Electrophoresis band velocity

Isotachophoresis. In isotachophoresis (ITP), or displacement electrophoresis or multizonal electrophoresis, the sample is inserted between two different buffers (electrolytes) without electroosmotic flow. The electrolytes are chosen so that one (the leading electrolyte) has a higher mobility and the other (the trailing electrolyte) has a lower mobility than the sample ions. An electric field is applied and the ions start to migrate towards the anode (anions) or cathode (cations). The ions separate into zones (bands) determined by their mobilities, after which each band migrates at a steady-state velocity and steady-state stacking of bands is achieved. Note that in ITP, unlike ZE, there is no electroosmotic flow and cations and anions cannot be separated simultaneously. Reference 26 provides a recent example of capillary isotachophoresis/zone electrophoresis coupled with nanoflow ESI-MS. 

If a further purification is desired, then the supernatant (above) can be subjected to ammonium sulfate fractionation, gel filtration on Sephadex G-200, and finally on a-aminopropane-agarose affinity column. On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, the final product migrated as a single band with an estimated molecular weight of 113,000. Upon sedimentation equilibrium velocity ultracentrifugation, an estimated molecular weight value of near 117,000 was obtained. 

Isotachophoresis. A variation of electrophoresis set up such that ionic species move with equal velocity in an ionic band through the medium. However, within this band, the most mobile ions form the leading edge and the least mobile ions form the trailing edge, resulting in the separation of ions based on their characteristic mobility under the specified conditions. 

A last variant we mention is capillary zone electrophoresis (Gordon et al. 1988). It employs an electroosmotically driven flow in a capillary, arising from an electric field applied parallel to the capillary, which is charged when in contact with an aqueous solution (Section 6.5). The flow has a nearly flat velocity profile (Fig. 6.5.1), thereby minimizing broadening due to Taylor dispersion of the electrophoretically separated solute bands. 

CapUlary electrophoresis (CE) is a routine analytical technique for fast and efficient separation of charged species. Under the influence of an electric field, the ionic species in a sample that is introduced as a plug (or zone) into an electrolyte at one end of a capillary will be separated into discrete bands when they migrate to the other end of the capillary at different electrophoretic velocities. However, Joule heating is an inevitable phenomenon in CE that limits the performance of electrophoretic separation. 

In zone electrophoresis, the analytes and other sample components migrate as zones or bands with different velocity. 

When the electric field is first applied in an isota-chophoretic separation, analyte ions migrate as in zone electrophoresis, each ion with its unique velocity given by the product p., . This difference in migration rates results in the separation of the various analyte species into adjacent bands, with fastest species located in a band directly adjacent to the leading buffer and the slowest just ahead of the terminal buffer. After the bands have formed, they then move at the same velocity. The reason that the bands have the same velocity is that the electric field becomes smaller for the more mobile bands and greater for the slower bands, so that the current is the same, as it must be, in all parts of the buffer. The ionic current that results from the flow of ions in the buffer is analogous to the dc current in a circuit consisting of several resistors connected in series to a battery. Flere, the current must be identical in all of the resistors. Hence, the potential across each of the resistors must vary in such a way that Ohm s law is obeyed. 

When equilibrium is reached in an isotachophoretic experiment, each sample component is migrating in a band sandwiched between a band that contains the next-slower-moving ions and the next-faster-moving band, as shown in Figure 30-9b, The boundary between bands is sharp. If a solute species starts to move into the next-faster band, it encounters a lower field, which reduces its velocity until it drops back into its original band. Note in Figure 30-9b that, in contrast to zone electrophoresis or elution chromatography, the analyte bands are immediately adjacent to one another and are not separated by bands of the buffer. 

Currently, analytical approaches are still the most preferred tools for model reduction in microfluidic research community. While it is impossible to enumerate all of them in this chapter, we will discuss one particular technique - the Method of Moments, which has been systematically investigated for species dispersion modeling [9, 10]. The Method of Moments was originally proposed to study Taylor dispersion in a circular tube under hydrodynamic flow. Later it was successfully applied to investigate the analyte band dispersion in microfluidic chips (in particular electrophoresis chip). Essentially, the Method of Moments is employed to reduce the transient convection-diffusion equation that contains non-uniform transverse species velocity into a system of simple PDEs governing the spatial moments of the species concentration. Such moments are capable of describing typical characteristics of the species band (such as transverse mass distribution, skew, and variance). 

Philpot (1940) obtained such a result in terms of t = Ljvz. The number of different charged species which can therefore be separated in a ffee-flow electrophoresis device is limited by solute dispersion in the x-direction, amongst other things. The peak-to-peak distance between two species may be estimated from equation (7.3.7). A more exact solution, which includes the effect of dispersion in the z-direction, has been provided by Reis etal. (1974) for a parabolic velocity profile Vziy) in the y-direction effectively, the Philpot (1940) model underestimates x-directional band broadening substantially. The base width of the profile described by equation (7.3.9) depends on t at z values less than L, the base width will be smaller. Note, however, the separation of Figure 7.3.1 is essentially at steady state. The time coordinate used here allows a specification of position along the z-axis. 

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