Directed control of electroosmotic flow in nonaqueous electrolytes using poly(ethylene glycol) coated capillaries

Poly(ethylene glycol) (PEG)-coated capillaries exhibit unique properties in nonaqueous electrolytes. Immobilized PEG interacts significantly with different cations present in nonaqueous electrolytes. This can induce a positive surface charge on PEG-coated capillaries and results in an adjustable anodic electroosmotic flow (EOF) in nonaqueous electrolytes whereas a reduced cathodic EOF is observed in aqueous electrolytes. The EOF can reversibly be adjusted by the variation of the electrolyte constitution, namely the type of the solvent used and the nature and concentration of background cations. In methanol and especially in acetonitrile electrolytes the magnitude and also the direction of EOF is strongly dependent on the water content. Using different alkali metal cations, the EOF can be increased, reduced, or even reversed depending on the nature of the cation. The directed manipulation of EOF in methanolic electrolytes using PEG-coated capillaries was applied for optimization of nonaqueous capillary electrophoretic separations of acidic compounds with regard to reproducibility, resolution, and analysis time.     

Non-uniform surface charge distributions in CE: theoretical and experimental approach based on Taylor dispersion

The control of the EOF direction and magnitude remains one of the more challenging issues for the optimization of separations in CE. In this work, we investigated the possibility to use non-uniform surface charge distribution for the modulation of the EOF in CE. Non-uniform zeta potentials were obtained by modifying a section of the capillary surface using adsorption of polyelectrolytes. Three different methods were studied: (i) partial polycation coating on a fused silica capillary, (ii) partial polycation (or polyanion) coating on polyelectrolyte multilayers, and (iii) partial polycation coating on a capillary previously modified with poly(ethylene oxide). The magnitude and the direction of the EOF as a function of the coated capillary length were first studied. The stability of the EOF and the separation performances were also considered taking two dialanine diastereoisomers as model compounds. In partially coated capillaries, the average solvent flow is the sum of two contributions: a non-dispersive electroosmotic contribution related to the capillary surface charge, and a dispersive hydrodynamic contribution that depends on the difference of surface charge between the coated and the non-coated capillary zones. To get a better insight into the influence of the hydrodynamic contribution to the total peak dispersion, the peak variances corresponding to the Taylor dispersion, the injection plug, and the axial diffusion were calculated. This work demonstrates that peak dispersion in a capillary partially coated by the inlet end is different from that obtained when the coating is performed by the outlet end. Experimentally, the combination of a partially coated capillary with a large volume sample stacking preconcentration step can be used for injecting up to 95% of the capillary volume. This approach leads to a preconcentration factor of 60 compared with CZE with classical injection.     

Separation of living and dead polymers in synthetic polypeptide mixtures by nonaqueous capillary electrophoresis using differences in ionization states

The complexity in the mechanisms of polymerization of N-carboxyanhydrides requires the development of new analytical techniques able to separate mixtures of synthetic polypeptides. This work focuses on the separation of poly(N(epsilon)-trifluoroacetyl-L-lysine) (PTLL) mixtures by nonaqueous capillary electrophoresis (CE). The main goal of this work was to find electrophoretic conditions that permit the separation and the quantification of the dead polymer families that were previously identified in the samples. The influence of the pH of the electrolyte on the selectivity of the separation was carefully investigated. The mechanisms of separation of the PTTLs are discussed as a function of their ionization state. The separations obtained on a noncovalently coated capillary were compared with those obtained on a fused-silica capillary. Finally, using two different electrolytes, it is possible to quantify the three families of PTLLs, namely, the living PTLLs, the dead PTLLs with N-formyl end group and the dead PTLLs with a carboxylic end group. These results confirm the importance of CE for the separation of synthetic organic polymers in nonaqueous electrolytes.     

Nonaqueous capillary electrophoresis with alcoholic background electrolytes: separation efficiency under high electrical field strengths

The effect of high voltage on capillary electrophoresis (CE) separations of anionic analytes in nonaqueous separation media was investigated. Methanol, ethanol, 1-propanol, and 1-butanol were tested as background electrolyte (BGE) solvents. Experiments were carried out with a laboratory-built CE instrument suitable for high-voltage separations. Potentials up to 60 kV were applied with reversed polarity to generate unusually high field strengths (e.g. 2000 Vcm-1) and so achieve fast and efficient separations. Highest separation efficiencies were obtained with propanol as BGE solvent, and the dependency of the efficiency on the separation voltage was more or less linear. With the other alcohols, separation efficiency decreased or remained roughly constant with increasing absolute voltage. The separation efficiencies are discussed in terms of longitudinal diffusion, Joule heating, and analyte interaction with the capillary wall. Capillary preconditioning had a varied effect on the separations in the different BGEs as the BGE and the conditioning process affected the electroosmotic flow (EOF) velocity and direction.     

Nonaqueous capillary electrophoresis-mass spectrometry

Nonaqueous background electrolytes broaden the application of capillary electrophoresis displaying altered separation selectivity and interactions between analytes and buffer additives compared to aqueous background electrolytes. In addition, nonaqueous capillary electrophoresis (NACE) appears to be ideally suited for online coupling with mass spectrometry due to the high volatility and low surface tension of many organic solvents. Despite these advantages and an increasing use of nonaqueous background electrolytes in CE, coupling of NACE to mass spectrometry has not yet been applied very often to date. The present review summarizes the applications of online NACE-MS with regard to the analysis of drugs, stereoisomers, peptides, alkaloids, polymers and others. A brief discussion of solvent effects in NACE and pH of nonaqueous background electrolyte systems is also presented.     

毛细管电泳中影响径向电场控制电渗的主要因素

利用自制的二维电场毛细管电泳系统研究了不同因素对径向电场控制电渗能力的影响,发现缓冲液的ｐＨ值、浓度、种类以及管壁表面状态、管径等对电渗的电场调控有关键性的影响。有趣的是,添加剂不影响电场的调控能力,而杯芳烃涂层毛细管却能提高电渗对径向电场的响应能力。利用这种涂层效应有可能实现较高ｐＨ值下电渗的电场调控。     

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.     

Influence of polyelectrolyte capillary coating conditions on protein analysis in CE

CE of biomolecules is limited by analyte adsorption on the capillary wall. To prevent this, monolayer or successive multiple ionic‐polymer layers (SMILs) of highly charged polyelectrolytes can be physically adsorbed on the inner capillary surface. Although these coatings have become commonly used in CE, no systematic investigation of their performance under different coating conditions has been carried out so far. In a previous study (Nehmé, R., Perrin, C., Cottet, H., Blanchin, M. D., Fabre, H., Electrophoresis 2008, 29, 3013–3023), we investigated the influence of different experimental parameters on coating stability, repeatability and peptide peak efficiency. Optimal coating conditions for monolayer and multilayer (SMILs) poly(diallyldimethylammonium) chloride/ poly(sodium 4‐styrenesulfonate) coated capillaries were determined. In this study, the influence of polyelectrolyte concentration and ionic strength of the coating solutions, and the number of coating layers on coating stability and performance in limiting protein adsorption was carried out. EOF magnitude and repeatability were used to monitor coating stability. Coating ability to limit protein adsorption was investigated by monitoring variations of migration times, time‐corrected peak areas and separation efficiency of test proteins. The separation performance of polyelectrolyte coatings were compared with those obtained with bare silica capillaries.     

Ionic liquids as electrolytes for nonaqueous capillary electrophoresis

Acetonitrile is a well-suited medium for nonaqueous capillary electroseparations and enables extending the range of applications of capillary electrophoresis (CE) techniques to more hydrophobic species. In this study, the dialkylimidazolium-based low temperature melting organic salts know as "ionic liquids" (ILs) are used as electrolytes. At room temperature these liquids are miscible with acetonitrile which makes it easy to use them for adjustment of analyte mobility and separation. The anionic part as well as the concentration of an IL influence the general electrophoretic mobility of the buffer system. The separation of different analytes is achieved because they become charged in the presence of ILs in separation media. There is also a possibility for a complex formation between the solute and the electrolyte which alters the mobility of the solute. A selected application of separations of phenols and aromatic acids will be discussed.     

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.     

Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.     

Analysis of double-stranded DNA by capillary electrophoresis using poly(ethylene oxide) in the presence of hexadecyltrimethylammonium bromide

The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by capillary electrophoresis in conjunction with laser-induced fluorescence (CE-LIF) detection using poly(ethylene oxide) (PEO) solution is described. The use of CTAB for improved separation reproducibility and efficiency of DNA has not been demonstrated although it is widely used for controlling the magnitude and direction of electroosmotic flow in CE. With increasing CTAB concentration, the interactions of DNA with ethidium bromide (EtBr) and with the capillary wall decrease. For the separation of DNA fragments with the sizes ranging from several base pairs (bp) to 2,176 bp, a polymer solution consisting of 0.75% poly(ethylene oxide), 100 mM TB buffer (pH 8.0), 25 microg/mL EtBr, and 0.36 microg/mL CTAB is proper. Using the PEO solution, we separated a mixture of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BglI digest and pBR 328/HinfI digest) within 8 min at -375 V/cm, with the limit of detection of 2.0 ng/mL based on the peak height for the 18-bp DNA fragment. The method is highly efficient (>10(6)plate/m), repeatable (RSD of the migration times <1.5%), and sensitive. In addition, it is convenient to fill a capillary (75 microm in diameter) with such a low-viscosity PEO solution by syringe pushing.     

Use of coated capillaries for the electrophoretic separation of stereoisomers of a growth hormone secretagogue

The diastereoisomeric separation of peptidomimetics of hexarelin, a strong growth hormone secretagogue, in CE has been studied. Highly sulfated‐γ‐CD was found to be an appropriate selector for the separation of the stereoisomers. However, non‐repeatable analyses were obtained on bare fused silica capillary due to the progressive adsorption of the analytes on the capillary wall. Two types of polyelectrolyte coating agents were tested to prevent this phenomenon. Coating with neutral polyethylene oxide was found to be efficient but resulted in a very long analysis time (about 40 min). Coating with cationic poly(diallyldimethylammonium) chloride was found both to prevent analyte adsorption, reduce analysis time and alter separation selectivity. EOF measurement revealed that the highly sulfated‐γ‐CDs were strongly adsorbed on the poly(diallyldimethylammonium) chloride coating surface yielding a stable strong cathodic EOF, which considerably reduced analysis time (about 12 min). Very good repeatability of analysis was obtained (RSD_{migration time}<1%).     

Electroosmotic flow changes due to interactions of background electrolyte counter-ions with polyethyleneimine coating in capillary zone electrophoresis of proteins

The properties and behavior of polyethyleneimine (PEI) covalently coated capillaries with respect to different background electrolytes used in capillary zone electrophoresis (CZE) are described. The coating stability and changes of inner surface charge in the capillary were followed by measurement of electroosmotic flow (EOF). Interest was focused mainly on conjugate bases of carboxylic acids as anionic background electrolyte components (acetate, citrate, malate, malonate, tartrate, and succinate). An interesting phenomenon was observed in PEI-coated capillaries: The direction (and the magnitude) of EOF depends on the composition of the background electrolyte and at a certain pH it can undergo reversible change. Ionic complex formation was suggested as a hypothesis to explain this behavior. With this knowledge, the PEI-coated capillary was used for the separation of basic proteins in the above-mentioned background electrolytes. A standard protein mixture of cytochrome c, ribonuclease A, and lysozyme at a concentration of 0.25 mg/mL each was chosen as model sample.     

Control of the EOF in CE using polyelectrolytes of different charge densities

The control of the EOF direction and magnitude remains one of the more challenging issues for the optimization of separations in CE. In this work, we investigated the possibility to use variously charged polyanions for a fine-tuning of the EOF using polyelectrolyte multilayers. For that purpose, polyanions of poly(acrylamide-co-2-acrylamido-2-methyl-1-propanesulfonate) (PAMAMPS) with different chemical charge rates varying between 3 and 100% were used. These copolymers are statistic hydrophilic copolymers of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS). The study of the influence of the chemical charge rate (AMPS molar proportion in the copolymer) on the electroosmotic mobility (mu(eo)) of a capillary modified by a polyelectrolyte bilayer (polycation/PAMAMPS) revealed that the fine-tuning of the EOF was possible, at least for cathodic or slightly anodic EOF (micro(eo) from -5 x 10(-5) to +35 x 10(-5) cm(2)V(-1)s(-1)). Electroosmotic mobility values were compared with the free-draining electrophoretic mobilities of the PAMAMPS constituting the last layer of the capillary coating. The stability of the EOF is discussed in detail on the basis of successive determinations of electroosmotic mobility and migration times. The application to the separation of a model peptide mixture demonstrated the interest (and the simplicity) of this approach for optimizing resolution and analysis time. Experimental resolutions were compared to the theoretical ones that we would obtain on a fused-silica capillary having the same EOF as the coated capillary.     

Capillary electrophoresis using copolymers of different composition as physical coatings: a comparative study

In this work, a comparative study on the use of different polymers as physically adsorbed coatings for CE is presented. It is demonstrated that the use of ad hoc synthesized polymers as coatings allows tailoring the EOF in CE increasing the flexibility of this analytical technique. Namely, different polymers were synthesized at our laboratory using different percentages of ethylpyrrolidine methacrylate (EpyM) and N,N-dimethylacrylamide (DMA). Thus, by modifying the percentage of EpyM and DMA monomers it is possible to manipulate the positive charge of the copolymer, varying the global electrical charge on the capillary wall and with that the EOF. These coated capillaries are obtained by simply flushing a given EpyM-DMA aqueous solution into bare silica capillaries. It is shown that by using these coated capillaries at adequate pHs, faster or more resolved CE separations can be achieved depending on the requirements of each analysis. Moreover, it is demonstrated that these coated capillaries reduce the electrostatic adsorption of basic proteins onto the capillary wall. Furthermore, EpyM-DMA coatings allow the reproducible chiral separation of enantiomers through the partial filling technique (PFT). The EpyM-DMA coated capillaries are demonstrated to provide reproducible EOF values independently of the pH and polymer composition with%RSD values lower than 2% for the same day. It is also demonstrated that the coating procedure is reproducible between capillaries. The compatibility of this coating protocol with CE in microchips is discussed.     

On-column concentration and separation of double-stranded DNA by gradient capillary electrophoresis

We describe the separation of dsDNA by capillary electrophoresis in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO). Using 1.0% PEO, the separation of DNA fragments with sizes ranging from 51 bp to 23 kbp has been achieved in less than 12 min, which is better than conventional methods (in the absence of EOF) in terms of speed and resolution. In order to concentrate and separate the DNA sample, gradient changes in the concentrations of PEO and ethidium bromide (EtBr) have been conducted. Different concentrations of PEO solutions are injected to the polyethylene tubes by pressure, where they enter the capillary by EOF. Because the large DNA fragments migrate faster towards the cathode end under counterflow conditions, the introduction sequence is from low to high concentrations of PEO solutions after sample injection. Using the gradient CE approach, the separations of the DNA sample injected at 30 cm height for times up to 120 s have been demonstrated. The linearity between injection time and peak height shows that the DNA fragments stacked during migration from the sample zone to PEO. We found that stacking efficiency is greater when the analysis was performed by simultaneously changing the PEO and EtBr concentration, compared to individual changes in PEO concentration.     

Cationized hydroxyethylcellulose as a novel, adsorbed coating for basic protein separation by capillary electrophoresis

We present cationized hydroxyethylcellulose (cat-HEC) synthesized in our laboratory as a novel physically adsorbed coating for CE. This capillary coating is simple and easy to obtain as it only requires flushing the capillary with polymer aqueous solution. A comparative study with and without polymers was performed. The adsorbed cat-HEC coating exhibited minimal interactions with basic proteins, providing efficient basic protein separations with excellent reproducibility. Under broad pHs, the amine groups are the main charged groups bringing about a global positive charge on the capillary wall. As a consequence, the cat-HEC coating produced an anodal EOF performance. A comparative study on the use of hydroxyethylcellulose (HEC) and cat-HEC as physically adsorbed coatings for CE are also presented. The separation efficiency and analysis reproducibility proved that the cat-HEC polymer was efficient in suppressing the adsorption of basic proteins onto the silica capillary wall. The long-term stability of the cat-HEC coating in consecutive protein separation runs has demonstrated the suitability of the coating for high-throughput electrophoretic protein separations.     

Polymer solutions as a pseudostationary phase for capillary electrochromatographic separation of DNA diastereomers

The solutions of linear polymers traditionally used for DNA separation have been employed for the capillary electrophoresis (CE) of diastereomers of chemically modified DNA. The selectivity of diastereomeric separation of the phosphorothioate (PS) and 2'-O-methylated (2-OMe) PS oligonucleotides depends on the nature of the polymer additive in the CE background electrolyte. The selectivity of separation for different polymers increases in the line: linear polyacrylamide < polyethylene glycol < polyvinyl pyrrolidone. The separation of oligomer diastereomers was shown to be primarily based on the hydrophobic interaction with the polymer network that acts as a pseudostationary phase. While lowering the temperature resulted in improved separation, the addition of organic modifiers such as formamide, methanol or acetonitrile counteracts the solute adsorption on the polymer network, and decreases the selectivity of DNA diastereoseparation. The effect of molecular mass and concentration of the polymer on the separation selectivity was investigated.