Stabilization Against Convection

When performing a run in practice, the remixing of separated zones, due to undesired convection and other movements in the liquid electrolyte system, has to be prevented. The liquid system must be stabilized against convection in some way. In electrophoresis such stabilization can be achieved in many ways. Valmet (53) has made the following summary on methods used  

Capillary system (filterpaper, powder, gel) in columns, thin layer or 

Multichamber cells, using permeable or semi-permeable membranes to 

Electrophoresis convection, where the undesirable thermal convection is eliminated because separation takes place in a relatively narrow channel. 

Forced-flow electrophoresis, where stabilization is achieved by a forced, laminar flow of liquid superimposed perpendicular to the direction of electrophoretic migration of the proteins. 

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We found earlier that transient (nonsteady-state) sedimentation required density gradients to stabilize against convection. Isopycnic sedimentation relies on density gradients not only for anticonvective purposes but also as the secondary gradient needed to establish steady-state conditions. The difference in the two cases is found in the magnitude of the density gradient and in the degree to which components are allowed to approach their steady-state condition. The equipment is similar the zonal rotor developed by Anderson is used for isopycnic as well as transient zonal separations [45]. 

As both the phases of ATPSs are electrically conductive, application of electric fields in these systems gives rise to electrokinetic mass transfer of charged species. Thus ATPS was shown as a medium for electrophoretic separation with two aqueous phase interfaces providing the stability against convection and facilitating product recovery [126]. Proteins have been directed into either the... 

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. 

There are no foreign additives, such as sugar or gel, used to stabilize against convection. 

Column apparatus using sucrose gradients to stabilize against convection. 

Leaback and Rutter (37) reported trials with other ways of stabilizing against convection for example, cellulose acetate and methylated paper. These trials failed due to electro-end-osmosis. The authors measured the end-osmosis in polyacrylamide gel and found it to be 1-2 cm/16 hr. 

Over the last two years, isoelectric focusing has certainly proved its importance as a refined separation technique in biochemistry. Electrofocusing hinges on three main principles the pH-gradient, the medium for stabilization against convection, and the result measuring technique used. The development of these three main principles will decide the future progress of the technique. 

The method of stabilization against convection determines the character of the equipment. Gels, density gradients or zone convection indeed give the equipment different images. Many of the techniques used in electrophoresis for stabilization today have not yet been studied and tested for electrofocusing. Further developments based on those techniques can be expected. 

The Biostream rotationally stabilized free-flow electrophoresis device, based on the Philpot-Harwell design (Figure 4), uses an annular geometry stabilized against radial convection by rotation of the outer cylinder. Carrier buffer and feed are injected at the base of a vertical annulus and move axially upward to fraction collectors at the top. 

Instant coagulation also excludes methods that require a stable dispersion, such as electrophoresis. The manufacturers of certain types of zetameters claim that their instruments are suitable to perform measurements in electrolyte solutions up to about 1 M. However, in order to use a zetameter, one has to prepare a stable dispersion first, and this may be problematic. Electro-osmosis does not require stability against sedimentation, but other problems, such as low absolute values of the potential (which may be smaller than the scatter of results) and the production of heat, convective currents, or electrolysis products (acids, bases, and gases), severely limit the application of classical electrokinetic methods (including electrophoresis) in measurements at ionic strengths greater than 0.1 M. Very few publications report potentials obtained by classical electrokinetic methods at higher electrolyte concentrations, and the results are controversial. 

In all electrofocusing, some method must be used to stabilize the liquid against convection currents. The usual methods employ sucrose (density gradient) or polyacrylamide (gel). These substances are present in much larger quantities than the actual substance being tested. This always involves the risk of disturbances. Sucrose must be removed by dialysis at the end of the fractionation. It is also difficult to separate the protein from the polyacrylamide. 

As for all other electrophoretic techniques described thus far, electrofocussing also needs provision for stabilization of separating protein zones against convective flow in the solution. Three ways are in use (1) density gradient, (ii) gel, and (iiO zone convection electrofocussing. The last one is not as popular as the other two, but is discussed as a special case at the end. 

Placing the hot zone above the cold will create a thermal gradient that will tend to stabilize the melt against convective flows, although the radial thermal gradients will still cause some mixing. If the rejected component of the alloy system is more dense than the... 

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