Velocity sedimentation centrifugation

By accelerating deposition with a slowly rotating ultracentrifuge, Stokes law [Eq. (4.1)] is modified in uniform circular motion to an equilibrium distribution along the axis of rotation (0 to x), as a function of solute mass (mt). At higher centrifugal velocities, sedimentation succeeds the equilibrium distribution. Sedimentation equilibrium and sedimentation velocity provide a means to determine M. 

Regardless of the rotor speed and maximum velocity, sedimentation (or flotation) will not occur in a solution of equal density to the sample. Iso-density methods use this lack of movement in a manner comparable to a pH gradient in iso-electric focusing techniques. The methods are a combination of sedimentation and flotation, achieved by using a density gradient that straddles the density of the particles concerned. On centrifugation, the particles sediment until they reach a solvent zone with the same density. This results in the development of a zone for each type of particle present in the sample. 

In rate-zonal centrifugation (or velocity sedimentation) the sample is loaded as a thin layer on the top of a density gradient medium. During centrifugation, the sample separates into bands, and the particles are separated on the basis of their different sedimentation coefficients (s). For biological particles, the coefficient s is mainly related to the size of the particles. This process is illustrated in Figure 13.7(a). 

Microsomes are small spherical membranous vesicles with attached ribosomes. They sediment, during differential sedimentation, only in the late stages of a preparation when very high centrifugal velocities are used. They don t appear in electronmicrographs of a cell. From where do they arise ... 

Two approaches are in vogue for determination of molecular weights through centrifugation, the sedimentation velocity method and the sedimentation equilibrium method, the former being more popular. A third method, the approach to equilibrium method is also used. 

The introduction of centrifugal sedimentation makes the technique capable of determining the distribution of particles below 5 pm. The lower limit depends on the centrifugal velocity. The time of analysis is reduced drastically and multiple samples in cells can be analyzed simultaneously. 

Thermodynamic data from the ultracentrifuge experiment ean be obtained either from flie sedimentation velocity (sedimentation coefficient) or from the sedimentation-diffusion equilibrium since the centrifugal forces are balanced by the activity gradient. The determination of sedimentation and diffusion coefficients yields the virial coefficients by ... 

In isopycnic centrifugation, particles sediment in a density gradient until such time as they reach a region of density equal to their own density, as can be seen from equation (1), the sedimentation velocity becomes zero under these conditions. The experimental procedure is summarized in Figures 4 and S. 

V is the velocity of sedimentation and a is the acceleration of free fall or centrifugation. 

A larger sedimentation force can be developed by centrifuging the suspension. At an angular velocity of CO radians pet second the centrifugal... 

Factors Influencing Centrifugal Sedimentation. The sedimentation velocity of a particle is defined by equations I and 2. Each of the terms therein effects separation. 

Continuou.s Centrifugal Sedimentation Theory The Stokes settling velocity of a spherical particle under centrifugal field is given... 

For the same case of n = 1200 rpm and r = 0.5, we obtain u,/Ug = 800, whereas for the turbulent regime the ratio was only 28. This example demonstrates that the centrifugal process is more effective in the separation of small particles than of large ones. Note that after the radial velocity u, is determined, it is necessary to check whether the laminar condition. Re < 2, is fulfilled. For the transition regime, 2 < Re < 500, the sedimentation velocity in the gravity field is ... 

We have an emulsion of oil in water that we need to separate. The oil droplets have a mean diameter of lO " m, and the specific gravity Of the oil is 0.91. Applying a sedimentation centrifuge to effect the separation at a spedd of 5,000 rpm, and assuming that the distance of a droplet to the axis of rotation is 0.1 m, determine the droplet s radial settling velocity. 

Determine the settling velocity of a particle (d = 4 x 10" m and Pp = 900 kg/m ) through water in a sedimentation centrifuge operating at 4,000 rpm. The particle velocity is a function of distance from the axis of rotation, as shown by the following data ... 

Centrifugation methods separate macromolecules on the basis of their characteristic densities. Particles tend to fail through a solution if the density of the solution is less than the density of the particle. The velocity of the particle through the medium is proportional to the difference in density between the particle and the solution. The tendency of any particle to move through a solution under centrifugal force is given by the sedimentation coefficient, S ... 

Sedimentation coefficients are a measure of the velocity witli which a particle sediments in a centrifugal force field. Sedimentation coefficients are typically expressed in Svedbergs (.symbolized S), named to honor The Svedberg, developer of the ultracentrifuge. One S equals 10 . sec. 

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