Pelleting (Differential Separation) - Beckman Coulter Avanti J Series Instructions For Use Manual

Pelleting (Differential Separation)

Pelleting separates particles of different sedimentation coefficients, the largest particles in the
sample traveling to the bottom of the tube (or bottle) first. Differential centrifugation is the
successive pelleting of particles of decreasing sedimentation velocities, using increasingly higher
forces and/or long run times. The relative pelleting efficiency of each rotor is measured by its
k factor (clearing factor):
where ω is the angular velocity of the rotor in radians per second (2πRPM/60, or ω = 0.10472 × rpm),
is the maximum radius, and r
r
max
After substitution,
This factor can be used in the following equation to estimate the time t (in hours) required for
pelleting:
where s is the sedimentation coefficient
values in seconds are such small numbers, they are generally expressed in Svedberg units (S), where
1 S is equal to 10
based on sedimentation in water at 20°C. Clearing factors can be calculated at speeds other than
maximum rated speed by use of the following formula:
Run times can also be calculated from data established in prior experiments when the k factor of the
previous rotor is known. For any two rotors, a and b:
where the k factors have been adjusted for the actual run speed used.
Figure 1.3 lists sedimentation coefficients for some common biological materials. The k factors at
rated speeds for Beckman Coulter J series rotors are provided in the table of general specifications
in each rotor use section.
* s = dr/dt x l/ω
PN JR-IM-10AG
k =
is the minimum radius.
min
(
2.533
------------------------------------------------------------------------ -
k =
*
-13
seconds). It is usual practice to use the standard sedimentation coefficient s
k =
adj
2
r, where dr/dt is the sedimentation velocity.
13
( ⁄ )
ln r r
10
max min
×
---------------------------------- - ----------- -
ω
2 3600
11
× ) ( r ⁄ )
10 ln r
min
max
2
rpm
k
-- -
t =
s
of the particle of interest in Svedberg units. (Because s
2
⎛ rated speed of rotor ⎞
------------------------------------------------ -
k
⎝ ⎠
actual run speed
t k
a a
---- - ------
=
t k
b b
1
Rotors
Rotor Selection
EQ 1
EQ 2
EQ 3
20,w
EQ 4
EQ 5
1-11