analytical ultracentrifugation 06
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Christiane - October 2006
1. Theory
2. Equipment
3. Practical Considerations
4. Data
• invented in 1920s by Theodor Svedberg
• spin sample at high speed • monitor in real time (UV-VIS or interference / refractive index) • distribution: concentration versus radius
• determine molecular mass • sample homogeneity
• shape of sample • study self-associating systems
How it works:
What you get:
• study interactions
When a solute particle in solution is subjected to a gravitational field, three forces act it:
Sedimenting Force Fs
Frictional Force Ff
Constant velocity
Buoyant Force Fb
These three forces come into balance: Fs + Fb + Ff = 0
€
c(r ) = c0 ⋅ eM (1−υ ⋅ ρ ) ⋅ω 2
2⋅R ⋅T⋅ r 2 −r0
2( )
Sedimentation Diffusion
Equilibrium: Concentration Distribution is only dependent on Molecular Mass, corrected for effects of buoyancy (M*(1-υρ))
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?
Sedimentation Equilibrium:
• lower angular velocity than sedimentation velocity
• equilibrium between diffusion and sedimentation
• concentration distribution: exponential with the sq of radial position
• reaching eq depends on length of column (3mm ~ 18h)
Some useful diagnostics when you get started:
• ideal case: only one homogeneous component
• Plot: ln A/A0 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
For example a Beckman XL-I ultracentrifuge
• 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)
Cell 1 Cell 2 Cell 3
What the raw data looks like… (UV scan across chamber with three sample slots)
- 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)
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