CENTRIFUGATION 85 



1.6 



\J_____ 



Fig. 40. The situation in a CsCl density gradient in which the solution den- 

 sity varies from 1.6 at the inner edge of the tube to 1.8 at the outer edge of the 

 tube. The point of density 1.7 is indicated, and particles of that density will be 

 found in a band at that point. If particles were to the left of the band, they 

 would be more dense than the solution and thus would be moved to the right 

 by the centrifuging force. If to the right of the band, they would be less dense 

 than the solution and thus would be floated inward to the left. Thus, after 

 many hours of spinning the mixture of particles and CsCl solution, the situation 

 sketched will be obtained. 



be presented below. This is called the method of the sedimentation 

 velocity. 



(c) If the centrifuge is run for a sufficiently long time (days), all the 

 particles will tend to be at the bottom of the tube. But, due to the 

 Brownian (heat) motion, some of the particles will actually go backward 

 toward the axis of rotation. When these two processes (centrifugal sedi- 

 mentation and backward diffusion) have come to equilibrium, there will 

 be a gradient of particle concentration from essentially zero at the inner 

 edge of the tube to a maximum at the outer edge. This gradient can be 

 made visible by the optical methods referred to above. The bigger the 

 particles, the more they will tend to be concentrated near the outer edge 

 of the tube. From a measurement of the actual concentration gradient, 

 then, the molecular weight of the particles may be directly deduced. This 

 is called the method of sedimentation equilibrium. 



(d) If a low molecular weight salt were in solution, the sedimentation 

 equilibrium method would result in a very gradual gradient. Indeed, it 

 should be obvious that a salt could be chosen w r ith so low a molecular 

 weight that it is hardly sedimented at all. Table salt (NaCl), for exam- 

 ple, would hardly be expected to be recovered from a solution by this 

 method. But it will in fact give a measurable gradient. In practice, for 

 example, using a certain concentration of CsCl instead of NaCl, we can 

 set up a salt gradient so that at the inner edge the density is 1.6 gm/ml 

 and rises essentially uniformly to about 1.8 gm/ml at the outer edge. 

 Other gradients may be obtained with other CsCl concentrations. 



Now, if particles of an intermediate density, say 1.7 gm/ml, are placed 

 in t; e solution before centrifuging, they will all end up in a band at their 

 own characteristic density, as indicated in Fig. 40. The reasoning is that 

 when the salt equilibrium has been established, those particles finding 



