Types of Electrophoresis

Using different media in different physical formats and a variety of instrumental configurations, several different types of electrophoretic techniques are used for the separation of a diverse range of analytes. 

The pH gradient is created with carrier ampholytes, a group of amphoteric polyaminocarboxylic acids, which have slight differences in pKa value and molecular weights of 300 to 1000. Mixtures of 50 to 100 different compounds 

Two-dimensional (2-D) electrophoresis is extensively used in the field of proteomics to study families of proteins and search for genetic- or disease-based differences or to study the protein content of cells of various types.It has also been applied to the study of human gene mutations and the DNA of various bacteria and tumor cells as a means to earlier diagnosis. By combining charge-dependent IFF in the 

The 2-D electrophoresis method of O FarreU uses PAGE-IEF in 130 X 2.5-mm (internal diameter) tubes for the first dimension and covers a pH range of 3 to 10 units. The anodal compartment is in contact with lOmmol/L phosphoric acid and the cathodal compartment witli 20 mmol/L NaOH. After electrophoresis is complete, the gel is extruded from the tube and placed in contact with a thin, polyacrylamide gradient gel slab that incorporates SDS. At the end of the process, the polypeptides are detected by one of several different methods. 

In the O Farrell method, SDS is used in the second dimension, -mercaptoethanol in the first. Others have used SDS in both dimensions and in sample preparation. The use of p-mercaptoethanol and SDS denatures proteins to polypeptides by reducing disulfide bonds and depolymeriz-ing proteins. When native proteins, such as enzymes, are desired for further study, nondenaturing sample preparation and electrophoresis conditions must be used. 

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Another difference between other types of electrophoresis and disc electrophoresis is that the molecules in a sample do not start to significantly separate until entering the separating gel. A discontinuous gel system may be used with almost any type of 2one electrophoresis appHcation. 

Paper Electrophoresis. Paper (qv) as an electrophoretic matrix was employed in some of the first electrophoretic techniques developed to separate compounds. Paper is easier than a gel matrix because the paper matrix requires no preparation. Besides being easy to obtain, paper is a good medium because it does not contain many of the charges that interfere with the separation of different compounds. Two types of paper employed in this type of electrophoresis are Whatman 3 MM (0.3 mm) and Whatman No. 1 (0.17 mm). 

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... 

After the release of the oligosaccharides, they must be purified by a variety of methods before structural analyses can be undertaken. Ion-ex-change chromatography, gel-filtration chromatography, or some type of electrophoresis is usually used for the purification. At this point, structural analyses may begin. 

There are many combinations of separations techniques and methods of coupling these techniques currently employed in MDLC systems. Giddings (1984) has discussed a number of the possible combinations of techniques that can be coupled to form two-dimensional systems in matrix form. This matrix includes column chromatography, field-flow fractionation (FFF), various types of electrophoresis experiments, and more. However, many of these matrix elements would be difficult if not impossible to reduce to practice. 

Application of new analytical methods, in particular of different types of electrophoresis, has also provided new data on the molecular properties of these enzymes and has set more rigorous criteria of their purity and homogeneity. 

This technique is a variant of CZE. A cationic or anionic surfactant compound, such as sodium dodecylsulphate, is added to the mobile phase to form charged micelles. These small spherical species, whose core is essentially immiscible with the solution, trap neutral compounds efficiently by hydrophylic/hydrophobic affinity interactions (Fig. 8.7). Using this type of electrophoresis, optical purity analysis can be conducted by adding cyclodextrins instead of micelles to the electrolyte. This is useful for separating molecules that are not otherwise separable. Under such conditions, the enantiomers form inclusion complexes of different stability with cyclodextrin (cf. 3.6). 

The type of electrophoresis we have been discussing so far is called capillary zone electrophoresis. Separation is based on differences in electrophoretic mobility. If the capillary wall is negative, electroosmotic flow is toward the cathode (Figure 26-20) and the order of elution is cations before neutrals before anions. If the capillary wall charge is reversed by coating it with a cationic surfactant (Figure 26-24) and the instrument polarity is reversed, then the order of elution is anions before neutrals before cations. Neither scheme separates neutral molecules from one another. 

The two most widely used types of electrophoresis tests are (i) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and (ii) isoelectric focusing (IEF). 

All types of electrophoresis are governed by the single set of general principles illustrated by Equation 4-1 ... 

There has been a revival of the slab gel techniques originally used when starch was the principal support medium. For this type of electrophoresis, acrylamide is polymerized into a thin square or rectangular slab between two glass plates (11,12). Sample holes or wells are made at one end of the gel by placing a comb-shaped jig like that shown in Figure 6-12 into the... 

Most types of electrophoresis using supporting media are simple to carry out and the apparatus can be easily constructed, although inexpensive equipment is commercially available. High-resolution techniques such as two-dimensional electrophoresis and capillary electrophoresis require more sophisticated equipment, both for separation and analysis (see later). 

Specific types of electrophoresis that are used for Hb analysis include isoelectric focusing, electrophoresis, and capiUaiy electrophoresis. 

Until the appearance of capillary electrophoresis, electrophoretic separations were not canned out in columns but were performed in a flat stabilized medium such as paper or a porous semisolid gel. Remarkable separations were realized in such media, but the technique was slow, tedious, and required a good deal of operator skill. In the early 1980s,. scientists began to explore the feasibility of performing these same separations on micro amounts of sample in fused silica capillary tubes. Their results proved promising in terms of resolution, speed, and potential for automation. As a consequence, capillary electrophoresis (CE) has become an important tool for a wide variety of analytical separation problems and is the only type of electrophoresis that we will consider. 

In general, the fractionation obtained by this traditional type of electrophoresis is rather disappointing and usually inferior to that which could be obtained on the basis of molecular weight by the techniques outlined in the previous section of this chapter. As a result, the use of electrophoresis with humic substances has declined somewhat in popularity. 

Information resulting from starch-gel and other types of electrophoresis is desirable, but in itself cannot be interpreted with the precision that is possible in combination with the biochemical information. 

Electrophoresis can be carried out in different types of electrophoresis cells. For geometric reasons, these cells have to be miniaturized and they must be cylindrical. They consist of two concentric chambers. An outer glass or plastic centrifuge tube forms the anodal buffer chamber (cf. Fig. 8.2(a)). The volume of the buffer chambers must be large enough to minimize the increasing cell resistance. 

Electrophoretic methods are used to separate substances based on their charge-to-mass ratios, using the effect of an electric field on the charges of these substances. These techniques are widely used for charged colloidal particles or macro-molecular ions such as those of proteins, nucleic acids, and polysaccharides. There are several types of electrophoresis, zone electrophoresis being one of the most conunon. 

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