Sedimentation and Velocity
The first method starts with a well defined boundary, or layer, of solution near the center of rotation and follows the movement of this layer toward the outside of the cell as a function of time. Such a method is termed a sedimentary velocity experiment.
A particle of mass m at a distance x from center of rotation experiences a force given by
ƒ_{centrif} = m’xω^{2}
Where w is the angular velocity in radiation per second m’ is the distance effective mass of the solute particle, i.e. the actual mass corrected for the new effect of the solvent.
To express this buoyancy effect, we first recognize that v the specific volume of the solute, is the mass of 1 g of the solute. The volume of m g of solute is mv, and the mass of this volume of solvent is m of the solute is m - mv_{p} = mj (1 – v_{p}). We now can rewrite equation as:
Centrif = m (1 – v_{p}) xω^{2}
Equating these two force expressions leads us to the constant drif velocity. A rearrangement of the equality:
M (1 – v) xω^{2} = 6∏rή dx/dt
Equating these two force expressions leads that collect the dynamic variables gives:
Dx/dt/xω^{2} = m (1 – v_{p})/6∏rή
The collection of dynamic terms on the left side of equation describes the results of sedimentation velocity experiments. This collection (dx/dt) xw^{2} can be looked on as the velocity with which the solute moves per unit centrifugal force. The sedimentation coefficient S is introduced as:
S = dx/dt/xω^{2}
The experimentation results can therefore be tabulated as values of S. the value of S for many macromolecules is of the order of 10^{-13} has therefore been introduced, called a Svedberg, in honor of T. Svedberg, who did much of the early work with the ultracentrifuge.
Molar mass: s = dx/dt/xω^{2} = m )1 – v_{p})/ 6∏rή
Rearrangement and multiplication by Avogadro’s number give:
M = Nm = 6∏rήNS/ 1- vp
Now the troublesome terms involving ή and r can be replaced by their effective values appear in the measurable values D of equation, to give the desired result:
M = RTS/ D (1 – vp)
Thus measurements of the substances of the sedimentation and diffusion coefficients and of the solvent and solute allow the deduction of the molar mass for a few macromolecules. The necessary data for such calculations for a few macromolecular materials are included.
A particular advantage of the sedimentation velocity technique is that a macromolecular solution containing two or more types of macromolecules is separated according to the molecular masses of the components. The type of sedimentation diagrams obtained for a system containing a number of macromolecular species.
Density gradient: better resolution can be obtained by allowing the sedimentation to occur in a density gradient solution, prepared, for example, by filling the centrifuge tube layer by layer with solutions of decreasing sucrose concentration. As the macromolecular substance or mixture of substances is centrifuged, it moves through a solvent with gradually increasing density. The result is more stable macromolecular zones and a better “spectrum” of the components. The technique is thus a modification of the sedimentation velocity method.
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