Paper and Cellulose Polyacetate Electrophoresis

 

Filter paper and other porous media were found to be an ideal medium with which to develop electrophoresis. It had both economic and technical advantages. The apparatus was simple and could be easily fabricated in the laboratory. Alternatively, because it was simple, it was much less expensive to buy than the complicated moving boundary apparatus. Even more important as complete separation between the components was achieved more information was obtainable. Boundary stabilization could be achieved in the capillary spaces where significant hydraulic flow is severely restrained and, in contrast, electrophoretic migration and diffusion is virtually unimpeded. In addition the cross section of the liquid flow-path is exceedingly small and so ohmic heating is restrained and, thus, high voltages could be used without introducing significant thermal effects. Finally the accurate location of the separated material could be identified by simple staining techniques without the use of involved optical systems and furthermore, by using specific stains different types of compounds could be selectively identified.

 

Unfortunately, migration velocities can only be used with considerable difficulties to determine absolute mobilities. The supporting material (e.g. paper) can carry charges that can lead to electro-osmotic flow that is often acting in direct opposition to the electrophoretic flow. This can be confirmed experimentally by using electrically neutral markers such as glucose, dextran etc. Regrettably, evaporation of volatile buffers can take place and cause apparent convective flow and can also change the conductivity of the electrolyte, which, in turn, will also affect the migration rate.

 

An example of a paper electrophoresis apparatus is shown in figure 7

 

 

Figure 7. Apparatus for Paper Electrophoresis

 

The apparatus shown in figure 7 is unique in that the paper rests on a series of plastic spikes (hence the term Fakir bed) that allowed free movement of the ions with out one side of the paper being in contact with a surface.

 

One problem that arises with the use of paper is the fibre structure of the medium, which complicates quantitative measurements. Attempts were made to soak the paper in oil that had the same refractive index as cellulose; some papers were calibrated with known quantities of protein. Another problem arose that resulted from the adsorptive character of paper with regard to certain proteins. These problems were partly solved by the use of cellulose acetate films as an alternative to sheet paper. 

 

Cellulose acetate membranes have now largely replaced the use of filter paper and other types of treated paper. The advantages of cellulose acetate membranes are that apparatus is again more simple and thus, less expensive; the membranes can be fabricated with more uniform porosity and, consequently, give much sharper boundaries providing shorter migration times and significantly improved resolution; finally they can be made completely transparent after staining that results in far more accurate results. The membranes are brittle dry and floppy and limp when wet and are, thus, somewhat deleicate and are, thus, often supported on a sheet of rigid transparent plastic.

 

Another form pf paper and cellulose acetate electrophoretic apparatus is shown in figure 8.

 

 

Figure 8 A Diagram of the Durrum Paper Electrophoresis Cell

 

A series of paper strips are draped over a glass rod the ends being in contact with a suitable wick that dips into the electrolyte bath. One of the more common buffers employed are 0.05 N veronal, pH 8.6 which is very similar to that used in moving boundary electrophoresis.

 

An example of the separation of a serum using a cellulose acetate membrane is shown in figure 9.

 

 

 

Figure 9. Electrophoretic Density Curve Obtained in the Separation of Serum Components