Electrophoresis general discussion

For example, Barlow and Margoliash [33] showed that phosphate, chloride, iodide, and sulfate, in decreasing order of effect, reduced the electrophoretic mobihty of human cytochrome c at pH 6.0 by up to a factor of 2. The cations lithium, sodium, potassium, and calcium had no effect. It is possible to account for the binding equilibria of these counterions so that the titration and electrophoresis results can be compared however, in many of the early electrophoresis experiments these data were not available and relevant conditions were not recorded or controlled. For general discussions on the extensive field of ligand binding to proteins, see Cantor and Schimmel [60] and van Holde [403]. 

The protein components of a membrane can be readily visualized by SDS-polyacrylamide gel electrophoresis. As discussed earlier (Section 4.1.4). the electrophoretic mobility of many proteins in SDS-containing gels depends on the mass rather than on the net charge of the protein. The gel-electrophoresis patterns of three membranes—the plasma membrane of erythrocytes, the photoreceptor membrane of retinal rod cells, and the sarcoplasmic reticulum membrane of muscle—are shown in Figure 12.16. It is evident that each of these three membranes contains many proteins but has a distinct protein composition. In general, membranes performing different functions contain different repertoires of proteins. 

In this section, instrumentation, general electrophoretic operations, technical and practical considerations, and types of conventional electrophoresis are discussed. 

Electrophoresis is the most powerful method available for separation and analysis of complex mixtures of charged biopolmers. This chapter provides an overview of modern electophoresis as a general introduction to the chapters which follow. The basic electrophoretic operating modes and formats for these modes are described. Means for detection of separated zones are reviewed. Finally, an approach to fully instrumental electrophoresis is discussed. 

There are broader exceptions to simple stretched-exponential behavior. These exceptions serve to test the generalization that stretched-exponential behavior is donfinant. For electrophoresis, as discussed in Chapter 3, a transition to power-law behavior with increasing P and E appears to correspond to the onset of nonlinear transport in which /i depends on the applied field. For the low-shear viscosity, the solufionfike-meltlike transition is sometimes seen as discussed in Section 9.10 and Chapter 11, this transition occurs simultaneously with the appearance of a light... 

The standard Rodbard-Ogston-Morris-Killander [326,327] model of electrophoresis which assumes that u alua = D nlDa is obtained only for special circumstances. See also Locke and Trinh [219] for further discussion of this relationship. With low electric fields the effective mobility equals the volume fraction. However, the dispersion coefficient reduces to the effective diffusion coefficient, as determined by Ryan et al. [337], which reduces to the volume fraction at low gel concentration but is not, in general, equal to the porosity for high gel concentrations. If no electrophoresis occurs, i.e., and Mp equal zero, the results reduce to the analysis of Nozad [264]. If the electrophoretic mobility is assumed to be much larger than the diffusion coefficients, the results reduce to that given by Locke and Carbonell [218]. 

Thin-layer chromatography (TLC) is a type of liquid chromatography in which the stationary pease is in the form of a thin layer on a flat surface rather than packed into a tube (column). It is a member of a family cf techniques that include some types of electrophoresis and paper chromatography, more generally referred to as planar chromatccraphy. Since we will not discuss electrophoresis in this section, and since TLC has virtually superseded paper chromategr pby in most analytical... 

Gel electrophoresis is widely used in the routine analysis and separation of many well-known biopolymers such as proteins or nucleic acids. Little has been reported concerning the use of this methodology for the analysis of synthetic polymers, undoubtedly since in many cases these polymers are not soluble in aqueous solution - a medium normally used for electrophoresis. Even for those water-soluble synthetic polymers, the broad molecular weight dispersities usually associated with traditional polymers generally preclude the use of electrophoretic methods. Dendrimers, however, especially those constructed using semi-controlled or controlled structure synthesis (Chapters 8 and 9), possess narrow molecular weight distribution and those that are sufficiently water solubile, usually are ideal analytes for electrophoretic methods. More specifically, poly(amidoamine) (PAMAM) and related dendrimers have been proven amendable to gel electrophoresis, as will be discussed in this chapter. 

This chapter discusses general considerations for improving capillary electrophoresis (CE) method performance from a robustness angle. Several method parameters are discussed and examples are offered of how CE procedures are handled to obtain optimal performance. The purpose of this chapter is to raise the awareness and help the development of more robust and sensitive methods. 

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. 

The first four methods are described in Refs. [81,253,254] and the electroacoustical methods in [130,255-257]. Of these, electrophoresis finds the most use in industrial practice. The electroacoustic methods are perhaps the best suited to studying concentrated suspensions and emulsions without dilution [258], In all of the electro-kinetic measurements, either liquid is made to move across a solid surface or vice versa. Thus the results can only be interpreted in terms of charge density (a) or potential (zeta potential, ) at the plane of shear. The location of the shear plane is generally not exactly known and is usually taken to be approximately equal to the potential at the Stern plane, = W d), see Figure 4.9. Several methods can be used to calculate zeta potentials [16,81,253], Some of these will be discussed here, in the context of electrophoresis results. 

As discussed in Chapters 1-7, diffusion, Brownian motion, sedimentation, electrophoresis, osmosis, rheology, mechanics, interfacial energetics, and optical and electrical properties are among the general physical properties and phenomena that are primarily important in colloidal systems [12,13,26,57,58], Chemical reactivity and adsorption often play important, if not dominant, roles. Any physical chemical feature may ultimately govern a specific industrial process and determine final product characteristics, and any colloidal dispersions involved may be deemed either desirable or undesirable based on their unique physical chemical properties. Chapters 9-16 will provide some examples. 

The achievements discussed in this chapter also underline that such systems are more than just separation systems. They should rather be regarded as generally applicable fluid handling systems for small volume samples. In particular in the case of electric field controlled systems, the fluid handling requires only control of electric potentials and can be easily automated. Electrophoresis experiments on the level of single DNA molecules have already been demonstrated... 

Capillary electrophoresis is a general term that is used to describe a number of different separation techniques. Capillary zone electrophoresis (CZE) is the classic technique and is therefore usually referred to as just CE. Other techniques include micellar electrokinetic chromatography (MEKC), capillary isoelectric focusing, and capillary isotachophoresis. CZE and MEKC are the predominant techniques and are those used herein, so only they will be discussed in detail here. 

Most practical applications of electrophoresis require a thorough understanding of the boundary effects and particle interactions. These effects have also been discussed in detail in this article. In general, both the boundary effects and particle interactions are found to be weaker than the corresponding cases in sedimentation because the velocity disturbance caused by a particle undergoing electrophoresis decays faster than that by a settling particle. For some cases, the boundary effects even enhance the electro-... 

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