Analytical centrifugation is a type of ultracentrifugation.

1. ANALYTICAL
CENTRIFUGATION
Presented by:
Megha Varshini Gowda B R

2. Content
• Introduction
• Comparison between preparative and AUC
• Instrumentation
• Working principle
• Types of analysis using AUC
1. Sedimentation velocity method
2. Sedimentation equilibrium method
• Comparison between SV and SE method
• Applications
• References

3. Introduction
• Theodor Svedberg invented
ultracentrifuge in 1923 and was
awarded Nobel Prize in 1926 for his
research on colloids and proteins using
ultracentrifuge.
• Ultracentrifugation is a achieved
through rapid spinning, which imposes
high centrifugal forces on suspended
particles/molecules and causes
separation of such matter on the basis
of differences in weight.
• Ultracentrifugation can be categorized as
Preparative and Analytical centrifugation.
Picture 1: Theodor Svedberg

4. Figure 2 : Analytical ultracentrifuge instrument. Model- Proteome XL-1
• The sample can be monitored
in real time through a optical
detection system.
• It can run at about 120,000-
150,000 rpm and RCF as high as
625,000g.
• Rotor chambers are
evacuated to reduce friction.
• Equipped with self balance
system and microprocessor
control.
Analytical centrifugation

5. AUC technology can provide more answers questions such as
• Shape: How spherical is my protein?
• Diameter: What’s the size of my particle?
• Mass: What’s the molecular weight of my protein or complex in
solution?
• Stoichiometry: How many subunits comprise my protein?
• Purity: Are there other particles in my sample?
• Heterogeneity: Is my protein bound to other molecules, and what’s
the configuration of the complex?
• Association: Does my protein associate and/or dissociate with other
proteins?
• Conformation: Does the conformation of my protein change upon
binding to a ligand?
Advantages of AUC

6. Comparison between preparative and analytical
ultracentrifugation
Preparative ultracentrifugation
• Large sample size can be
used.
• No optical read-out. collect
fractions and analyze them
after the run.
• Less pure sample can be
used.
• Used to estimate
sedimentation coefficient
and MW.Generally used to
separate organelles and
molecules.
Analytical ultracentrifugation
• Uses small sample size (less
than 1ml).
• Built in optical system to
analyze progress of
molecules during
centrifugation.
• Uses relatively pure samples.
• Used to precisely determine
sedimentation coefficient
and MW of molecules.

7. Instrumentation and working
• The main parts of an analytical centrifugation are:
A rotor
An optical system
A data acquisition and
analysis system

8. Rotors and cells
• The rotor is solid with holes to hold the sample cells. Each cell
contains a centerpiece with channels to hold liquid samples.
• The centerpiece in turn is sealed between windows to permit the
passage of light.
• Centerpieces are available that hold either 4 or 8 cells.
• Different analytical cells are available with volume capacities
between 0.02 to 1.0 cm3.
Fig.3: Analytical centrifuge rotor and sample double sector-
shaped cell.
Fig.4: Rotor

9. Optical system and Data acquisition
• As the rotor spins, each cell passes through the optical paths of
detectors capable of measuring the concentration of molecules at
closely spaced radial intervals in the cell.
• There are three commercially available optical detectors to
measure the concentration distributions:
1. Absorbance spectrophotometer
2. Rayleigh interferometer
3. Fluorescence detector.
Absorbance spectrophotometer: usable over wavelength range
190-800nm. Under conditions where Beer-Lambert law holds,
the absorbance signal is directly proportional to the solute
concentration.

10. Fig.6: Schematic diagram of the optical system of an analytical ultracentrifuge.

11. Rayleigh interferometer: signal consists of equally spaced
horizontal fringes and does not rely on chromophore, hence
colorless compounds (Polysaccharides and lipids) can be
characterized.
Fluorescence detector: laser light and suitable labels are used
in this.
• A detector collects light absorption data, which a computer
digitizes and records.
• Data obtained will be interpreted using softwares such as
ULTRASCAN, SEDANAL, HETEROANALYSIS,
SEDFIT/SEDPHAT, SEDNTERP and result is given out.

12. Principle
Basic principle remains the same as that of the preparative
ultracentrifugation.
• Mass will redistribute in a
gravitational field until the
gravitational potential energy
exactly balances the chemical
potential energy at each radial
position.
• If we monitor the rate at which
boundaries of molecules move
during this redistribution, then it is
sedimentation velocity experiment.
• If we determine the concentration distribution after equilibrium is
reached, then it is a sedimentation equilibrium experiment.
Fig.7: Different forces acting on particle.

13. Factors affecting sedimentation
• More density - faster sedimentation
• Denser biological buffer system – slower movement of the particle
• More frictional coefficient - slower movement
• More centrifugal force- faster sedimentation
• Sedimentation rate=0. If, density of particle =density of the
surrounding medium.
Sedimentation coefficient: the velocity of a particle per unit
centrifugal field.
Where,
s = sedimentation coefficient
v = velocity of a particle
ω = angular velocity of rotation
r = distance of a particle to the rotor axis
v
ω2 r
s =

14. Types of experiments in AUC
• The two most common types of analysis performed with
analytical ultracentrifuges are:
1. Sedimentation velocity experiments.
2. Sedimentation equilibrium experiments.

15. • Sedimentation velocity experiments are performed at high speed
to overcome the effect of diffusion.
• An initially uniform solution is placed in a cell and a sufficiently
high angular velocity is applied to cause rapid sedimentation of
solute towards the cell bottom.
• As a result there is a depletion of solute near the meniscus,
causing a characteristic spectrum .
• It is performed using 2 sector cells which requires 420
μL/sample.
• The ultracentrifuge, detector and computer record the time course
of the sedimentation process, yielding information about the
shape, mass, and size of the molecules.
Sedimentation velocity method

16. Determination of molecular
weight:
M= RTs
D(1-ʋρ)
Where,
M: anhydrous molecular weight of the
macromolecules
R: Gas constant
T: Absolute temperature
s: sedimentation coefficient of the molecule
D: Diffusion coefficient of the molecule
ʋ: partial specific volume of the molecule
ρ : density of the medium
Fig.8: Graph obtained after SV experiment.

17. Sedimentation equilibrium method
• Sedimentation equilibrium experiments involve studying the
steady-state equilibrium of the sample in solution. And requires
lower rotor speed than sedimentation velocity experiments.
• Solute particles do not pellet at the bottom of the cell, but instead
the process of diffusion opposes the process of sedimentation
until after a period of time, the two opposing forces reach
equilibrium and the apparent concentration profile does not
change.
• SE are performed in 6 sector centerpieces which require
110μL/channel.
• Sedimentation equilibrium provides the same type of information
about the solution molar masses, stoichiometries, association
constants and solution nonideality.

18. Fig.9: Concentration profile in
sedimentation-diffusion equilibrium.
Determination of molecular
weight:
M= 2RT ln (C2/C1)
ω2 (1-ʋρ) (r1
2-r2
2 )
Where,
R: Gas constant
ω : angular velocity
T: Absolute temperature
ʋ: partial specific volume of the molecule
ρ : density of the solvent
C2 and C1 : concentration of solute at distances
at r2 and r1

20. Comparison between the two types of experiment in AUC
Sedimentation velocity Sedimentation equilibrium
Angular velocity
Large
(chosen according to the particles’
sedimentation properties)
Small
Sample required 420μL 110μL/channel
Analysis As a function of time
(3-6 hours)
At equilibrium
(after 24 hours)
Measurement
Forming a boundary
(sedimentation profile as function
of time)
The particle distribution in
the cell
Calculated parameters Shape, mass composition Mass composition
Graph obtained

21. Applications
With Analytical Ultracentifugation (AUC) the following
characteristics of a molecule can be determined:
• Native Molecular Mass
• Stoichiometry
• Assiociation
• Assembly Models
• Conformation & Shape
• Diffusion & Sedimentation

22. Native Molecular Mass
• AUC is the only technique with which you can determine
accurately the native molecular weight of a sample.
• AUC is applicable over a wide range of molecular weights from
approx. 2.5 kDa up to 1.5 MDa.
• A typical experiment takes about 16 hours and should be done
overnight.
Stoichiometry
• The stoichiometry of a molecule can be calculated from the
determined molecular mass.
• with high quality data it can be easily established whether the
native conformation of a protein is a hexa- or a heptamer.
Association
• The sedimentation equilibrium method is particularly sensitive for
the study of relatively weak associations with associations
constants (K) in the order of 10-100 M.

23. Assembly Models
• The assembly of a protein complex can be calculted from the
determined molecular mass.
• Ligand binding can also be analyzed using sedimentation velocity
methods if the ligand and acceptor differ greatly in their
sedimentation coefficients.
• Alternatively, a thermodynamic analysis may be made using
sedimentation equilibrium methods.
Conformation & Shape
• Information about the shape and the conformation of a protein as
well as the interaction between macromolecules can be obtained
from the sedimentation and diffusion coefficients obtained from a
sedimentation velocity experiment.
• Sedimentation coefficients are particular useful for monitoring
changes in conformation of a protein.

24. References
• Wilson, K., Walker, J. Principles and Techniques of Practical
Biochemistry.7th edition 2010. Cambridge university press, New
York. Pp: 95-99.
• Upadhyay, A., Upadhyay, K. Biophysical chemistry. Himalaya
publishing house, Delhi. Pp: 321-330.
• Serdyuk, I.N., Zaccai, N.R. Methods in Molecular Biophysics.
2007. Cambridge university press, UK. Pp: 339-385.
• Talluri, S. Bioanalytical Techniques. IK ineternational
publishing house Pvt.Ltd, New Delhi. Pp:22-28.
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711687/#!po=7
5.7813