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   

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.     

Application of dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium in wall modification for capillary electrophoresis separation of proteins

A zwitterionic surfactant, dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium (C12H25N+(CH3)2CH2CHOHCH2SO3-), named dodecyl sulfobetaine (DSB), was used as a novel modifier to coat dynamically capillary walls for capillary electrophoresis separation of basic proteins. The DSB coating suppressed the electroosmotic flow (EOF) in the pH range of 3-12. At high DSB concentration, the EOF was suppressed by more than 8.8 times. The DSB coating also prevented successfully the adsorption of cationic proteins on the capillary wall. Anions, such as Cl-, Br-, I-, SO4(2-), CO3(2-), and ClO4-, could be used as running buffer modifiers to adjust the EOF for better separation of analytes. Using this dynamically coated capillary, a mixture of eight inorganic anions achieved complete separation within 4.2 min with the efficiencies from 24,000 to 1,310,000 plates/m. In the presence of ClO4- as EOF adjustor, the separation of a mixture containing four basic proteins (lysozyme, cytochrome c, alpha-chymotrypsinogen A, and myoglobin) yielded efficiencies of 204,000-896,000 plates/m and recoveries of 88%-98%. Migration time reproducibility of these proteins was less than 0.5% relative standard deviation (RSD) from run to run and less than 3.1% RSD from day to day, showing promising application of this novel modifier in protein separation.     

Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis

To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis.     

Co‐electroosmotic capillary electrophoresis of basic proteins with 1‐alkyl‐3‐methylimidazolium tetrafluoroborate ionic liquids as non‐covalent coating agents of the fused‐silica capillary and additives of the electrolyte solution

The paper reports the results of a study carried out to evaluate the use of three 1‐alkyl‐3‐methylimidazolium‐based ionic liquids as non‐covalent coating agents for bare fused‐silica capillaries and additives of the electrolyte solutions (BGE) for CE of basic proteins in the co‐EOF separation mode. The three ionic liquids are differentiated from each other by the length of the alkyl group on the imidazolium cation, consisting of either an ethyl, butyl or octyl substituent, whereas tetrafluoroborate is the common anionic component of the ionic liquids. Coating the capillary with the ionic liquid resulted in improved peak shape and protein separation, while the EOF was maintained cathodic. This indicates that each ionic liquid is effective at masking the protein interaction sites on the inner surface of the capillary, also when its adsorption onto the capillary wall has not completely neutralized all the negative charges arising from the ionization of the silanol groups and the ionic liquid is not incorporated into the BGE employed for separation. Using the coated capillaries with BGE containing the ionic liquid employed for the coating, at concentration low enough to maintaining the EOF cathodic, both peak shape and protein separation varied to different extents, based on the particular ionic liquid used and its concentration. Fast and efficient separation of the model basic protein mixture in co‐electroosmotic CE is obtained with the 1‐butyl‐3‐methylimidazolium tetrafluoroborate coated capillary and 100 mM acetate buffer (pH 4.0) containing 4.4 mM 1‐butyl‐3‐methylimidazolium tetrafluoroborate as the BGE.     

Effect of quasi‐interpenetrating network of polyacrylamide and poly(N,N‐dimethylacrylamide) on separation of dsDNA fragments and basic protein using CE

Quasi‐interpenetrating network (quasi‐IPN) of linear polyacrylamide (LPA) with low molecular mass and poly(N,N‐dimethylacrylamide) (PDMA), which is shown to uniquely combine the superior sieving ability of LPA with the coating ability of PDMA, has been synthesized for application in dsDNA and basic protein separation by CE. The performance of quasi‐IPN on dsDNA separation was determined by polymer concentration, electric field strength, LPA molecular masses and different acrylamide (AM) to N,N‐dimethylacrylamide (DMA) ratio. The results showed that all fragments in Φ×174/HaeIII digest were achieved with a 30 cm effective capillary length at –6 kV at an appropriate polymer solution concentration in bare silica capillaries. Furthermore, EOF measurement results showed that quasi‐IPN exhibited good capillary coating ability, via adsorption from aqueous solution, efficiently suppressing EOF. The effect of the buffer pH values on the separation of basic proteins was investigated in detail. The separation efficiencies and analysis reproducibility demonstrated the good potentiality of quasi‐IPN matrix for suppressing the adsorption of basic proteins onto the silica capillary wall. In addition, when quasi‐IPN was used both as sieving matrix and dynamic coating in bare silica capillaries, higher peak separation efficiencies, and better migration time reproducibility were obtained.     

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.     

Highly efficient protein separations in capillary electrophoresis using a supported bilayer/diblock copolymer coating

A surfactant/polymer wall coating consisting of the doubly chained cationic surfactant dimethyldioctadecylammonium bromide (DODAB) and polyoxyethylene (POE) 40 stearate is investigated. The coating is formed by simply rinsing a capillary with a solution containing DODAB and POE 40 stearate. The resultant coating is semi-permanent--demonstrating stable electroosmotic flow (EOF) even after a 60 min high pressure rinse with buffer. The EOF (-0.45+/-(0.23) x 10(-4) cm(2) V(-1) s(-1) at pH 7.4) is suppressed by more than a factor of ten compared to that observed for DODAB alone. Model protein mixtures were separated over a pH range of 3-10 with efficiencies of up to greater than 1 million plates/m for the basic proteins cytochrome c, lysozyme, ribonuclease A and alpha-lactalbumin, and the acidic proteins insulin chain A, trypsin inhibitor, and alpha-chymotrypsinogen A. Migration time reproducibility was 0.5-4.0% from run to run and 0.6-4.3% from day to day. Protein recoveries with this coating ranged from 84% to 97%.     

Effect of coating electrolytes on two-tailed surfactant bilayer coatings in capillary electrophoresis

Surfactants such as dioctadecyldimethylammonium bromide (DODAB) form semi-permanent coatings that effectively prevent adsorption of cationic proteins onto the fused silica capillary in capillary electrophoresis (CE). The bilayer coating is generated by flushing the capillary with a 0.1 mM surfactant solution. However, formation of the bilayer is strongly dependent on the coating electrolyte. The effect of counter-ions, electrolyte concentrations and buffer co-ions were monitored based on: the separation of basic model proteins; the adsorption kinetics of DODA⁺ onto fused silica; and dynamic light scattering (DLS) to determine vesicle size. Low concentrations (≤10.0 mM) and/or weakly associating buffers such as phosphate (pH 3.0), acetate (pH 4.0) and chloride should be used for DODAB coating solutions. Dissolving the surfactant in strongly associating electrolyte, such as phosphate at pH 7.0, results in poor coating of the capillary surface. Effective cationic bilayer coatings are formed if the buffer conditions favor formation of vesicles with diameters < 300 nm. Monitoring turbidity at 400 nm provides a convenient means of verifying vesicle diameter variation of <5 nm; that is, that the coating solution is effective.     

Separation of acidic and basic proteins by capillary electrophoresis using gemini surfactants and gemini-capped nanoparticles as buffer additives

This paper demonstrated simultaneous separation of acidic and basic proteins using cationic gemini surfactants as buffer additives in capillary electrophoresis. We showed that even at a low concentration (0.1 mmol·L-1) of alkanediyl-α,ω-bis(dimethyloctadecylammonium bromide) (18-s-18), the wall adsorption of both acidic and basic proteins could be effectively suppressed under acidic conditions. Smaller micelle size (e.g., s=5-8) is more effective for the separation of acidic proteins than larger micelle siz...     

Novel cationic coating agent for protein separation by capillary electrophoresis†

A novel positively charged surfactant N‐dodecyl‐N,N‐dimethyl‐(1,2‐propandiol) ammonium chloride was used for the dynamic coating of the inner wall of a silica capillary. This paper covers the evaluation of dynamic coating and study of the influence of the analysis conditions for the magnitude and direction of electroosmotic flow as well as for the effective and selective separation of chosen proteins (ribonuclease A, cytochrome c, lysozyme, and myoglobin). The concentration of 0.1 mM of N‐dodecyl‐N,N‐dimethyl‐(1,2‐propandiol) ammonium chloride enabled the reversal of the electro‐osmotic flow, however, to separate basic as well as neutral proteins the higher concentration of the studied surfactant was necessary. The final conditions for the separation of studied proteins were set at 100 mM sodium acetate pH 5.5 with 10.0 mM of the studied surfactant. The results were also compared with those of two commercially available cationic surfactants, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide. Additionally, the developed method for protein separation was applied for the determination of lysozyme in a cheese sample. The limits of detection and quantification of lysozyme were 0.9 and 3.0 mg/L, respectively. The mean concentration of lysozyme found in the cheese sample was 167.3 ± 10.3 mg/kg.     

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.     

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.     

Silanizing agent for semipermanent wall coating in micellar electrokinetic capillary chromatography

In this paper, the long-chained, silanizing agent chloro(dodecyl)dimethylsilane (CDDS) was investigated as a semipermanent coating in micellar electrokinetic capillary chromatography (MEKC). CDDS coating had great stability due to the formation of covalent bonding with the silanol groups on the surface of fused-silica capillary and remained stable for over 100 min after removal of the rinse step of CDDS solution. Anionic surfactant sodium dodecyl sulfate (SDS) could aggregate at this CDDS coating by the hydrophobic group and formed a SDS layer which could increase the electroosmotic flow (EOF). The separation was performed with the running buffer composed of 60mM sodium tetraborate, 12 mM SDS at pH 9.9, with the applied voltage of 20 kV and capillary temperature 25 degrees C. The effect of the coating agent was investigated by the analysis of amino acids. Compared with previous no-coating method, the EOF increases from 4.34 x 10(-4)cm(2)V(-1)s(-1) to 7.02 x 10(-4)cm(2)V(-1)s(-1). Migration time reproducibility was less than 0.97% R.S.D. from run to run and less than 1.56% R.S.D. from day to day.     

Poly-N-hydroxyethylacrylamide as a novel, adsorbed coating for protein separation by capillary electrophoresis.

We present the polymer poly-N-hydroxyethylacrylamide (PHEA) (polyDuramide) as a novel, hydrophilic, adsorbed capillary coating for electrophoretic protein analysis. Preparation of the PHEA coating requires a simple and fast (30 min) protocol that can be easily automated in capillary electrophoresis instruments. Over the pH range of 3-8.4, the PHEA coating is shown to reduce electroosmotic flow (EOF) by about 2 orders of magnitude compared to the bare silica capillary. In a systematic comparative study, the adsorbed PHEA coating exhibited minimal interactions with both acidic and basic proteins, providing efficient protein separations with excellent reproducibility on par with a covalent polyacrylamide coating. Hydrophobic interactions between proteins and a relatively hydrophobic poly-N,N-dimethylacrylamide (PDMA) adsorbed coating, on the other hand, adversely affected separation reproducibility and efficiency. Under both acidic and basic buffer conditions, the adsorbed PHEA coating produced an EOF suppression performance comparable to that of covalent polyacrylamide coating and superior to that of adsorbed PDMA coating. The protein separation performance in PHEA-coated capillaries was retained for 275 consecutive protein separation runs at pH 8.4, and for more than 800 runs at pH 4.4. The unique and novel combination of hydrophilicity and adsorptive coating ability of PHEA makes it a suitable wall coating for automated microscale analysis of proteins by capillary array systems.     

Highly efficient dynamic modification of plastic microfluidic devices using proteins in microchip capillary electrophoresis

New dynamic coating agents were investigated for the manipulation of electroosmotic flow (EOF) in poly(methylmethacrylate) (PMMA) microchips. Blocking proteins designed for enzyme-linked immunosorbent assay (ELISA) applications (e.g. Block Ace and UltraBlock), and egg-white lysozyme were proposed in this study. The EOF could be enhanced, suppressed or its direction could be reversed, depending on the buffer pH and the charge on the proteins. The coating procedure is simple, requiring only filling of the microchannels with a coating solution, followed by a rinse with a running buffer solution prior to analysis. One major advantage of this method is that it is not necessary to add the coating agent to the running buffer solution. Block Ace and UltraBlock coatings were stable for at least five runs in a given microchannel without the need to condition the coating between runs other than replenishing the buffer solution after each run, i.e. the RSD values of EOF (n=5) were less than 4.3%, and there was no significant change in the EOF after 5 runs. The reproducibility of the coating procedures was found from the channel-to-channel RSD values of the EOF, and were less than 5.0% when using HEPES-Na buffer (pH 7.4) as the running buffer. Several examples of electrophoretic separations of amino acids and biogenic amines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) are demonstrated in this paper. The dynamic coating method has the potential for a broad range of applications in microchip capillary electrophoresis (microchip CE) separations.     

Studies on the analytical performance of a non-covalent coating with N,N-didodecyl-N,N-dimethylammonium bromide for separation of basic proteins by capillary electrophoresis in acidic buffers in 25-and 50-μm capillaries

Capillaries (25-and 50-μm inner diameter) coated with a double-alkyl-chain cationic surfactant N,N-didodecyl-N,N-dimethylammonium bromide (DDAB) were used for the separation of four basic standard proteins in buffers of pH 4 at various ionic strengths. The choice of buffer is critical for the analytical performance. Ammonium ions must be avoided in the buffer used in the non-covalent coating procedure owing to competition with the surfactant. Phosphate buffer gave a better separation performance than some volatile buffers; the reason seems to be a complex formation between the proteins and dihydrogenphosphate ions, which decreases tendencies for adsorption to the capillary surface. The DDAB coating was easy to produce and stable enough to permit, without recoating, consecutive separations of the proteins for up to 100 min with good precision in migration times and peak areas. A strong electroosmotic flow gives rapid separations, which is of special importance when commercial instruments are used, since the choice of the length of the capillary is restricted. Figure EOF stability in 25 micrometer i.d. capillaries. Consecutive injections of mesityloxide performed after an initial coating with 1.0     

Capillary electrophoresis of seed albumins fromVicia species using uncoated and surface-modified fused silica capillaries

Summary Capillary zone electrophoresis has been developed for the separation of seed albumins fromVicia faba using both uncoated and polyoxyethylene ether (Brij-35) coated octadecylsilane derivatized capillaries. Optimal separation conditions were found by studying the effect of pH, buffer composition and applied voltage. The nonionic surfactant/C₁₈ coated capillary significantly reduced albumin adsorption and electroosmotic flow (EOF). A gradual washing out of the surfactant from the coated capillary during use altered not only the magnitude of the EOF, but also its reproducibility. The introduction of hydrophilic polymer solutions between analyses for dynamic modification of the Brij/C₁₈ coated capillary surface prevented desorption of coating material, allowed optimization of resolution and ensured stability of the EOF. CE with surface-modified capillaries was then used to compare seed albumin profiles of severalVicia species. This technique appears to provide a powerful tool for use in taxonomic investigations.     

Capillary electrophoresis of seed albumins fromVicia species using uncoated and surface-modified fused silica capillaries

Summary Capillary zone electrophoresis has been developed for the separation of seed albumins fromVicia faba using both uncoated and polyoxyethylene ether (Brij-35) coated octadecysilane derivatized capillaries. Optimal separation conditions were found by studying the effect of pH, buffer composition and applied voltage. The nonionic surfactant/C₁₈ coated capillary significantly reduced albumin adsorption and electroosmotic flow (EOF). A gradual washing out of the surfactant from the coated capillary during use altered not only the magnitude of the EOF, but also its reproducibility. The introduction of hydrophilic polymer solutions between analyses for dynamic modification of the Brij/C₁₈ coated capillary surface prevented desorption of coating material, allowed optimization of resolution and ensured stability of the EOF. CE with surface-modified capillaries was then used to compare seed albumin profiles of severalVicia species. This technique appears to provide a powerful tool for use in taxonomic investigations.     

Spermine-graft-dextran non-covalent copolymer as coating material in separation of basic proteins and neurotransmitters by capillary electrophoresis

Spermine-graft-dextran (Spe-g-Dex) copolymer was synthesized and used as a non-covalent coating for the separation of proteins and neurotransmitters by capillary electrophoresis. The coating was obtained via flushing the capillary with 1.0% Spe-g-Dex copolymer solution for 2 min. Electroosmotic flow (EOF) was strongly suppressed, ranging from −1.60 × 10⁻⁹ to 3.65 × 10⁻⁹ m² V⁻¹ s⁻¹. Effect of experimental conditions, such as the copolymer concentration, the concentration and pH of the background electrolyte (BGE), on the Spe-g-Dex coating was investigated. Separation of lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen yielded high separation efficiencies ranging from 141 000 to 303 000 plates/m and recoveries from 85.4% to 98.3% at pH 4.0 (284.0 mM sodium acetate–acetic acid buffer, I = 50 mM). Run-to-run repeatabilities and day-to-day, and capillary-to-capillary reproducibilities were all below 1.7%. In addition, Spe-g-Dex coating allowed the successful separation of five neurotransmitters, 5-hydroxytryptamine, dopamine, epinephrine, isoprenaline, dobuamine at pH 4.0 with high separation efficiencies of 290 000–449 000 plates/m.