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04/24/23Biophysical Tools '02 - Sedimentation 1
SedimentationSedimentation
04/24/23Biophysical Tools '02 - Sedimentation 2
PrinciplePrinciple
2 2m r - r(m ) vf
centrifugal force - buoyancy = frictional force
angular velocityr: distance from the center of spinningm: mass of the molecule partial specific volume: density of solutionv: velocity of sedimentationf: frictional coefficient
2
v m(1- ) r
sf
Sedimentation coeff. = velocity per unit centrifugal field
04/24/23Biophysical Tools '02 - Sedimentation 3
Analytical CentrifugeAnalytical Centrifuge
04/24/23Biophysical Tools '02 - Sedimentation 4
Boundary sedimentationBoundary sedimentation
equivalent2
d ln(r )1sdt
2
v m(1- ) r
sf
04/24/23Biophysical Tools '02 - Sedimentation 5
Application: Application: HomogeneityHomogeneity
04/24/23Biophysical Tools '02 - Sedimentation 6
Application: Molecular weight determination Application: Molecular weight determination
•Sedimentation Equilibrium– Sedimentation at low speeds so that the sedimentation is counterbalanced by
diffusion
1 2E E- 1 kT
2
c ec
1
2
21 2 r
m (1- )rdrr
E E
222
1ln)-(1
2RT Marc
c
a
r
04/24/23Biophysical Tools '02 - Sedimentation 7
Application: Shape determinationApplication: Shape determination
6o o of R
Stokes
6.7 S 6.2 S 5.4 S
04/24/23Biophysical Tools '02 - Sedimentation 8
Application: PurificationApplication: Purification
04/24/23Biophysical Tools '02 - Sedimentation 9
Zonal - Density CentrifugationZonal - Density Centrifugation
04/24/23Biophysical Tools '02 - Sedimentation 10
Practical tipsPractical tips
• K-factor:
• Pelleting time (t):
• But if you spin at speed less than the maximum speed (wmax) k is smaller:
• g force
sk t
k k2
maxadj
2
000,11.12r g
135 max min max min
2 2
2.53x10 ln(r / ) ln(r / ) 10( /1000) 3600
r rkRPM