Electrophoresis discussion

Our research on responsive gels has been carried out with applications in separations and drug delivery in mind. Except for the slurry dewatering process described in the next section, however, we have not focused on the development of specific applications. One novel idea which we pursued, however, was the me of temperature-sensitive PNIPAAm for the preparative gel electrophoresis, discussed in Sect. 7.2. 

The type of electrophoresis discussed so far is called capillary zone electrophoresis, in which separation is based on different electrophoretic velocities of different ions. Electroosmotic flow of the bulk fluid is toward the cathode (Figure 23-16b). Cations migrate faster than the bulk fluid and anions migrate slower than the bulk fluid. Therefore the order of elution is cations before neutrals before anions. If electrode polarity is reversed, the order of elution is anions before neutrals before cations. Neither scheme separates neutral molecules from one another. 

Voltages used fall in the range of 10-30 kV. This translates in currents in the range of 20-100 mA. Higher currents are avoided usually because they produce too much heat inside the capillary (see "Factors Affecting Electrophoresis" discussed earlier in this chapter). This can result in peak broadening and loss of resolution. 

In particular, in polar solvents, the surface of a colloidal particle tends to be charged. As will be discussed in section C2.6.4.2, this has a large influence on particle interactions. A few key concepts are introduced here. For more details, see [32] (eh 13), [33] (eh 7), [36] (eh 4) and [34] (eh 12). The presence of these surface charges gives rise to a number of electrokinetic phenomena, in particular electrophoresis. 

This chapter will first cover the nature of electrophoretic separations, especially those concerning capillary electrophoresis. Comprehensive multidimensional separations will then be defined, specifically in terms of orthogonality and resolution. The history of planar and non-comprehensive electrodriven separations will then be discussed. True comprehensive multidimensional separations involving chromatography and capillary electrophoresis will be described next. Finally, the future directions of these multidimensional techniques will be outlined. 

In considering the applicability of preparative classical electrophoretic methods to chiral separations, it should be noted that practitioners in the art of classical electrophoresis have been particularly inventive in designing novel separation strategies. For instance, pH, ionic strength and density gradients have all been used. Isoelectric focusing and isotachophoresis are well-established separation modes in classical electrophoresis and are also being implemented in CE separations [7, 8]. These trends are also reflected in the preparative electrophoretic approaches discussed here. 

The author would like to acknowledge R S Technologies, Inc. (Wakefield, RI, USA) for the loan of the continuous free flow electrophoresis system, and Cerestar, Inc. for the donation of the sulfated cyclodextrin. The author would also like to thank Drs. Chris Welch and Prabha Painuly for helpful discussions. 

One must be very careful in reviewing the older, and some more recent, literature in consideration of the tortuosity and constriction factors some work has attempted to separate these two factors however, more modem developments show that they cannot be strictly decoupled. This aspect will be particularly important when reviewing the barrier and tortuous-path theories of electrophoresis, as discussed later. 

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 volume averaging approach discussed in the section on diffusive transport can also be extended to account for electrophoresis [215] and hydrodynamic flow [215,436]. Locke [215] considered the application of volume averaging to the determination of the effective... 

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]. 

Presented below are four increasingly stringent confirmatory techniques for PCR and a brief discussion of considerations, limitations and advantages of each. These four techniques are agarose gel electrophoresis, restriction analysis. Southern blotting and sequencing. 

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... 

The use of 2D gel electrophoresis and mass spectrometry to identify proteins was discussed in Chapter 2. Protein expression mapping involves the use of these methodologies to compare expression patterns in different cell types or in the same cell type that has been exposed to different... 

Table 1 summarizes several of the experimental methods discussed in this chapter. A need exists for new or revised methods for transport experimentation, particularly for therapeutic proteins or peptides in polymeric systems. An important criterion for the new or revised methods includes in situ sampling using micro techniques which simultaneously sample, separate, and analyze the sample. For example, capillary zone electrophoresis provides a micro technique with high separation resolution and the potential to measure the mobilities and diffusion coefficients of the diffusant in the presence of a polymer. Combining the separation and analytical components adds considerable power and versatility to the method. In addition, up-to-date separation instrumentation is computer-driven, so that methods development is optimized, data are acquired according to a predetermined program, and data analysis is facilitated. 

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