Electrophoresis microelectrophoresis

Of the electrokinetic phenomena we have considered, electrophoresis is by far the most important. Until now our discussion of experimental techniques of electrophoresis has been limited to a brief description of microelectrophoresis, which is easily visualized and has provided sufficient background for our considerations to this point. Microelectrophoresis itself is subject to some complications that can be discussed now that we have some background in the general area of electrical transport phenomena. In addition, the methods of moving-boundary electrophoresis and zone electrophoresis are sufficiently important to warrant at least brief summaries. 

Zone electrophoresis is influenced by adsorption and capillarity, as well as by electroosmosis. Therefore evaluation of mobility (and f) from this type of measurement is considerably more complex than from either microelectrophoresis or moving-boundary electrophoresis. Nevertheless, zone electrophoresis is an important technique that is widely used in biochemistry and clinical chemistry. One particularly important area of application is the field of immunoelectrophoresis, which is described briefly in Section 12.11. Additional information on zone electrophoresis may be obtained from Probstein (1994) and Hunter (1981) and the references given there. Variants of zone electrophoresis also exist see, for example, Gordon et al. (1988) for information on a variant known as capillary zone electrophoresis and Righetti (1983) for information on what is known as isoelectric focusing. 

On the other hand, the preparation of the substrate is far from easy and demands considerable technical skill. Another drawback is the strong electroendosmosis which amounts to half the main electrophoretic mobility and drives (1- and y-globulins toward the cathode. This means that the application zone lies in the middle of the run and disturbs photometric scanning. Microelectrophoresis, according to Scheidegger (SI), is among the promising developments in this field of electrophoresis. With this substrate it becomes possible to perform an electrophoresis on an ultramicroscale with as little as 0.1-1 (ig protein, which needs about 3 minutes (Wl). 

Good descriptions of practical experimental techniques in conventional electrophoresis can be found in Refs. [81,253,259]. For the most part, these techniques are applied to suspensions and emulsions, rather than foams. Even for foams, an indirect way to obtain information about the potential at foam lamella interfaces is by bubble electrophoresis. In bubble microelectrophoresis the dispersed bubbles are viewed under a microscope and their electrophoretic velocity is measured taking the horizontal component of motion, since bubbles rapidly float upwards in the electrophoresis cells [260,261]. A variation on this technique is the spinning cylinder method, in which a bubble is held in a cylindrical cell that is spinning about its long axis (see [262] and p.163 in Ref. [44]). Other electrokinetic techniques, such as the measurement of sedimentation potential [263] have also been used. 

This test, called the Comet Assay or single-cell gel-electrophoresis assay, allows the degree of DNA damage to be determined within a nucleic cell population. The principle of the method is based on the microelectrophoresis of nuclei of isolated cells, under basic conditions, on agarose gel (the whole being observed under a fluorescence microscope). 

Zeta potential was the first, experimentally available value characterizing edl. The potential of the solid particles in the electrolyte solutions may be determined on the basis of one of the four following phenomena microelectrophoresis, streaming potential, sedimentation potential and electroosmosis. The most popular of them and the best described theoretically and methodically is the electrophoresis. Other papers, concerning the electrophoretic mobility, stationary level determination and the theory of the charged particles transportation in the electric field are still published. 

Possible Effect of Charged End-groups. A possible reason for differences between samples might be the process used for polymerising the original polyvinyl acetate. Emulsion polymerisation is likely to introduce a proportion of ionic (sulphate or carboxyl) end-groups which would not be expected if bulk polymerisation with benzoyl peroxide had been used. An Antweller Microelectrophoresis apparatus was used to measure rates of electrophoresis of polyvinyl alcohols in solution in a pH 7.8 phosphate buffer. No significant difference was observed between... 

The term microelectrophoresis, or even better, microscopic electrophoresis, refers to a technique in which the motion of individual particles is followed, usually ultramicroscoplcally. Other terms refer to alternative ways of measurement, such as moving boundary electrophoresis, paper electrophoresis, laser-Doppler electrophoresis, gel electrophoresis, capillary electrophoresis, etc. see secs. 4.5 and 10. 

Regarding the determination of particle velocities, two approaches are possible. In the first, known as microelectrophoresis. Individual particles are made visible and their translation is monitored. This is the most basic procedure because the variation of the velocities over the particles can be measured. When microelectrophoresis is not feasible, the collective displacement of a large number of particles can be studied. This technique is known as moving boundary electrophoresis. We shall now discuss these modes of operation. 

Few issues of the analytical journals go by without the appearance of a new design for gel electrophoresis apparatus, and there are no doubt many satisfactory alternatives to the three versions that we have discussed. Microelectrophoresis and preparative electrophoresis pose special problems, and will be considered separately ( 7.S.2.2 7.S.2.3 ch. 9). 

From the point of view of the electrophoretic cell, microelectrophoresis is a capillary electrophoresis. Experimenting in capillaries is complicated in that a streaming potential (comp. B) occurs, and the applied voltage causes the liquid in the cell to move. This movement, called electro-osmosis, occurs because the glass walls of the cell are negatively charged. There is only one particular point between the cell wall and the center of the cell, the stationary layer at a distinct distance x (x = r/y/2), where the particles move with their true velocity, which is due solely to their own charge. To get a true result, the microscope must be focused in this layer and the particles measured must be in focus. 

A number of methods for the determination of electrophoretic velocity and electrokinetic potential of particles have been developed. These methods include the moving boundary method (a direct study of motion of the boundary between the disperse system and the free dispersion medium due to the applied potential difference), microelectrophoresis (a direct observation of moving particles using a microscope or ultramicroscope), electrophoresis in gels, paper electrophoresis, etc [ 13]. These methods are broadly used to study disperse systems formed with low molecular weight substances, as well as polymers, especially those of natural origin. Electrophoretic methods allow one to separate and analyze mixtures of proteins, and thus are effectively used in scientific research and medical diagnostic applications. 

Let us now discuss in some detail the peculiarities of particle motion during electrophoresis and some other electrical properties of free disperse systems. Electrophoresis usually takes place in a stationary liquid. In a moving fluid the motion of particles occurs only in thin flat gaps and capillaries (microelectrophoresis), where the fluid motion is caused by electroosmosis. If fairly large non-conducting particles are dispersed in a rather dilute electrolyte solution, the ratio of particle radius to the double layer thickness may be substantially greater than 1, i.e., r/8 = kt 1. The streamlines of outer electric field surround the particle and are parallel to most of its surface, as shown in Fig. V-9. In this case the particle velocity, v0, can be with good precision described by Helmholtz-Smoluchowski equation. 

Electrophoresis The motion of colloidal species caused by an imposed electric field. The term replaces the older term cataphoresis. The species move with an electrophoretic velocity that depends on their electric charge and the electric field gradient. The electrophoretic mobility is the electrophoretic velocity per unit electric field gradient and is used to characterize specific systems. An older synonym, no longer in use, is kataphoresis. The term microelectrophoresis is sometimes used to indicate electrophoretic motion of a collection of particles on a small scale. Previously, microelectrophoresis was used to describe the measurement techniques in which electrophoretic mobilities are determined by observation through a microscope. The recommended term for these latter techniques is now microscopic electrophoresis (see reference 1). 

Electrophoresis is a powerful tool in the separation and analysis of colloids, proteins, and nucleic acids. There are three major electrophoretic techniques as well as variations, each with its own name. Which one is applicable or most appropriate depends on the size and characteristics of the dissolved or suspended material and the type of information desired. The three principal techniques are microelectrophoresis, moving boundary electrophoresis, and zone electrophoresis (Shaw 1980). 

Figure 7.3.1 Vertically mounted flat microelectrophoresis cell. [Shaw, D.J. 1969. Electrophoresis. New York Academic. With permission.]...

Particle electrophoresis, also sometimes known as microscope electrophoresis or microelectrophoresis, is one of the easiest and most useful techniques for investigating the electrical properties of colloidal particles. If the system of interest is in the form of a reasonably stable dispersion of particle size observable by light microscopy (say, larger than 200 nm for practical application), the electrokinetic behavior of the system can be observed and measured directly. Several commercial instruments are available for the purpose. For smaller particles, laser scattering instruments are now readily available. 

Attempts to discover higher-resolution and faster electrophoresis separation techniques and media started in the 1960s, when miniaturized methodswere described as microelectrophoresis, but imaging technologies were not yet quite as adequate to be able to capture separations on such a minute scale. Later, polyacrylamide gels contained... 

The most popular method of measuring electrophoretic mobility is micro-electrophoresis. In microelectrophoresis, particles are placed in a closed capillary with electrodes at either end. When an electric field is applied the particles migrate towards the electrode and their velocities are measured. Because the capillary walls are charged the applied electric field will also induce an electro-osmotic flow. However, since the capillary is closed a back pressure creates a net zero flow in the tube (see Fig. 3). The particle velocity is a combination of the electrophoretic motion and the fluid flow. To obtain the true electrophoretic mobility the particles must be tracked along the stationary layer where the fluid velocity is zero. 

Microelectrophoresis measurement is the conventional and direct way to determine electrophoretic mobility. It can be traced back to two centuries ago when Reuss performed the first electrophoresis experiment on clay particles as shown in Figure 6.3 [22]. 

Jiang, D. Sims, C. E. Allbritton, N. L. Microelectrophoresis platform for fast serial analysis of single cells. Electrophoresis 2010, 31, 2558-2565. 

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