Microelectrophoresis

The 2eta potential (Fig. 8) is essentially the potential that can be measured at the surface of shear that forms if the sohd was to be moved relative to the surrounding ionic medium. Techniques for the measurement of the 2eta potentials of particles of various si2es are collectively known as electrokinetic potential measurement methods and include microelectrophoresis, streaming potential, sedimentation potential, and electro osmosis (19). A numerical value for 2eta potential from microelectrophoresis can be obtained to a first approximation from equation 2, where Tf = viscosity of the liquid, e = dielectric constant of the medium within the electrical double layer, = electrophoretic velocity, and E = electric field. 

In order to obtain an estimate of the surface potential, the C potential of individual liposomes can be measured (>0.2 ym) by microelectrophoresis (e.g., Crommelin, 1984). This technique also offers the opportunity to detect the presence of structures with deviating electrophoretic mobility and, therefore, deviating composition. 

However, the equilibrium of the indicator adsorbed at an interface may also be affected by a lower dielectric constant as compared to bulk water. Therefore, it is better to use instead pH, the interfacial and bulk pK values in Eq. (50). The concept of the use at pH indicators for the evaluation of Ajy is also basis of other methods, like spin-labeled EPR, optical and electrochemical probes [19,70]. The results of the determination of the Aj by means of these methods may be loaded with an error of up to 50mV [19]. For some the potentials determined by these methods, Ajy values are in a good agreement with the electrokinetic (zeta) potentials found using microelectrophoresis [73]. It is proof that, for small systems, there is lack of methods for finding the complete value of A>. 

Figure 6.2 The effect of pH on the zeta potential of cellulosic fines and fibres as measured by streaming potential and microelectrophoresis (figures in brackets are negative).

Microelectrophoresis (electrophoretic mobility) . This involves the measurement of particle charge in an applied field. For paper furnishes, the supernatant solution—which contains finely divided colloidal matter, is usually removed and used to conduct the measurement. It must be questioned therefore as to how reflective this is of the charge characteristics of the larger particles and fibres which settle. However, as it is the colloidal fraction which requires to be flocculated to assist retention during drainage, it is still a useful measurement. 

The content of vaccine within the small liposomes is estimated as in the section Estimation of Vaccine Entrapment in Dehydration-Rehydration Vesicles Liposomes for both microfluidized and sucrose liposomes and expressed as percentage of DNA and/or protein in the mixture subjected to freeze drying as in the section Preparation of Vaccine-Containing Small Liposomes by the Sucrose Method in the case of sucrose small liposomes or in the original DRV preparation (obtained in the section Estimation of Vaccine Entrapment in DRV Liposomes ) for microfluidized liposomes. Vesicle size measurements are carried out by PCS as described elsewhere (6,8,17). Liposomes can also be subjected to microelectrophoresis in a Zetasizer to determine their zeta potential. This is often required to determine the net surface charge of DNA-containing cationic liposomes. 

The generation of colloidal charges in water.The theory of the diffuse electrical double-layer. The zeta potential. The flocculation of charged colloids. The interaction between two charged surfaces in water. Laboratory project on the use of microelectrophoresis to measure the zeta potential of a colloid. 

A good example of the use of microelectrophoresis experiments is supplied by the study of ferric floes, which are widely used in municipal water treatment plants. The zeta potentials shown below were derived from the measured floe electromobilities using the Smoluchowski equa-... 

The stability of most colloidal solutions depends critically on the magnitude of the electrostatic potential ( /o) at the surface of the colloidal particle. One of the most important tasks in colloid science is therefore to obtain an estimate of /o under a wide range of electrolyte conditions. In practice, the most convenient method of obtaining /q uses the fact that a charged particle will move at some constant, limiting velocity under the influence of an applied electric field. Even quite small particles (i.e. <1 xm) can be observed using a dark-field illumination microscope and their (average) velocity directly measured. The technique is called microelectrophoresis . 

Figure 6.17 Photograph of the Rank Bros MK2 microelectrophoresis instrument.

From microelectrophoresis measurements on a spherical colloid particle, the observed elctromobility L7e is directly related to the zeta potential by the equation ... 

Two conditions must be met to justify comparisons between f values determined by different electrokinetic measurements (a) the effects of relaxation and surface conductivity must be either negligible or taken into account and (b) the surface of shear must divide comparable double layers in all cases being compared. This second limitation is really no problem when electroosmosis and streaming potential are compared since, in principle, the same capillary can be used for both experiments. However, obtaining a capillary and a migrating particle wiih identical surfaces may not be as readily accomplished. One means by which particles and capillaries may be compared is to coat both with a layer of adsorbed protein. It is an experimental fact that this procedure levels off differences between substrates The surface characteristics of each are totally determined by the adsorbed protein. This technique also permits the use of microelectrophoresis for proteins since adsorbed and dissolved proteins have been shown to have nearly identical mobilities. 

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. 

Microelectrophoresis depends on the visibility of the migrating particles under the microscope. As such, it is inapplicable to molecular colloids such as proteins. By adsorbing the protein molecules on suitable carrier particles, however, the range of utility for microelectrophoresis can be extended. Dark-field illumination (see Section 1.6a. lc) can sometimes be used to advantage to extend microelectrophoresis observations to small, high-contrast particles. 

FIG. 12.9 Schematic illustration of a microelectrophoresis cell with a rectangular working compartment. 

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. 

Newman, R. A. and Harrison, R. 1973. Characterisation of the surface of bovine milk fat globule membrane using microelectrophoresis. Biochim. Biophys. Acta 298, 798-809. 

The multilamellar dispersions or vesicles were formed in the conventional manner (4). Drying down the lipid and suspending it in the desired aqueous solution (0.1M NaCl, 0.01M tris, pH 7.5) yielded, on gentle agitation, vesicles of the appropriate size for microelectrophoresis... 

Size exclusion/molecular sieve chromatography Ultra Itration/dia lysis Ultracentrifugation Fluorescent probes Spin label EPR NMR probes Calorimetry Microelectrophoresis Zeta potential... 

Figure 7.5 A vertically mounted flat particle microelectrophoresis cell93 (By courtesy of Academic Press Inc.)...

Figure 7.6 Possible arrangement for a thin-walled particle microelectrophoresis cell...

M. Stelzle, R. Miehlich, and E. Sackmann, 2-Dimensional microelectrophoresis in supported lipid bilayers, Biophys. J. 63, 1346-1354 (1992). 

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). 

Microelectrophoresis is the most common technique for electrokinetic measurements in colloidal systems. Here individual particles can be observed, in their normal environment, under the microscope. Very dilute dispersions can be studied and very small particles, down to about 0.1 pm diameter, can be observed using the dark-field microscope (ultramicroscope). High magnifications allow minimization of observation times, and in polydisperse systems a given size range of particles can be studied to the exclusion of others. 

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. 

Curtis, D.R. Microelectrophoresis. In Physical Techniques in Biological Research. Vol. V. Electro-physiological Methods. Part A. pp. 144-190. Academic Press, New York 1964. 

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