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!