III. CENTRIFUGATION 91 



to /?7?aj2 (where /; represents the height, measured radially, of the fluid 

 column, R is its average distance from the axis of rotation, and co is 

 the angular velocity) and has made a comparison of actual values for 

 different machines. For general work with most proteins and other 

 particles of comparable size it is desirable that the resolving power be 

 as high as possible and, as already stated, it has been found that op- 

 timum conditions are generally realized with comparatively large 

 rotors operating at speeds in the nieghborhood of 60,000 to 70,000 

 r.p.m. Limitations on the resolving power and the accuracy of 

 measurement can be appreciated by reference to Figure 1. First, 

 while the boundary is still near the meniscus during an early part of 

 a run, the diffusion process is not a normal one and measurements 

 cannot be started until the boundary begins to clear the meniscus. 

 Also the portion of the cell over which measurements of the boundary's 

 midposition may be continued is quite limited. Some idea of resolv- 

 ing power can be had from the observation that, with the vacuum ul- 

 tracentrifuge already described spinning at 60,000 r.p.m., it is barely 

 possible to differentiate two equally concentrated "globular" pro- 

 teins having molecular weights of about 18,000 and 40,000. In such 

 an instance, where resolution of boundaries is not obtained until the 

 faster is approaching the bottom of the cell, it is necessary in the de- 

 termination of sedimentation rate to know the effective starting time 

 of the boundary at the meniscus. For reasons explained below, this 

 corresponds to the moment of reaching full speed in the oil-driven 

 ultracentrifuge or, in the case of the vacuum ultracentrifuge, to the 

 moment when the rotor has reached about two-thirds of operating 

 speed, assuming a constant rate of acceleration. 



A second requirement is that the centrifuge rotor be able to ac- 

 commodate fluid columns of relatively great thickness (at least 1 

 cm.) so that measurements can be made with low concentrations. 

 Also adequate means must be provided for measuring the temperature 

 of the fluid to within a few tenths of one degree Centigrade. In the 

 case of the vacuum ultracentrifuge rotor, which is not very responsive 

 thermally to its surroundings and for which the temperature rise 

 during a run is not more than a few degrees, the mean of the starting 

 and final temperatures may be used for purposes of computation {6). 

 In many cases it is desirable that the ultracentrifuge be capable of 

 operation near 0°C. (cell temperature) so as to furnish comparable 

 experimental conditions for those substances whose diffusion con- 

 stants must be determined at reduced temperatures for best results. 



