Electrophoretic separation

Electrophoresis is the movement of charged particles under the action of an electric field, known as migration. In a mixture of different particles, they migrate according to their respective mobilities as per the following equation (see Section 5.4.1)  

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McKillop and associates have examined the electrophoretic separation of alkylpyridines by CZE. Separations were carried out using either 50-pm or 75-pm inner diameter capillaries, with a total length of 57 cm and a length of 50 cm from the point of injection to the detector. The run buffer was a pH 2.5 lithium phosphate buffer. Separations were achieved using an applied voltage of 15 kV. The electroosmotic flow velocity, as measured using a neutral marker, was found to be 6.398 X 10 cm s k The diffusion coefficient,... 

When fluids heat unevenly, the hot part of the fluid tends to rise with respect to the cooler part of the fluid because of differences in density. The flow is driven by gravity, and distorts resolution in electrophoretic separations. 

The abihty to remove heat from electrophoretic systems has severely limited the maximum capacity of these systems in terms of how large or thick the systems can be. Electrophoretic separations have been performed on space flights because the effect of gravity in outer space is small and mixing from heating is negligible. Whereas electrophoresis in outer space has been accompHshed (10), the economics for a scaleable process have not (see Space processing). 

The heating effect is the limiting factor for all electrophoretic separations. When heat is dissipated rapidly, as in capillary electrophoresis, rapid, high resolution separations are possible. For electrophoretic separations the higher the separating driving force, ie, the electric field strength, the better the resolution. This means that if a way to separate faster can be found, it should also be a more effective separation. This is the opposite of most other separation techniques. 

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. 

Charged macromolecules, such as proteins or polymers, are often separated elec-trophoretically. The rate of migration through an electric field increases with net charge and field strength. Molecular size of analytes and viscosity of separation media both have inverse relationships with rate of migration. These variables must all be taken into account in order to optimize the conditions for an efficient electrophoretic separation. 

Ultimately, however, it should be noted that these examples of classical gel electrophoretic separations are batch processes and therefore limited in sample throughput. To achieve true preparative-scale separations by electrophoresis, it becomes necessary to convert to continuous processes. 

Recent innovations [19] have circumvented the heat dissipation and sample stream distortion inherent in most of the previous designs. In one apparatus, developed by R S Technologies, Inc. (Wakefield, RI, USA), Teflon capillary tubes are aligned close to each other in the electrophoretic chamber. Coolant is pumped through the Teflon capillary tubes during the electrophoretic run while the electrophoretic separation is accomplished in the interstitial volume between the Teflon tubes. 

FIGURE 3-24 Electrophoretic separation of catechols with end-column detection. Detection potential, +0.8 V separation capillary, 20 kV The peaks correspond to 4.6 fmol dopamine (1), 4.1 fmol isoproterenol (2), and 2.7 fmol catechol (3). (Reproduced with permission from reference 60.)... 

Fig. 3. Agarose gel after electrophoretic separation of amplified DNA of Chlamydia trachomatis without (A), and with the addition of Ce-AR (B) in the electrophoretic system at exposure by transilluminator at 254 nm for 5 (1), 30 (2), 300 (3) and 600 (4) seconds, and profiles of the electrophoretic mobility (C).

There are other, nonhydrogel, new materials for chromatographic and electrophoretic separations [7,8,103,164,199,214,377,407], Eor example, Volkmuth and Austin [407] proposed electrophoretic studies in microlithographic arrays of posts and channels etched into sihcon wafers. This material may be useful for studying fundamental transport characteristics of macromolecules in defined media, and many recent studies have been conducted to develop chromatography and electrophoresis on silicon wafers with micron-scale channels... 

Rossier, IS Schwarz, A Reymond, F Ferrigno, R Bianchi, F Girault, HH, MicroChannel Networks for Electrophoretic Separations, Electrophoresis 20, 727, 1999. 

SaviUe, D Palusinski, OA, Theory of Electrophoretic Separations Part 1 Eormulation of a Mathematical Model, AIChE Journal 32, 207, 1986. 

Yiu, Y Locke, BR Van Winkle, DH Rill, RL, Optimizing Capillary Gel Electrophoretic Separations of Oligonucleotides in Liquid Crystalline Pluronic F127, Journal of Chromatography A817, 367, 1998. 

Periodic acid-Schiff reagent Detects glycoproteins as pink bands after electrophoretic separation. 

Incubation of cultured cells with glycoproteins as radioactive bands Leads to detection of a radioactive sugar after electrophoretic separation. 

Skude, 6. Electrophoretic separation, detection, and variation of amylase isoenzymes. Scand. J. Clin. Lab. Invest. (1975), 35, 41-47. 

Specificity of the antisera was assessed by Western blotting. Electrophoretically separated proteins from culture filtrates were transferred to 0.45 fim nitrocellulose membranes. After transfer of proteins, membranes were... 

Other purification methods include a liquid phase chromatography, electrophoretic separation by mass spectroscopy, separation using magnetic properties, and so on. These separation methods are limited only for the metal nanoparticles having a special property useful for these purification methods. 

Separation and detection methods Very refined chromatographic and electrophoretic separation techniques have been developed for metallothioneins. The detection is commonly based on the retention time and UV detection. Other researchers measured the element with e.g. ICP-MS to quantify the compotmd. Combination with electrospray MS-MS leads to the unequivocal identification of the species. 

This SRM contains human cells from two cell culture lines from which DNA can be extracted, genomic DNA from those two cell lines plus eight individuals, PCR-amplified DNA from the two cell lines plus four of the eight individuals, a D1S80 allelic ladder for characterization of amplified DNA, and a DNA size marker to assure proper electrophoretic separations. 

P. Juo and G. Stotzky, Electrophoretic. separation of proteins from roots and root exudates, Canadian Journal of Botany 48 1 7i (1970). 

H. Heuer. M. Krsek, P. Baker, K. Smalla, and E. M. H. Wellington, Analysis of actinomycete communities by specific atnplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl. Environ. Microbiol. 6.1 3233 (1997). 

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