Ultracentrifugation

Objectives

• Sedimentation and diffusion

• Sedimentation velocity (SV) and sedimentation equilibrium (SE)

• Instrumentation: Analytical and preparative ultracentrifuge

• Operation

• Applications

References:

• “Analytical Ultracentrifugation Instrumentation, Software, and Applications” by Susumu. Uchiyama Fumio Arisaka; Walter F Stafford; Tom Laue Tokyo : Springer Japan 2016

• You can download the above book from our U of M library http://link.springer.com.uml.idm.oclc.org/book/10.1007/978-4-431-55985-6/page/1

Centrifugation

• To separate particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed

• Higher density sink (sediment); so, lighter float to the top.

• Difference in density

• If there is no difference in density of solvent (Isopycnic conditions), the particles stay steady.

• Tiny differences in density Apply “centrifugal force”

• Molecular weight (MW), hydrodynamic and thermodynamic properties of a protein or other macromolecule

• Sedimentation is firmly based in thermodynamics of solute in solution

Types of Centrifuges

1. Analytical ultracentrifuge: To determine physical properties: massand shape of macromolecules or protein complexes, or the time course of sedimentation

2. Preparative ultracentrifuge: pelleting small particulate materials such as viruses, membranes, and organelles, or smaller particles such as DNA or RNA, and gradient separations

Analytical centrifugation

Instrumentation: Analytical ultracentrifuge

• High angular velocities (≃ 60000rpm)

• Evacuated chambers

• Temperature control

• Rotor with light passage and higher mechanical and corrosion stability

• If rotor can go up to 2,50,000g then it delivers 250Kg for 1g

Ref: http://www-bioc.rice.edu/bios576/AU/AU%20Page_files/image022.jpg

Double-sector centerpiece

Sedimentation• Pollens grains from honey to identify

the origin (location) of Honey

Fig.: http://2.bp.blogspot.com/-_xOs3YCHvyo/UDr_CLJ6pgI/AAAAAAAABjI/w5ZGXkGjfVQ/s1600/cl9SciCH2Fig5.jpg

Typical sedimentation

methods

Gravity

Takes couple of hours

Centrifuge

Within few mins

Theory of sedimentation

• The force of gravity, FG :

• Force due to buoyancy, FB :

• Force due to viscous drag, FD :

FDFB

FG

Radius of the sphereDensity of the sphere

Density of the displaced fluid

L

Viscosity of the liquid Velocity of sphere

Terminal velocity of the particle

The Stokes’ law

Sedimentation in a gravitational field

• Sedimentation coefficient (s) depends on the properties of the particle

• “s” has dimensions of seconds. For many substances, the value of “s” lies between 1

and 100 × 10-13 seconds

• Svedberg unit (abbreviation S) is defined as 10-13 seconds

• Densities of the solute and solvent are equal, (1 - vρ) = 0 No net movement

Diffusion

• Diffusion coefficient 𝐷 =𝑅𝑇

𝑁𝐴𝑓

• Svedberg’s equation:

• Molar mass can be obtained if we know ‘s’ and ‘D’

Frictional coefficient

Relation between sedimentation coefficient and

diffusion coefficient

• Diffusion causes the sedimenting boundary to spread with time.

Two basic types of experiments

1. High centrifugal force and the analysis of the time course of the sedimentation process, termed sedimentation velocity (SV); and

2. Low centrifugal force that permits the diffusion to balance the sedimentation such that a time-invariant equilibrium gradient can be observed, termed sedimentation equilibrium (SE).

Sedimentation velocity

• Lamm Equation:

Sedimentation coefficient Diffusion coefficient

Time course of the sedimentation along with diffusion

Sedimentation Equilibrium

• Consider this molecule rotating under a centrifugal force.

• Therefore after a rotating the solution at high speed for a period of time (equilibration) our original uniform solution is converted to a solution with a concentration gradient.

• The gradient is given by

Centrifugal forceThermal diffusion

2RT

])r()r[('exp

)c(r

)c(r 222

21

2

1 mConc at r1

Conc at r2

Buoyant mass

Distance of point 1 or 2 from rotating axis

Gas constant& temp

Angular velocity

Preparative Ultracentrifuge

RCF and RPM

radiusRCF RPM

http://cdn.biologydiscussion.com/wp-content/uploads/2015/10/clip_image00231.jpgwww.corning.com/discoverylabware

Nomograph

Instrumentation

Thermo Scienti corp. Sorvall WX+ Ultracentrifuge Instruction Manual 50145792-a • 09 / 2014

Rotors• Made from Aluminum/Titanium/

fiber-reinforced composites

• Titanium is stronger and more chemical resistant than Aluminium

• Selection factors: sample volume, number of sample components to be separated, particle size, desired run time, desired quality of separation, type of separation, and the centrifuge

Fixed angle rotor

• general-purpose

• pelleting subcellular particles and in short-column banding of viruses and subcellular organelles

• 20 to 45 degrees to the axis of rotation

• tube angle shortens the particle path length

Swinging bucket rotors • pelleting,

• isopycnic studies (separation as a function of density), and

• rate zonal studies (separation as a function of sedimentation coefficient

• best for rate zonal studies (for maximum resolution of sample zones)

• pelleting in the exact center

Vertical tube rotors

• parallel to the axis of rotation

• bands separate across the diameter of the tube

• Isopycnic, rate zonal separations when run time reduction is important

Near vertical tube rotors

• for gradient centrifugation

• reduced tube angle of these rotors significantly reduces run times from the more conventional fixed angle rotors

•

Particle separation and path length

Pelleting (differential separation)

• The short path length means less distance for particles to travel in the portion of the tube

• Selection of rotor plays important role here

Sedimentation Coefficients (in Svedberg Units) for Some Common Biological Materials

Pelleting by differential centrifugation

• Used for harvesting cells or producing crude subcellular fractions from tissue homogenate• E.g., nuclei, mitochondria, lysosomes, and membrane vesicles• Limitations: differential centrifugation suffers from contamination and poor recoveries• How to fix: resuspension and repeating the centrifugation steps

Rate zonal separations

• Sample is layered as a narrow zone on the top of a density gradient (B).

• Separation as a function of the particles’ sedimentation coefficient (density, size, and shape) and viscosity of the gradient material

• Sucrose is most common

https://www.sigmaaldrich.com/technical-documents/articles/biofiles/centrifugation-separations.html

Isopycnic separation

• also called buoyant or equilibrium separation

• separated solely on the basis of their density

• density of the gradient > particles to be separated

• particles will not sediment to the bottom of the tube, regardless of centrifugation time

• preparative techniques commonly use a discontinuous gradient

Density gradient medium and application area

Operation of ultracentrifuge

• Balancing tubes (at least 0.1g)

• Balance rotor

• Vacuum

• Temperature

• Selection of run type

You have to be punctual at every stages!!

• Mechanical failure rotor

• Balance

• Vacuum

• Temperature

• Vibration free place

Balancing rotor

Balanced Vs unbalanced load

ApplicationsAnalytical ultracentrifuge

• Examination of sample purity

• Molecular weight determination

• Sedimentation and diffusion coefficients— detection of conformation changes

• Ligand binding

Preparative ultracentrifuge

• Density gradient fractionation

• Subcellular fractionation

• Separation of Proteins and macromolecules

• DNA, RNA, organelles

Subcellular fractionation

http://images.slideplayer.com/25/8061636/slides/slide_21.jpg

Centrifugation method for separating cellular

components

http://4.bp.blogspot.com/-f8IQnZi6M8k/UQEeSenr4xI/AAAAAAAAALA/wB1ZNLvrC_8/s1600/Subcellular+Fractionation.jpg

https://proscientific.com/nprot.2006.478-F1.jpg

Further exercise

• Determination of Molecular Weight of Glycoproteins by Analytical Ultracentrifugation S. J. Shire, Genentech, Inc., South San Francisco, CA 94080

Thanks!