A presentation with some basic info about AUC. Hope it can be useful. Double-check for mistakes!

1. Christiane - October 2006

2. 1. Theory
2. Equipment
3. Practical Considerations
4. Data

3. • invented in 1920s by Theodor Svedberg
How it works:
• spin sample at high speed
• monitor in real time (UV-VIS or interference / refractive index)
• distribution: concentration versus radius
What you get:
• sample homogeneity
• determine molecular mass
• shape of sample
• study self-associating systems
• study interactions

4. When a solute particle in solution is subjected
to a gravitational field, three forces act it:
Frictional Force Ff Buoyant Force Fb
Sedimenting Force Fs
These three forces come into balance:
Constant
F s + F b + Ff = 0
velocity

5. M (1−υ ⋅ ρ )⋅ω 2
(
⋅ r 2 −r02 )
c(r ) = c0 ⋅ e 2⋅R ⋅T
Diffusion Sedimentation
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Equilibrium:
Concentration Distribution is only dependent on Molecular Mass, corrected
for effects of buoyancy (M*(1-υρ))

6. Sedimentation Velocity:
• Rapidly sediment solute
• depletion of solute near meniscus: formation of sharp boundary
(the plateau)
• measure rate of movement of this boundary (radius --> time)
• determine sedimentation coefficient s --> m, f
• measure spreading of the boundary: diffusion coefficient
• single boundary: homogeneity?

7. Sedimentation Equilibrium:
• lower angular velocity than sedimentation velocity
• equilibrium between diffusion and sedimentation
• reaching eq depends on length of column (3mm ~ 18h)
• concentration distribution: exponential with the sq of radial position

8. Some useful diagnostics when you get started:
• ideal case: only one homogeneous component
• Plot: ln A/A
0 r^2-r0^2 linear
• diagnostic plots from beckman handout.
• possible complications
heterogeneity --> average molecular weight
non-ideality --> e.g. charge effects, reduces apparent MW
self association --> different average MV at each point
• possible results
(apparent) MW (of monomer or complex)
assess whether eq has been attained
stoichiometry, eq constrant, measure of non-ideality

12. For example a
Beckman XL-I ultracentrifuge

13. • applicable to MW from 100’skDa to Millions kDa
• sample needed: 0.01-1g/L (absorbance of 0.5)
• check absorbance of buffer at wavelength you are going to use
• ideally dialyse your sample into buffer for reference cell
(having identical buffers is EXTREMELY important)

14. Cell 1 Cell 2 Cell 3
What the raw data looks like…
(UV scan across chamber with three sample slots)

15. - Beckman software (version of origin)
- Ultrascan (version 8.0 is best 9.0 crashes a lot)
(how to use that, check my documents on slideshare)
- SedFit (sedimentation velocity)
- SetPhat (equilibrium)