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)