IX. ELECTROPHORESIS 295 



electric current has been allowed to pass for a time will indicate, as 

 separate peaks in the pattern, the presence of more than one bound- 

 ary. The number of boundaries observed corresponds to the number 

 of electrophoretically different components present in the prepara- 

 tion. The area under the curve for each boundary compared to the 

 sums of the areas for the total boundary will yield the relative 

 amount of each such component in the mixture. Thus, this method 

 can be used to analyze a protein preparation as to the number and 

 relative amounts of electrophoretically different and independent 

 components present and as to the electrical mobility of each. Re- 

 quirement d, above, is very adequately met in this apparatus. 



Requirement e in the above list, i.e., the means for estimating the 

 voltage drop per centimeter or field strength under which the bound- 

 ary is migrating, can be approximated best by the method already re- 

 ferred to in the discussion of the microelectrophoresis method. If the 

 legs of the U tube are of uniform cross section and the area, A (cm.^) 

 of cross section is measured, the voltage drop per centimeter, E, in the 

 cell will be given by the relationship E = I/(\A), where / is the cur- 

 rent density (amp.) passing through the system (which can be cal- 

 culated from the voltage drop occurring across a standard resistance 

 placed in series with the electrical circuit to the cell) and X is the spe- 

 cific conductivity (mho) of the cell contents in the region of the bound- 

 ary. It is here, however, that an important uncertainty associated 

 with the moving-boundary method arises. 



4. Limitations 



Because the two solutions that meet at the boundary cannot be of 

 identical composition, it is to be expected that the two will vary to a 

 more or less marked degree in conductivity, pH, and buffer salt con- 

 centration as well as in the concentration of the component that dis- 

 appears in the boundary and whose electrophoretic properties are 

 being investigated. These circumstances give rise to certain bound- 

 ary anomalies such as (a) the nonmoving concentration gradients 

 that appear at the site of the initial boundaries (the so-called 5 and € 

 peaks in the electrophoresis patterns), (6) a difference in the distance 

 traveled, in unit time by the rising boundary and by the descending 

 boundary, and (c) differences in the shapes (sharpness) of the rising 

 and descending peaks. 



That the mobilities calculated from the distances moved by the 

 rising boundary (where the colloid component is moving into the 



