New adsorbed coatings for capillary electrophoresis

New acrylic polymers bearing oxirane groups were synthesized to be used in the production of coated capillaries. A fully automated coating procedure was devised based on the use of diluted water solutions of these polymers. The whole procedure required less than 30 min. The new polymers rapidly adsorbed from water onto the capillary wall, thus suppressing electroosmotic flow (EOF) to a negligible value. The adsorbed coatings were stable for hundreds of hours at high pH, temperature, and in the presence of 8 M urea. Efficient separations of acidic and basic proteins were achieved in the new phases.     

Polymer wall coatings for capillary electrophoresis

This review article describes the preparation of dynamic and static polymeric wall coatings for capillary electrophoresis. Properties of bare fused-silica surfaces and methods for the characterization of capillary coatings are summarized. The preparation and basic properties of neutral and charged wall coatings are considered. Finally, advantages and potential applications of various coatings are discussed.     

Connections between structure and performance of four cationic copolymers used as physically adsorbed coatings in capillary electrophoresis

In this work, the properties of four cationic copolymers synthesized in our laboratory are studied as physically adsorbed coatings for capillary electrophoresis (CE). Namely, the four copolymers investigated were poly(N-ethyl morpholine methacrylamide-co-N,N-dimethylacrylamide), poly(N-ethyl pyrrolidine methacrylate-co-N,N-dimethylacrylamide), poly(N-ethyl morpholine methacrylate-co-N,N-dimethylacrylamide) and poly(N-ethyl pyrrolidine methacrylamide-co-N,N-dimethylacrylamide). Capillaries were easily coated using these four different macromolecules by simply flushing into the tubing an aqueous solution containing the copolymer. The stability and reproducibility of each coating were tested for the same day, different days and different capillaries. It is demonstrated that the use of these coatings in CE can drastically reduce the analysis time, improve the resolution of the separations or enhance the analysis repeatability at very acidic pH values compared to bare silica columns. As an example, the analysis of an organic acids test mixture revealed that the analysis time was reduced more than 6-times whereas the separation efficiency was significantly increased to nearly 10-times attaining values up to 595,000 plates/m using the coated capillaries. Moreover, it was shown that all the copolymers used as coatings for CE allowed the separation of basic proteins by reducing their adsorption onto the capillary wall. Links between their molecular structure, physicochemical properties and their performance as coatings in CE are discussed.     

Brush-like copolymer as a physically adsorbed coating for protein separation by capillary electrophoresis

A brush-like copolymer consisting of poly(ethylene glycol) methyl ether methacrylate and N,N-dimethylacrylamide (PEGMA-DMA) was synthesized and used as a novel static physically adsorbed coating for protein separation by capillary electrophoresis for the first time, in order to stabilize electroosmotic flow (EOF) and suppress adsorption of proteins onto the capillary wall. Very stable and low EOF was obtained in PEGMA-DMA-coated capillary at pH 2.2-7.8. The effects of molar ratio of PEGMA to DMA, copolymer molecular mass, and pH on the separation of basic proteins were discussed. A comparative study of bare capillary with PEGMA-DMA-coated capillary for protein separation was also performed. The basic proteins could be well separated in PEGMA-DMA-coated capillary over the investigated pH range of 2.8-6.8 with good repeatability and high separation efficiency because the copolymer coating combines good protein-resistant property of PEG side chains with excellent coating ability of PDMA-contained backbone. Finally, the coating was successfully applied to the fast separation of other protein samples, such as protein mixture and egg white, which reveals that it is a potential coating for further proteomics analysis.     

Fast and easy coating for capillary electrophoresis based on a physically adsorbed cationic copolymer

In this work, a new copolymer synthesized in our laboratory is used as physically adsorbed coating for capillary electrophoresis (CE). The copolymer is composed of ethylpyrrolidine methacrylate (EPyM) and methylmethacrylate (MMA). The capillary coating is easily obtained by simply flushing into the tubing an EPyM/MMA solution. It is demonstrated that the composition of the EPyM/MMA copolymer together with the selection of the background electrolyte (BGE) and pH allow tailoring the direction and magnitude of the electroosmotic flow (EOF) in CE. It is also shown that the EOF obtained for the EPyM/MMA-coated capillaries was reproducible in all cases independently on pH or polymer composition. Thus, RSD values lower than 1.9% (n=5) for the same capillary and day were obtained for the migration time, while the repeatability interdays (n=5) was observed to provide RSD values lower than 0.5%. The stability of the coating procedure was also tested between capillaries (n=3) obtaining RSD values lower than 0.6%. It is demonstrated with several examples that the use of EPyM/MMA coatings in CE can drastically reduce the analysis time and/or to improve the resolution of the separations. It is shown that EPyM/MMA-coated capillaries allow the separation of basic proteins by reducing their adsorption onto the capillary wall. Also, EPyM/MMA-coated capillaries provide a faster separation of samples containing simultaneously positive and negative analytes. Moreover, it is demonstrated that the use of EPyM/MMA-coated capillaries can incorporate an additional chromatographic-like interaction with nucleosides that highly improves the separation of this group of solutes.     

Microchannel wall coatings for protein separations by capillary and chip electrophoresis

The necessity for microchannel wall coatings in capillary and chip-based electrophoretic analysis of biomolecules is well understood. The regulation or elimination of EOF and the prevention of analyte adsorption is essential for the rapid, efficient separation of proteins and DNA within microchannels. Microchannel wall coatings and other wall modifications are especially critical for protein separations, both in fused-silica capillaries, and in glass or polymeric microfluidic devices. In this review, we present a discussion of recent advances in microchannel wall coatings of three major classes--covalently linked polymeric coatings, physically adsorbed polymeric coatings, and small molecule additives. We also briefly review modifications useful for polymeric microfluidic devices. Within each category of wall coatings, we discuss those used to eliminate EOF, to tune EOF, to prevent analyte adsorption, or to perform multiple functions. The knowledgeable application of the most promising recent developments in this area will allow for the separation of complex protein mixtures and for the development of novel microchannel wall modifications.     

New physically adsorbed polymer coating for reproducible separations of basic and acidic proteins by capillary electrophoresis

In this work, a new physically adsorbed coating for capillary electrophoresis (CE) is presented. The coating is based on a N,N-dimethylacrylamide-ethylpyrrolidine methacrylate (DMA-EPyM) copolymer synthesized in our laboratory. The capillary coating is simple and easy to obtain as only requires flushing the capillary with a polymer aqueous solution for 2 min. It is shown that by using these coated capillaries the electrostatic adsorption of a group of basic proteins onto the capillary wall is significantly reduced allowing their analysis by CE. Moreover, the DMA-EPyM coating provides reproducible separations of the basic proteins with RSD values for migration times lower than 0.75% for the same day (n = 5) and lower than 3.90% for three different days (n = 15). Interestingly, the electrical charge of the coated capillary wall can be modulated by varying the pH of the running buffer which makes possible the analysis of basic and acidic proteins in the same capillary. The usefulness of this coating is further demonstrated via the reproducible separation of whey (i.e. acidic) proteins from raw milk. The coating protocol should be compatible with both CE in microchips and CE-MS of different types of proteins.     

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   

Critical factors for reproducible capillary electrophoresis of Escherichia coli cells

Sharp peaks of Escherichia coli JM 109 (up to 1 300 000 theoretical plates) were recorded with either extremely diluted (< 5 mM) or extremely concentrated (ca. 150 mM) Tris-borate (TB) running buffers. However, under the conditions of yielding sharp peaks, migration time of E. coli was irreproducible. Critial factors influencing reproducibility were found to include bacterial growth phase, storage condition, cell pretreatment before injection, and concentration of running buffer. Buffer concentrations in the range of 20-100 mM TB were essential for reproducibility. E. coli JM 109 was shown to be sensitive to ultrasonification. Bacterial growth and storage conditions could be monitored by CE, with results comparable to those obtained with optical methods.     

Mixed cationic/anionic surfactants for semipermanent wall coatings in capillary electrophoresis

Mixtures of the cationic surfactant cetyltrimethylammonium bromide (CTAB) with the anionic surfactant sodium dodecyl sulfate (SDS) form more stable coatings in fused-silica capillaries than CTAB alone. The reversed electroosmotic flow (EOF) generated by CTAB/SDS mixtures remains stable for over 80 min after removal of the surfactants from the buffer. Enhanced stability (relative to CTAB alone) was found even when the ratio of SDS to CTAB was as low as 1%. This greater coating stability is attributed to the structural transition from adsorbed micelle to bilayer, which is induced by addition of SDS. Separation of a mixture of basic proteins yielded efficiencies of 364 000-562 000 plates/m and recoveries ranging from 85% to 98%. Migration time reproducibility was less than 0.9% relative standard deviation (RSD) from run to run and less than 2.6% RSD from day to day.     

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.     

Performances of new sugar-bearing poly(acrylamide) copolymers as DNA sieving matrices and capillary coatings for electrophoresis

We have synthesized two new sugar monomers, allylamine of gluconic and lactobionic acid, by opening the corresponding lactone ring with allylamine. These monomers were copolymerized with acrylamide leading to formation of copolymers with a relative molecular mass of 288000 and 180000 Da, respectively. Double-stranded DNA fragments were separated in entangled solutions of these linear polymers in capillary electrophoresis. Resolution, peak spacing and peak width were the parameters taken into account to evaluate the quality of the separation achieved with the new polymers. This work indicates that the copolymers of acrylamide and allyl gluconic acid have a high sieving capacity and provide a performance similar to that of hydroxyethylcellulose (HEC) of comparable viscosity. Unlike HEC, this copolymer selfcoats onto the capillary wall, allowing DNA fragments to be efficiently separated in an uncoated capillary.     

Non-covalent capillary coatings for protein separations in capillary electrophoresis

Adsorption of proteins, particularly basic proteins onto fused silica capillaries severely degrades capillary electrophoretic performance. This review provides a synopsis of the fundamentals underlying protein adsorption and its impact on CE performance. The efficacy of small molecule background electrolyte additives, surfactants, physically adsorbed polymers (dynamic and static), and successive multiple ionic-polymer layer coatings are evaluated using a number of performance metrics. Peak efficiency and migration time reproducibility are used as measures of reversible protein adsorption, while protein recovery, electroosmotic flow reproducibility and step changes in the baseline are used as indicators of irreversible protein adsorption.     

Evaluation of new adsorbed coatings in chiral capillary electrophoresis and the partial filling technique

When using chiral selectors and the partial filling technique in capillary electrophoresis, a suitable and reproducible suppression of the electroosmotic flow is still a challenging issue, and there are a number of reasons to find alternatives to the use of covalently coated capillaries for such a particular application. In this paper, new achiral, neutral, and water-soluble polymers are evaluated as adsorbed polymers for the suppression of electroosmotic flow (EOF) when employing chiral capillary electrophoresis and the partial filling technique. Four chiral selectors, namely a cationic cyclopeptide, vancomycin, human serum albumin and riboflavin binding protein have been chosen for this study and some analytes such as derivatized amino acids, promethazine and prilocaine have been used as test compounds. Reproducibility of migration times, resolution, and selectivity as well as efficiency are reported to critically evaluate the performance of the adsorbed coatings. Results are compared to parallel data obtained with fused-silica and polyvinyl alcohol-coated capillaries.     

Quaternized celluloses as new dynamic coatings in capillary electrophoresis for basic protein separation

In this work, a series of quaternized celluloses (QCs), homogeneously synthesized in the NaOH/urea aqueous solutions, were studied as dynamic coatings for capillary electrophoresis. Capillaries coated with these cationic cellulose derivatives at the concentration as low as 3 μg/mL were able to generate a stable, reversed electroosmotic flow. The effects of QC molecular parameters, such as the degree of cationic substitution and molecular weight, and the effect of buffer pH on the EOF mobility as well as the separation of basic proteins were investigated in detail. It was shown that the use of QC coatings in CE could drastically reduce the analysis time and improve the separation performance within a broad pH range. Five basic proteins, that is, lysozyme, ribonuclease A, cytochrome C, bovine pancreatic trypsin inhibitor, and chymotrypsinogen were baselinely separated even at pH 8.0. The separation efficiency and analysis reproducibility demonstrated that the QC coatings were efficient in minimizing the adsorption of basic proteins on the fused silica capillary. The successful performance was further demonstrated for biosample analysis.     

Novel cationic polyelectrolyte coatings for capillary electrophoresis

The use of bare fused silica capillary in CE can sometimes be inconvenient due to undesirable effects including adsorption of sample or instability of the EOF. This can often be avoided by coating the inner surface of the capillary. In this work, we present and characterize two novel polyelectrolyte coatings (PECs) poly(2‐(methacryloyloxy)ethyl trimethylammonium iodide) (PMOTAI) and poly(3‐methyl‐1‐(4‐vinylbenzyl)‐imidazolium chloride) (PIL‐1) for CE. The coated capillaries were studied using a series of aqueous buffers of varying pH, ionic strength, and composition. Our results show that the investigated polyelectrolytes are usable as semi‐permanent (physically adsorbed) coatings with at least five runs stability before a short coating regeneration is necessary. Both PECs showed a considerably decreased stability at pH 11.0. The EOF was higher using Good's buffers than with sodium phosphate buffer at the same pH and ionic strength. The thickness of the PEC layers studied by quartz crystal microbalance was 0.83 and 0.52 nm for PMOTAI and PIL‐1, respectively. The hydrophobicity of the PEC layers was determined by analysis of a homologous series of alkyl benzoates and expressed as the distribution constants. Our result demonstrates that both PECs had comparable hydrophobicity, which enabled separation of compounds with log P_o/w > 2. The ability to separate cationic drugs was shown with β‐blockers, compounds often misused in doping. Both coatings were also able to separate hydrolysis products of the ionic liquid 1,5‐diazabicyclo[4.3.0]non‐5‐ene acetate at highly acidic conditions, where bare fused silica capillaries failed to accomplish the separation.     

Dynamic capillary coatings for electroosmotic flow control in capillary electrophoresis

Control of the electroosmotic flow (EOF) is critical for achieving optimal separations by capillary electrophoresis. For instance, manipulation of the EOF can yield either high resolution separations or rapid analyses. Dynamic capillary coatings are a simple and cost-effective approach to altering the EOF. The normal EOF can be slowed using buffer additives such as Mg²⁺ and hexamethonium which ion exchange onto the surface silanols to lower the effective wall charge. Alternatively, cationic polyelectrolytes or cationic surfactants can be used to establish a cationic coating on the capillary wall, which results in a reversed EOF. Practical considerations such as pH stability and reproducibility obtainable with an EOF modifier will be discussed.     

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.     

Enhanced stability of surfactant-based semipermanent wall coatings in capillary electrophoresis using oppositely charged surfactant

Semipermanent surfactant coatings are effective for the prevention of wall adsorption of proteins in CE. However, they often suffer from their unsatisfactory coating stability as they essentially degrade from the capillary walls after the surfactants are removed from the buffer. In this paper, we proposed a facile and universal method to improve the stability of semipermanent surfactant coatings based on addition of an oppositely charged surfactant into the coating. Didodecyldimethylammonium bromide (DDAB) and a gemini surfactant, 18-6-18, were used as the model semipermanent coatings, and sodium dodecyl sulfate (SDS) was chosen as their oppositely charged surfactant. SDS can strongly alter the packing parameter P of the cationic surfactants, and consequently mediates the coating stability. With the increase of SDS concentration in coating, the coating stability first dramatically increases due to the enlarged P, and then decreases due to the weakness of electrostatic interaction between the capillary wall and surfactant coating. At the proper SDS concentration, very stable coatings can be obtained that, even after rinsing under 138 kPa for 60 min, the reversed electroosmotic flow (EOF) only decreases by 3.6%. These SDS-enhanced coatings show excellent stability and reproducibility in protein separation (RSD of migration time <1.1% for run-to-run assay, n=9). Also, the high separation efficiency (>500,000 plates/m) and fine recovery of tested proteins indicate that these coatings are powerful in wall adsorption suppression. Finally, we found that the separation efficiency of protein was a more exact indicator for the coating stability than the traditional EOF magnitude.