III. 



CENTRIFUGATION 



79 



respect to the rotor. The scale is photographed through the solu- 

 tion with a camera of long focal length. In the photographic image 

 of the scale the lines are found to be still equally spaced in the regions 

 corresponding to supernatant fluid and the plateau region. How- 

 ever, in the portion corresponding to a sedimenting boundary the lines 

 are found to be displaced with respect to some reference line photo- 

 graphed through the opening in the counterbalance. The amount of 

 displacement is proportional to the refractive index gradient and 

 through plotting line displacement as a function of the distance from 

 the axis of rotation one can obtain a curve that gives the refractive 



REFRACTIVE 

 INDEX METHOD 



ABSORPTION 

 METHOD 



Fig. 5. Sedimentation photographs of dissociated hemocyanin 

 showing double boundary and illustrating connection between absorp- 

 tion method and cylindrical lens method, which depends on refraction 

 of light by concentration gradients. Speed, 16,000 r.p.m.; time at full 

 speed, 2.25 hours; cell thickness, 1 cm.; protein concentration, 

 0.4%. 



index gradient and hence the concentration gradient at all radial posi- 

 tions in the cell. The correction and interpretation of such curves 

 are the same as those for the cylindrical lens method, which is dis- 

 cussed in detail below. The scale method when properly employed is 

 probably subject to the fewest optical errors of any refractive index 

 method. However, it does involve a great deal of tedious measure- 

 ment and computation, which can be avoided through use of the 

 cylindrical lens method. 



In contrast to the scale method, the cylindrical lens method gives 

 directly by photography a curve of refractive index gradient as a 

 function of radial position within the cell. A schematic representa- 



