzonase. Adenovirus particles were separated from matrix components such as cell debris, residual cell DNA, and/or proteins on a PVA-coated capillary using a BGE consisting of 125mM Tris, 338mM tricine and 0.2% v/v polysorbate-20 at pH 7.7. Full factorial design of experiments was used for method optimization as part of the analytical quality by design (AQbD) method development approach. The method was validated for the quantification of adenoviruses on five representative samples from the manufacturing process in the range of 0.5×1011–1.5×1011 adenovirus particles per ml (~80 to 250pmol/l). The CE method showed intermediate precision of 7.8% RSD on concentration and an accuracy (spiked recovery) of 95–110%. CE proved highly useful for process development support and is being implemented for in-process control testing for adenovirus vaccine manufacturing.

Gel electrophoresis in a polyvinylalcohol coated fused silica capillary for purity assessment of modified and secondary-structured oligo- and polyribonucleotides.

Application of a polyvinylalcohol-coated (PVA-coated) capillary in capillary gel electrophoresis (CGE) enables the selective separation of oligoribonucleotides and their modifications at high resolution. Quality assessment of shorter oligomers of small interfering RNA (siRNA) is of key importance for ribonucleic acid (RNA) technology which is increasingly being applied in medical applications. CGE is a technique of choice for calculation of chemically synthesized RNAs and their modifications which are frequently obtained as a mixture including shorter oligoribonucleotides. The use of CGE with a PVA-coated capillary to analyze siRNA mixtures presents an alternative to conventionally employed techniques. Here, we present study on identification of the length and purity of RNA mixture ingredients by using PVA-coated capillaries. Also, we demonstrate the use of PVA-coated capillaries to identify and separate phosphorylated siRNAs and secondary structures (e.g. siRNA duplexes).

Charge-based characterisation of high-molecular-weight glutenin subunits from common wheat by capillary isoelectric focusing

In this study, the capillary isoelectric focusing (CIEF) method for the separation and charge characterisation of the heterogeneity of high molecular-weight-glutenin subunits (HMW-GS) in common wheat (Triticum aestivum L.) using linear polyacrylamide (LPA) and polyvinyl alcohol (PVA) coated capillaries was developed. Particularly good repeatability and well-resolved charge isoform profiles were obtained by introducing a mixture of carrier ampholytes (pH 3–10 and pH 5–8), a high concentration of urea (6M) and SB3-12 as detergent in a sample solution during separation in a PVA-coated capillary. One major and one or two minor isoforms were observed for the individual HMW-GS. These isoforms were satisfactorily separated using a pH gradient into two groups: y-type isoforms and x-type isoforms encoded by the Glu-B1 locus with shorter migration times and remaining x-type isoforms with longer times. The method produced from eight to twelve isoforms of wheat HMW-GS with pI points in the range of 4.72–6.98. Generally, the minor isoforms were more acidic compared with the major isoform. The y-type subunits had an approximately neutral character (pI 6.70–6.98); however, x-types showed a weakly acidic character (pI 4.72–5.23), with the exception of subunits encoded by the Glu-B1 locus. The isoelectric point peak profiles were compared with capillary zone electrophoresis (CZE) electropherograms. Generally, the number of detected isoforms for the particular HMW-GS detected using both methods were similar.

Rapid separation of fatty acids using a poly(vinyl alcohol) coated capillary in nonaqueous capillary electrophoresis with contactless conductivity detection

The determination of fatty acids by nonaqueous CZE with capacitively coupled contactless conductivity detection was investigated. A new deoxycholate-based BGE, which had previously been found to give significantly improved baseline stability in the determination of lipophilic organic ammonium ions, was found to be similarly beneficial for the determination of the anions. The use of a PVA-coated capillary was required for suppression of the EOF and to obtain well reproducible results. The complete separation of 12 fatty acids could be achieved with 10 mM DOC in methanol within 6 min under optimized conditions. The PVA-coated capillary demonstrated outstanding stability over 300 runs with no sign of depletion of the PVA layer. Method validation showed a good linearity range from 0.75 to 25 μM with correlation coefficients between 0.9949 and 0.9979. The LOD was determined as 0.5 μM for all fatty acids. The developed approach was successfully demonstrated for the separation of free fatty acids in commercial and home-made edible oil.

Novel poly(vinyl alcohol)-based column coating for capillary electrophoresis of proteins

A novel and simple method for the preparation of chemically bonded poly(vinyl alcohol) (PVA) coating to silica capillary inner wall was developed, and the PVA-coated capillary columns were employed for capillary electrophoresis (CE). The coating procedure included pretreatment of the capillary inner wall, silanization, aldehyde group functionalization and PVA immobilization. Electroosmotic flow of the coated capillary was almost suppressed over a wide pH range (pH 3–10). High-efficiency separations of cationic proteins (including cytochrome c, lysozyme, α-chymotrypsinogen A) at pH 3.0–5.0 and of anionic proteins (including myoglobin and trypsin inhibitor) at pH 10.0 were achieved with the PVA-coated capillary. Moreover, a “dual-opposite-injection” approach was adopted for simultaneous separations of both cationic and anionic proteins at neutral pH with the prepared column. In this CE mode, positively charged proteins migrated from one end of the column to the detector while negatively charged proteins from the other end to the detection window. Good run-to-run repeatability was obtained in all of the protein CE separations performed in this work. The PVA-coated column can also afford long-term stable uses for protein separations, as demonstrated in 100 repeated uses using a single capillary with the relative standard deviation values of the retention times less than 0.9%. Moreover, good column-to-column reproducibility was demonstrated by protein CE separation with 10 different columns. The results indicate that the present method for PVA-coated capillary preparation is promising for protein CE applications.

CE detection of N‐(3‐dimethylaminopropyl)‐N‐carbodiimide/N‐hydroxysuccinimide‐coupled proteins after homo‐ and hetero‐crosslinking reactions

Protein-protein conjugates formed by carbodiimide crosslinking reactions have been analyzed for the first time using CE. Lysozyme and BSA were chosen as model proteins to study the efficacy of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide and N-hydroxysuccinimide as crosslinkers. Detection of the molecular mass increase was checked by SDS-PAGE. Commercially available, PVA-coated capillaries showed appropriate selection, while phospho-deactivated and dynamic PVA-coated capillaries did not give suitable resolution. CE was found to be an efficient tool to characterize homo- (lysozyme-lysozyme) and hetero- (lysozyme-BSA) protein coupling by suitable variations of electrophoretic mobilities.

Reproducible chiral capillary electrophoresis of methamphetamine and its related compounds using a chemically modified capillary having diol groups

Excellent reproducibility of chiral capillary electrophoresis for methamphetamine (MA) and its related compounds was obtained using a chemically modified capillary having diol groups. The data were compared with those using an untreated fused-silica capillary and a poly(vinyl alcohol) (PVA)-coated capillary. For five replicate analyses of a standard mixture, relative standard deviations (RSDs) of the migration times of analytes with diol or PVA-coated capillaries were less than 0.1%, which were lower than those obtained with an untreated fused-silica capillary (ca. 0.4%). The diol capillary gave reproducible migration times especially in analyses of real samples, such as crude human urine. In the analyses of five spiked urine samples, the RSD values of migration times of analytes with the diol capillary were not greater than 0.14%, which were much lower than those obtained with the PVA-coated capillary (ca. 1.5%). The proposed method was successfully used for chiral analyses of actual urine samples of MA addicts.

Electroosmotic pump-assisted capillary electrophoresis of proteins

A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt- c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt- c with the EOPACE system is demonstrated.

Influence of pH*-value of methanolic electrolytes on electroosmotic flow in hydrophilic coated capillaries

The dependency of EOF on the H +-concentration and the related so called pH* value of methanolic electrolytes has been examined with poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA) and uncoated capillaries. These results were compared with the pH dependency of EOF of these capillaries using aqueous buffers. In uncoated capillaries the dependency of EOF on the pH(*)-value is very similar for aqueous and methanolic electrolytes. The EOF increases with increasing H +-concentration and pH-hysteresis is observed. In PVA coated capillaries the EOF is strongly reduced over wide pH* or pH ranges for both methanolic electrolytes and aqueous buffers. The EOF in PEG coated capillaries is surprisingly directed to the anode with methanolic electrolytes whereas a reduced cathodic EOF is observed in aqueous electrolytes. The anodic EOF of PEG-coated capillaries in methanolic electrolytes is independent of the pH*-value. The usefulness of PEG- and PVA-coated capillaries for adjusting the EOF in non-aqueous electrolytes for the analysis of isomeric organic acids was demonstrated.

Poly(vinylamine)-coated capillaries with reversed electroosmotic flow for the separation of organic anions

A simple method for the preparation of poly(vinylamine) (PVA) coating on the inner surface of a fused-silica capillary for CE is reported. A high quality PVA was synthesized from acidic hydrolysis of N-vinyl- tert.-butylcarbamate. Based on the reactivity of PVA primary amino groups a coating procedure consisting of two steps: (1) dynamic adsorption of PVA from an aqueous solution, (2) cross-linking of the polymer amino functions with N,N′-methylenebisacrylamide and end-capping of the primary amino groups with an acrylamido derivative bearing quaternary amino functions was devised. A permanent coating that generates a strong anodic electroosmotic flow was produced which is fully compatible with the separation of polyanionic acids. A complete evaluation of reproducibility, efficiency and long-term stability of the coating was carried out using a mixture of eight organic mono-, di- and tricarboxylic acids. The usefulness of PVA-coated capillaries in the analysis of real samples containing UV-transparent organic acids was demonstrated.

Use of coated capillaries for nonaqueous capillary electrophoresis

The performance and stability of different hydrophilic capillary coatings have been examined using nonaqueous electrolytes. Coated capillaries can be successfully applied to nonaqueous capillary electrophoresis (CE) separations as well as in the normal aqueous mode. Capillaries coated with polyethylene glycol (PEG) and polyvinyl alcohol (PVA) show high stability toward a large number of methanol rinsing steps. The stability of PVA-coated capillaries in a long series of separations performed in methanol was superior to that of the PEG capillaries. The electroosmotic flow (EOF) characteristics of PVA, PEG, and uncoated fused silica capillaries have been examined using methanolic electrolytes. In PVA-coated capillaries the EOF was strongly reduced with aqueous and nonaqueous electrolytes. Surprisingly the EOF of PEG-coated capillaries in methanolic electrolytes was found to be anodic, whereas a reduced cathodic EOF is observed in aqueous systems as expected. The selectivity for the separation of weak electrolytes using nonaqueous media was influenced by the addition of acids or bases. ©1999 John Wiley & Sons, Inc. J Micro Sep 11: 209–213, 1999