Electro-osmotic flow , capillary electrophoresis

Capillary electrophoresis (CE) has several unique advantages compared to HPLC, snch as higher efficiency dne to non-parabolic fronting, shorter analytical time, prodnction of no or much smaller amounts of organic solvents, and lower cost for capillary zone electrophoresis (CZE) and fused-silica capillary techniques. However, in CZE, the most popular separation mode for CE, the analytes are separated on the basis of differences in charge and molecular sizes, and therefore neutral compounds snch as carotenoids do not migrate and all co-elute with the electro-osmotic flow. 

Tsuda, T., Modification of electro-osmotic flow with cetyltrimethylammonium bromide in capillary zone electrophoresis, /. High Resolut. Chromatogr., 10,622, 1987. 

Landers, J. P., Oda, R. P., Madden, B. J., and Spelsberg, T. C., High-performance capillary electrophoresis of glycoproteins the use of modifiers of electro-osmotic flow for analysis of microheterogeneity, Anal. Biochem., 205, 115, 1992. 

Variation of ion mobility with pH is only part of the story with regard to separation by capillary electrophoresis - the other major factor is electro-osmotic flow (EOF). 

In capillary electrophoresis, components of a mixture are separated according to two main factors electrophoretic mobility and electro-osmotic flow. These terms apply to ions, molecules or micelles. 

Another factor that controls the migration of the solute is the electro-osmotic mobility //EOS, which results in movement of the electrolyte or electro-osmotic flow. This flow is present in gel electrophoresis to a small extent and to a greater extent in capillary electrophoresis because of the internal wall of the capillary. 

In capillary electrophoresis instruments, the electro-osmotic flow is used to impose, on all charged species in the sample, a direction of migration that is oriented from the anode towards the cathode. An increase in the electro-osmotic flow vEOS decreases, at the detector, the gap in migration times of ions travelling in the same direction. The use of fused silica capillaries partially deactivated by coating the inner wall allows modulation of the electro-osmotic flow. A voltage gradient can also be used to this end. 

This mode of electrophoresis, in which the electrolyte migrates through the capillary, is the most widely used. The electrolyte can be an acidic buffer (phosphate, citrate, etc.) or basic buffer (borate) or an amphoteric substance (a molecule that possesses both an acidic and an alkaline function). The electro-osmotic flow increases with the pH of the liquid phase, or can be rendered non-existent. 

This technique represents the transposition of classical polyacrylamide or agarose gel electrophoresis into a capillary. Under these conditions, the electro-osmotic flow is relatively weak. In this approach, the capillary is filled with an electrolyte impregnated into a gel that minimises diffusion and convection phenomena. In contrast to its use for proteins that are fragile and thermally unstable, CGE is ideal for separating the more rugged oligonucleotides. 

Electrodriven separations, such as capillary electrophoresis (CE) and capillary electrochromatography (CEC), are based on the different electrophoretic mobilities in an electric field of the molecules to be separated. They provide a higher separation efficiency then conventional HPLC since the electrophoretic flow (EOF) has a plug-flow profile. Whereas the mobile phase in CE is driven only by the electro-osmotic flow, it is generated in CEC by a combination of EOF and pressure. CEC has a high sample capacity which favours its hyphenation with NMR. 

Electro-osmosis - the movement of liquid relative to a stationary charged surface (e.g. a capillary or porous plug) by an applied electric field (i.e. the complement of electrophoresis). The pressure necessary to counterbalance electro-osmotic flow is termed the electro-osmotic pressure. 

Magnuson, M.L., Creed, J.T. and Brockhoffl C.A. (1997) Speciation of arsenic compounds in drinking water by capillary electrophoresis with hydrodynamically modified electro-osmotic flow detected through hydride generation inductively coupled plasma mass spectrometry with a membrane gas-liquid separator./. Anal. At. Spectrom., 12, 689-695. 

Capillary electrophoresis separations are dependent on the relative mobilities of analytes under the influence of an electric field and do not depend on mobile phase/stationary phase interactions. A fused silica capillary is filled with a buffer and both ends submerged into two reservoirs of the buffer. A platinum electrode is immersed in each reservoir and a potential difference (5-30 kV) is applied across the electrode. An aliquot of sample of a few nanoliters is injected onto the capillary by either hydrostatic or electrokinetic injection, and the components migrate to the negative electrode. Separations of analytes arise from differences in the electrophoretic mobilities, which are dependent on the mass-to-charge ratio of the components, physical size of the analyte, and buffer/analyte interactions. An electro-osmotic flow (EOF) of the buffer occurs in the capillary and arises as a result of interactions of the buffer with dissociated functional groups on the surface of the capillary. Positive ions from the buffer solution are attracted to negative ions... 

I.6. Capillary electrophoresis [55] Simultaneous analysis of silicate with other ions (nitrite, nitrate, phosphate) is carried out by capillary electrophoresis with an indirect UV detection. The separation is achieved in a fused silica capillary filled with an electrolyte solution containing sodium chromate and an electro-osmotic flow modifier, trimethyltetrade-cylammonium bromide. 

Poison N. A., M. A. Hayes, 2000, Electro-osmotic flow control of fluids on a capillary electrophoresis microdevice using an applied external voltage. Anal. Chem. 72, 1088-1092. 

The solution contained within the capillary in which the separation occurs is known as the background electrolyte (BGE), carrier electrolyte, or, simply, the buffer. The BGE always contains a buffer because pH control is the most important parameter in electrophoresis. The pH may affect the charge and thus the mobility of an ionizable solute. The electro-osmotic flow (EOF) is also affected by the buffer pH. Table 1 contains a list of buffers that may prove useful in high-performance capillary electrophoresis (HPCE). As will be seen later, only a few of these buffers are necessary for most separations. 

As the mobile phase moves through the capillary containing the sorbent under the effect of this electro-osmotic flow (EOF), sample components partition between the two phases in sorption and diffusive mechanisms characteristic of liquid chromatography. Ions in the sample move both under the influence of EOF and by their added attraction toward the oppositely charged electrode (electrophoresis). Uncharged components, on the other hand, move only under the influence of EOF. Thus, sample components, in general, separate by chromatographic and, sometimes, electrophoretic processes. 

In capillary electrophoresis (CE), several criteria can be applied to classify solvents [e.g., for practical purposes based on the solution ability for analytes, on ultraviolet (UV) absorbance (for suitability to the UV detector), toxicity, etc.]. Another parameter could be the viscosity of the solvent, a property that influences the mobilities of analytes and that of the electro-osmotic flow (EOF) and restricts handling of the background electrolyte (BGE). For more fundamental reasons, the dielectric constant (the relative permittivity) is a well-recognized parameter for classification. It was initially considered to interpret the change of ionization constants of acids and bases according to Born s approach. This approach has lost importance in this respect because it is based on too simple assumptions limited to electrostatic interactions. Indeed, a more appropriate concept reflects solvation effects, the ability for H-bonding, or the acido-base property of the solvent. 

The reversal of the direction of the electro-osmotic flow by the adsorption onto the capillary wall of alky-lammonium surfactants and polymeric ion-pair agents incorporated into the electrolyte solution is widely employed in capillary zone electrophoresis (CZE) of organic acids, amino acids, and metal ions. The dependence of the electro-osmotic mobility on the concentration of these additives has been interpreted on the basis of the model proposed by Fuerstenau [6] to explain the adsorption of alkylammonium salts on quartz. According to this model, the adsorption in the Stern layer as individual ions of surfactant molecules in dilute solution results from the electrostatic attraction between the head groups of the surfactant and the ionized silanol groups at the surface of the capillary wall. As the concentration of the surfactant in the solution is increased, the concentration of the adsorbed alkylammonium ions increases too and reaches a critical concentration at which the van der Waals attraction forces between the hydrocarbon chains of adsorbed and free-surfactant molecules in solution cause their association into hemimicelles (i.e., pairs of surfactant molecules with one cationic group directed toward the capillary wall and the other directed out into the solution). 

Electro-osmotic Flow Nonuniformity Influence on Efficiency of Capillary Electrophoresis... 

There are two types of nonuniformities of electro-osmotic flow (EOF) that can contribute signihcantly to the solute peak broadening and are important for capillary electrophoresis (CE). The first is the transversal nonuniformity of the usual EOF in the capillary with the zeta potential of the walls and longitudinal electric field strength constant and independent of coordinates. The second one is the nonuniformity of EOF caused by the dependence of the zeta potential of the walls or electric field strength on coordinates. 

Gradient elution in HPLC is achieved using two pumps, two different solvent reservoirs, and a solvent mixer. In capillary electrophoresis (CE), electro-osmotic flow controls the flow of the mobile phase, which is, in most cases, an aqueous buffer and is used in place of a mechanical pump. 

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