A systematic studies of the electrophoresis technique of human serum proteins (in sodium barbiturate buffer at pH 8.6) using the open horizontal method ware carried out.
1. During electrophoresis, two kinds of the buffer flow occur on paper surface, one of which heads for the cathode on account of the electroendosmosis. The other one goes to the central part of paper from both of the anodic and cathodic buffer vessels in order to compensate for the relative dryness which occurs owing to the evaporation during electrophoresis. The apparent buffer flow on paper surface is the algebraic sums of these two kinds of buffer flow. The equlibrum points (zero points) of buffer flow are generally located on the cathodic part of the horizontal portion of paper.
2. These equlibrium points, however, displace to the central part of paper as voltage, ionic strength and electrophoresis hour incerease. It is because, in these cases, the buffer flow due to the evaporation augments more rapidly than the one due to the electroendosmosis.
3. The buffer flow on the more anodic part than the equlibrium points heads for the cathod and inversely on the more cathodic part. Their dimensions are proportional to the distances from the equlibrium points of buffer flow.
4. On account of the buffer flow on paper, 1) the electrophoretic patterns are blurred, especially on the anodic part of paper, and displaced, as a whole, to the anodic or cathodic parts, leaving some extent of tailing on paper. This is disadvantageous for the precise determination of serum fractions because one fraction may be superimposed on the other's talings.
5. As the buffer solution displaces under the continuous evaporation during electrophoresis, the ionic strength varies throughout on paper. To detect the variations of ionic strength, accumulated amounts of electrolytes on the dried papers, were examined by the high-frequency papyrography The ionic strength is proved to be small where the buffer flow is large. So, the ionic strength on paper decreases in proportion to the increases of distance from the equlibrium points of buffer flow.
6. The time-migration relationship of B. P. B, stained Alb, is not linear but of inversely S-shaped curve, the formation of which is expounded to be due to the variations of ionic strength on paper.
7. The most well separated electrophoretic patterns are obtained when serum is applied on the equlibrium points of buffer flow. It is because the applied serum migrates on the paper with relatively high ionic strength and relatively small buffer flow.
8. In drying papers, the electrophoretically separated fractions are displaced owing to the water displacement due to the evaporation.
9. The paper electrophoretic mobilities are only approximate in both of the closed and open method electrophoresis because the changes of buffer flow and ionic strength during electrophoresis, and the water displacement in drying papers do not take place uniformly on paper.