Density gradient isoelectric focusing

In some steady-state methods of separation (isoelectric focusing, density-gradient centrifugation, and sometimes elutriation), component zones approach a stationary configuration centered about different points in space. Separation occurs by virtue of the different steady-state positions of the various solutes. In other systems (field-flow fractionation, zone refining,... 

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. 

Two variations of the basic technique are isoelectric focusing and immuno-electrophoresis. The former offers improved resolution and sharper bands in the separation of weak acids, weak bases and ampholytes through the use of pH and density gradients superimposed along the potential gradient. The latter employs specific antigen-antibody interactions (Chapter 10) to visualize the separated components of serum samples. 

Figure 5. Isoelectric focusing of the Cx component of T. koningii in an ampholyte gradient covering the range pH 3.72-4.25. The ampholyte was supported in a sucrose density gradient in a 110-mL LKB electro-focusing column. The Ci component used was similar to one eluted as a single component from DEAE-Sephadex (Figure 1) and purified further by chromatography on DEAE-Sephadex (6). From Ref. 6, in part.

The final expression, Eq. 8.53, shows how the resolution of two species varies with their average molecular weight Af, the difference Aps in their density, the field strength G, and the density gradient dp/dx. Like Eq. 8.46 for isoelectric focusing, Eq. 8.53 can be used to quantitatively describe the parameters necessary for resolution in isopycnic sedimentation [50]. 

After 20 hours of focusing the gel tube is removed, equilibrated at room temperature, and immediately placed in an ISCO gel scanner (slits 0.25 mm) equipped with a UA 5 detector. The scan is shown in Fig. 47 as well as the temperature and paH gradients. From an 8-cm gel containing the protein, a zone of 0.6-0.7 cm (measured in the scanner densitogram at half optical density) is obtained after isoelectric focusing. 

The introduction of density gradients allowed several improvements in the analytical and preparative capabilities of centrifugal processes. First, it is possible to pour the sample in the top of a tube, in a more or less thin layer differences in density between sample and separation medium avoids convection and delays the homogenization process. Second, it is possible to diminish convection fluid movements in the tube convection can be produced by a difference in temperature between different parts of the centrifugal rotor. Third, it is possible to focus a particle when its reaches its own density, in a process analogous to isoelectric focusing. 

There are several drawbacks to density-gradient isoelectric focusing in comparison with other isoelectric fractionation methods. 

Many readers will by now have recognized that the procedure of isoelectric focusing has another analogue in density-gradient centrifugation discussed in Section 5.5. 

Isoelectric focusing and electrofocusing are names that have been accepted, and in current use, since 1967. Before that time the technique was called isoelectric separation, isoelectric fractionation, isoelectric condensation, isoelectric analysis and focusing electrophoresis, as well as stationary electrolysis. Terms such as density gradient electrofocusing or gel electrofocusing indicate the medium in which the experiments are carried out. 

A great advance in the equipment for isoelectric focusing has recently been made by Valmet (40). He has described a new principle for electrofocusing called zone convection electrofocusing. In this technique the stabilizing against convection does not call for any capillary system, density gradient or membranes. 

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