1 58 CHROMATOGRAPHY 



A chamber for electrophoresis might consist of a simple tube with an 

 electrode at each end, as in Fig. 11-5. Many elaborations may be intro- 

 duced into the structure of the chamber, such as a means of removing 

 components after separation, or an ingenious method of sliding sections 

 of tubing together so that individual components in the mixture move 

 as zones. 



Fig, 11-5. A simple chamber for electrophoresis. 



Electrophoresis is also conducted very conveniently on filter paper 

 or other supporting materials such as starch gel. A strip of filter paper 

 moistened (but not sopping wet) with a solvent is hung on a rack with 

 the two ends dipping into separate containers of the solvent. The solvent 

 is a salt solution, so that it will conduct an electrical current, and usually 

 its pH is controlled by the inclusion of a suitable buffer. The two elec- 

 trodes, leading from a direct current power supply, are placed in the 

 two containers of solvent. A spot of the mixture to be separated is placed 

 on the paper strip, the circuit is closed, and various components move 

 with the current. Positively charged molecules move in one direction, 

 negatively charged molecules in the other. All positively charged mole- 

 cules do not necessarilv move at the same rate because of differences 

 in molecular weight, particle shape, magnitude of charge, viscosity of 

 solvent, and several other physical properties. 



Proteins are readily separated by electrophoresis because of the nature 

 of the protein molecule. It is composed of a large number of amino 

 acids, some of which possess side chains having an acidic — COOH 

 group, others of which contain basic groups of one kind or another. Since 

 the dissociation of these side-chain groups will depend on pH, —COOH 

 groups could exist as —COOH or as — COO~. Basic groups, such as 

 — NH2, exist in this form at high pH but as — NH3+ in acidic solutions 

 of low pH. The ratio of acidic to basic side chains in the molecule and 

 the pH of the solution determine its net charge. Two distinct protein 

 types, even if the net electrical charge is of the same sign, would move 

 at rates depending on the magnitude of the charge. 



