Techniques of electrophoresis

each ampholyte migrates in the applied field until it reaches a position on the plate where the pH of the medium is equal to the component s pi. At this point the ampholyte is in its zwitterion form and is neutral and thus it loses its electrophoretic mobility and becomes focused in a narrow zone at this point. Regardless of the point of application on the plate the ampholyte always migrates to the location of its pi and then remains stationary. 

As spreading of the bands is minimised due to application of the applied field and the pH gradient, high resolution can be achieved and proteins that differ by as little as 0.01 pH units can be adequately resolved. Analytical applications of lEF are concerned with the determination of pi values, assay of purity and also preparative scale procedures for the isolation of purified fractions. 

A number of developments to further improve the resolution and specificity of the method have been reported [78,79], namely cross-over electrophoresis, rocket electrophoresis, two-dimensional Immunoelectrophoresis, and radioimmunoassay. 

In discontinuous disc electrophoresis, the system used comprises a vertical cylindrical column, containing two discrete gel layers each maintained initially at a specific pH. The upper third of the column contains a wide-pore acrylamide gel in a Tris-HCl buffer of 6.7 pH units. The lower two-thirds is packed with a small-pore acrylamide gel in Tris-HCl at pH 8.9. The gels are prepared and the buffer located in situ. 

The above is a simple quahtative description. More exhaustive treatments of this and the related technique of isotacheophoresis can be found in the following references [80-82]. 

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An effective way to isolate these fragments is through diagonal electrophoresis (Figure 5.26) (the basic technique of electrophoresis is described in... 

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. 

In this more recent technique of electrophoresis, an open-ended fused silica capillary with a small inner diameter (10 150 pm) is used to replace the supported gel (Fig. 8.3). The capillary, of length L that varies between 20 and 80 cm, is filled with the same aqueous buffer solution of electrolyte as the two reservoirs. A voltage of up to 30 kV is applied to the electrodes. The intensity of the current should not exceed 100 pA (corresponding to a maximum dissipated power of 3 W) to avoid overheating the capillary, which should be contained in a thermostated enclosure. 

D. Holme and H. Peck, Analytical Biochemistry, 3rd ed. (1998), Addison Wesley Longman (Essex), pp. 132-146. Introduction to all techniques of electrophoresis. 

D Holme and H. Peck, Analytical Biochemistry, 3rd ed. (1998), Addison Wesley Longman (Essex), pp 132-146 Introduction to all techniques of electrophoresis M Khaledi, High Performance Captllaiy Electrophoresis Theory, Techniques, and Applications (1998), John Wiley Sons (New York) Up-to-date coverage of CE W. Ruhr, Anal. Chem. 62, 403R-414R (1990) Capillary Electrophoresis ... 

Using the technique of electrophoresis on paper, Forfar et al. (F7) found that cholesterol was abnormally concentrated in the (3-lipoprotein fraction. Using reverse phase chromatography, an attempt was made to demonstrate sterols other than cholesterol, but this was unsuccessful. If they are actually present, such substances must have an Rf similar to that of cholesterol or else be present in quantities too small to be demonstrated by the technique employed. 

How can we tell whether a purification scheme is effective One way is to ascertain that the specific activity rises with each purification step. Another is to visualize the effectiveness by displaying the proteins present at each step. The technique of electrophoresis makes the latter method possible. 

In conclusion, capillary electrophoresis in carbohydrate analysis has advantages in both separation and detection over other techniques of electrophoresis, as well as chromatography. It allows high efficiency (up to a few million plate numbers) and very good sensitivities (up to femtomolar). In addition, CE permits analysis by a variety of separation modes simply by changing the electrolyte (capillary zone electrophoresis, MEKC, CGE). 

In CE, the classic techniques of electrophoresis are performed in small-bore (10 to 100 pm) fused sihca capillary tubes of 20 to 200 cm in length (see Chapter 5). It is a very efficient, rapid, sensitive, and versatile analytical technique that is used to analyze a diverse spectrum of analytes ranging from small ions to macromolecular proteins or nucleic acids. When coupled with sensitive detectors, it is capable of measuring femtomolar quantities of amino acids. 

A5. Ambert, J., Pechery, C., and Carpentier, C., Estimation of urinary amino acids. 1. In the normal subject. (Technique of electrophoresis at pH 5.4, two-dimensional chromatography on silica gel thin layers and electrochromatography.) Ann. Biol. Clin. (Paris) 24, 17-40 (1966). 

The more useful genetic markers are those which exhibit a large number of commonly occurring, alternate forms, which are relatively stable, and can be readily resolved and assayed. The analyses of the majority of genetically-controlled polymorphic systems of forensic Interest have utilized the technique of electrophoresis. Historically, the development of electrophoretic systems for forensic analyses has centered upon conventional electrophoretic techniques (1, 2). Such systems have... 

The technique has in particular been applied to the study of proteins in the blood. Careful pH control is required, because the pH has a strong effect on the zeta potential and therefore on the rates of movement. The technique of electrophoresis supplements the ultracentrifuge, which separates according to molecular weight and shape. In the ultracentrifuge, different macromolecules having the same molecular sizes and shapes behave identically, but they may have different electrical properties and hence can be separated by electrophoresis. 

The technique of electrophoresis involves the migration of charged substances in a conducting solution under the influence of an applied electrical field. Boundary electrophoresis refers to migration in free solution, whilst the term zone electrophoresis is applied to the process of migration in supported electrolytes. In the field of carbohydrate chemistry, the most commonly used support for the electrolyte is filter paper, although others have been used when this seemed desirable. " ... 

In the late 1980 s, Jorgensen and Lukacs popularized the use of capillary electrophoresis. This technique of electrophoresis has become very popular over the years because of the many advantages that it has over conventional electrophoresis techniques. These advantages are listed below. 

German meaning hybrid ion ). Since the net charge is 0, the zwitterion cannot migrate in an electric field, e.g. during the technique of electrophoresis. This pH is termed the isoelectric point (pl). 

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