Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins

A new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (p/s) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with p/s higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3-10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.     

Comparison of different capillary isoelectric focusing methods--use of "narrow pH cuts" of carrier ampholytes as original tools to improve resolution

Two capillary isoelectric focusing (CIEF) systems have first been optimized: one uses a bare silica capillary and 30% (v/v) of glycerol in the separation medium while the other uses a coated capillary and an aqueous background electrolyte. To perform permanent capillary coating, two neutral polymers have been compared: hydroxypropylcellulose (HPC) and polyvinylalcohol (PVA). HPC coating gave best results for electroosmotic flow (EOF) limitation on a wide pH range: as compared to a bare silica capillary, it allowed to decrease EOF by 96% at pH 7.2 after acidic and basic treatments, whereas PVA coating lead only to a 76% decrease. The glycerol CIEF system was more satisfying for the separation of model proteins classically used as pI markers. Finally, the use of "narrow pH cuts" of carrier ampholytes added to commercial ampholyte mixtures allowed increasing resolution up to a factor 2.4 at a chosen pH for the separation of pI markers and milk proteins.     

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.     

Use of quasi-isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes.     

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.     

Monomer surface modifications for rapid peptide analysis by capillary electrophoresis and capillary electrochromatography coupled to electrospray ionization-mass spectrometry

In this study, positively charged alkylaminosilyl monomers were used to modify the inner surface of fused silica capillaries, which subsequently were employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC). The obtained surfaces yield a reversed electroosmotic flow (EOF) and have varying carbon chain lengths, that interact with the analytes and give chromatographic retention. The coating procedure is very simple and fast. The performance of the modified capillaries was evaluated regarding pH influence on EOF and chromatographic interactions. The experiments were conducted with UV and mass spectrometry (MS) and applied to the separation of various neuropeptides. The derivatized surfaces showed a linear (R(2) approximately 0.99) pH dependence with isoelectric points (pI) at 8.6-8.8. Rapid separations of peptide standards and a protein digest with efficiencies as high as 5 x 10(5) plates/m were performed.     

Nonaqueous capillary electrophoresis using a titania-coated capillary

In this work, an ordered mesoporous titania film was introduced to coat a capillary by means of the sol-gel technique. Its electroosmotic flow (EOF) property was investigated in a variety of nonaqueous media (methanol, formamide and N,N'-dimethylformamide and mixtures of methanol and acetonitrile). The titania-coated capillary exhibited a distinctive EOF behavior, the direction and magnitude of which were strongly dependent on various parameters such as the solvent composition, apparent pH (pH*) and the electrolytes. The nonaqueous capillary electrophoresis separation of several alkaloids was investigated in the positively charged titania-coated capillary. Comparison of separation between coated and uncoated capillaries under optimal nonaqueous conditions was also carried out.     

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.     

pH-independent large-volume sample stacking of positive or negative analytes in capillary electrophoresis

In capillary electrophoresis, the short optical path length associated with on-column UV detection imposes an inherent detection problem. Detection limits can be improved using sample stacking. Recently, large-volume sample stacking (LVSS) without polarity switching was demonstrated to improve detection limits of charged analytes by more than 100-fold. However, this technique requires suppression of the electroosmotic flow (EOF) during the run. This necessitates working at a low pH, which limits using pH to optimize selectivity. We demonstrate that LVSS can be performed at any buffer pH (4.0-10.0) if the zwitterionic surfactant Rewoteric AM CAS U is used to suppress the EOF. Sensitivity enhancements of up to 85-fold are achieved with migration time, corrected area, and peak height reproducibility of 0.8-1.6%, 1.3-3.7%, and 0.8-4.9%, respectively. Further, it is possible to stack either positively or negatively charged analytes using zwitterionic surfactants to suppress the EOF.     

Control of electroosmotic flow in nonaqueous capillary electrophoresis by polymer capillary coatings

In aqueous capillary electrophoresis the electroosmotic flow (EOF) can be strongly suppressed or eliminated by coating the capillary surface silanols either by buffer additive adsorption or chemical modification. Hydrophilic coatings, e.g., polyvinyl alcohol (PVA) proved to be most efficient for EOF control in applications like DNA analysis. In nonaqueous capillary electrophoresis (NACE), however, the EOF cannot be totally suppressed with these capillaries and coating efficiency turned out to be solvent-depending. In this paper, fused-silica capillaries with monomeric and polymeric coatings differing in hydrophobicity and chemical properties (vinyl, vinyl acetate, vinyl alcohol and acrylates with different alkyl chain length) were investigated. Besides studying the EOF characteristics with different organic solvents and water, gas chromatography (GC) measurements were carried out to probe the silanol reduction via ether retention and the surface hydrophobicity by retention of nonane. Good correlations between GC results and EOF magnitude could be found. It could be demonstrated that the polymeric coating has to be solvatized by the buffer solvent to reduce the EOF. The PVA coating was optimal for aqueous systems but not effective for some nonaqueous buffers. On the other hand, polyvinyl acetate and polyethyl acrylate as polymeric coatings proved to be optimal to reduce the EOF in NACE.     

Preparation of a sulfonated fused-silica capillary and its application in capillary electrophoresis and electrochromatography

In the present paper, two new methods, sol-gel and chemical bonding methods, were proposed for preparation of sulfonated fused-silica capillaries. In the sol-gel method, a fused-silica capillary was coated with the sol solution obtained by hydrolysis of 3-mercaptopropyltrimethoxysilane (MPTS) and tetramethoxysilane, and followed by age; while in the chemical bonding method, a capillary was chemically bonded directly with MPTS. Then, both the resulting capillaries were oxidized with an aqueous solution of hydrogen peroxide solution (H2O2) (30%, m/m) to obtain the sulfonated capillaries. The electroosmotic flow (EOF) for the sulfonated capillaries was found to remain almost constant within the studied pH range, and greater than that of the uncoated capillary. However, the coating efficiency of the capillary prepared by chemical bonding method was higher than that by sol-gel method, by comparing their magnitude of the EOF, the degree of disguise of the silanol and reproducibility of preparation procedure. The effects of the electrolyte's concentration and the content of methanol (MeOH) on the EOF were also studied. Especially, the study of the apparent pH (pH*) on the EOF in a water-MeOH system was reported. Finally, capillary electrophoretic separation of seven organic acids was achieved within 6.5 min under optimal condition using the chemically bonded sulfonated capillary. Moreover, separation of four alkaloids on the sulfonated capillary was compared with that on uncoated capillary in different conditions. Ion-exchange mechanism was found to play a key role for separation of these four basic analytes on the sulfonated capillary.     

Capillary isoelectric focusing--reproducibility and protein adsorption

Capillary isoelectric focusing (CIEF) is an important tool for the quality assurance of biotechnologically maintained drugs and for proteome analysis. The critical performance parameters of this technique are the precisions of isoelectric point (pI) values and peak areas. Compared to capillary zone electrophoresis (CZE), where precise results can be obtained (e.g., 0.5% relative standard deviation (RSD) for peak areas, n = 60), only few data are available for CIEF experiments. So far, reproducible data of pI values (RSD = 0.5%) have been acquired, but peak areas show inferior results (about 3-15% RSD). Nonstable capillary coatings and protein adsorption have been discussed as possible reasons. Recent work of Righetti et al. [25, 27] has proven that the use of coated capillaries can reduce the adsorption of proteins by 50% but cannot prevent it. In our CIEF experiments irregular and poorly reproducible peak patterns have been observed. In a long-time experiment of 106 repeated runs, an overall RSD of 10% was obtained for peak areas, RSD of 2% only in series of about 10 consecutive replicates. Especially at higher concentrations the reproducibility deteriorates. This seems to be the result of a self-amplifying process, induced by adsorbed protein molecules, leading to further agglomerations. CZE control experiments in linear polyacrylamide (LPA)-coated capillaries proved a strong pH dependency of these effects within a small range. Compared to bare fused-silica surfaces, adsorption effects are reduced but not inhibited. An enhancement of reproducibility in CIEF experiments can be achieved only by controlling the interactions of proteins and capillary walls.     

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   

Enantiomeric separation by capillary electrophoresis with an electroosmotic flow-controlled capillary

Perfect control of electroosmotic flow (EOF) was achieved by dovetailing successive multiple ionic-polymer layer (SMIL) coated capillaries. The direction and magnitude of the EOF was perfectly controllable over the pH range 2-13. Zone diffusion was not observed, even if the inner wall of the dovetailed capillary was discontinuous, or if the sample zone passed through the connected part of the capillary because the RSDs of migration time, theoretical plates, symmetry factor and S/N of the marker were almost the same when seamless capillary and dovetailed capillary were compared. The dovetailed capillary was applied to cyclodextrin modified capillary zone electrophoresis. The control of the EOF enabled us to control both the resolution and the migration order of the enantiomers. The migration time was also controllable and, therefore, the best condition between separation and migration time could be determined by controlling the EOF. Partial filling affinity electrokinetic chromatography with a protein used as a chiral selector was also studied. The migration of the pseudostationary phase was controllable by EOF, and detection of the solute at 214 nm was possible. Therefore, the EOF-controlled dovetailed capillary has great potential to expand the application of the separation technique.     

Evaluation of capillary isoelectric focusing in glycerol-water media with a view to hydrophobic protein applications

Capillary isoelectric focusing (CIEF) separations are usually performed with neutral coated fused-silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (eta = 945 mPa x s at 25 degrees C), known to help stabilize any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to using commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 v/v glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused-silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their pI with a good agreement between experimental and literature aqueous pIs. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase, a complex mixture of protein isoforms. The good concordance with the separation obtained by the conventional CIEF system indicated the adequacy of this new system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form.     

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.     

Semipermanent capillary coatings in mixed organic-water solvents for CE

This report describes the creation of semipermanent capillary coatings that are compatible with organic-water solvent systems in CE. The coatings are created by simply rinsing the fused-silica capillary with long double-chain cationic surfactants, such as dimethyl-ditetradecyl ammonium bromide (2C(14)DAB), dihexadecyldimethyl ammonium bromide (2C(16)DAB), and dimethyldioctadecyl ammonium bromide (2C(18)DAB). These surfactants generate semipermanent bilayer coatings on the capillary surface, which display a high degree of stability in buffers containing up to 60% v/v of organic solvents, such as methanol and ACN. The coating stability increases with increasing hydrophobicity of the surfactant, i.e., with increasing chain length. For instance, the EOF changes by only 1.2% in a 2C(18)DAB-coated capillary after 130 capillary volumes of rinsing with 60% v/v methanol containing buffer. The bilayer coatings allow separations to be performed without the need to regenerate the coating between runs or to maintain the EOF modifier in the run buffer. Rapid separations (<2 min) of anions and basic drugs with migration time reproducibility of less than 0.5% RSD and efficiencies of 0.4-0.6 million plates/m are obtained. In addition, selectivity changes for small anions and cationic drugs are also observed when the organic solvent content is adjusted.     

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.     

Modification of capillary electrophoresis capillaries by poly(hydroxyethyl methacrylate), poly(diethylene glycol monomethacrylate) and poly(triethylene glycol monomethacrylate)

Modification of capillary electrophoresis (CE) capillaries by poly(hydroxyethyl methacrylate) (poly(HEMA), poly(diethylene glycol monomethacrylate) (poly(DEGMA) and poly(triethylene glycol monomethacrylate) (poly(TEGMA), was studied. Methods based on physical adsorption of the modifier and on its chemical binding were compared on the basis of the electroosmotic flow (EOF) reproducibility, the EOF dependence on the pH, the symmetry of the peak of positively charged tyramine, the stability of the coating and the separation of standard and milk proteins in the modified capillaries. Reproducible coatings were obtained by chemical binding of the polymers to the capillary walls and by coating with a solution of a polymer, as also demonstrated by the atomic force microscopy.     

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.