Centrifugation

Page 1: Introduction to Centrifugation

  • Course Information

    • BCH3B04 - Techniques in Biochemistry

    • Presenter: Rosemol Jacob M.

    • Affiliation: Department of Biochemistry, Sree Krishna College, Guruvayur

  • Topics Covered

    • Principle of sedimentation technique

    • Relationship between RPM and radius of rotation

    • Relative centrifugal force (RCF) and centrifugal force (expressed as multiples of g)

    • Types of centrifuges and rotors

Page 2: Introduction to Sedimentation

  • Key Concept

    • Macromolecules’ insensitivity to gravitational settling due to low gravitational forces acting on small particles.

    • Random bombardment of surrounding medium molecules supersedes gravity.

  • Implication of Gravity

    • Concentration gradients only develop when solutions remain undisturbed for long periods.

    • To accelerate sedimentation, gravitational potential energy can be increased using high-speed rotation.

Page 3: Effect of Centrifugal Force

  • Particle Sedimentation

    • Centrifugal force added to gravitational force accelerates particle sedimentation.

  • Historical Context

    • Svedberg and Nicols (1923) first used centrifuge for measuring particle sizes.

  • Biological Relevance

    • Biological centrifugation crucial for separating and purifying cellular components, such as organelles and macromolecules.

Page 4: Basic Principles of Sedimentation

  • Sedimentation Rates

    • Earth’s gravitational field (G = g = 9.81 m/s²) vs increased sedimentation rates in a centrifugal field (G > 9.81).

  • Example

    • Sand sediments slowly in still water but faster when the container is spun.

  • Observation

    • Biological structures also show increased sedimentation rates under centrifugal force.

  • Relative Force Measurement

    • Relative centrifugal field (RCF) expressed as multiples of gravitational acceleration.

Page 5: Factors Affecting Sedimentation Rate

  • Designing a Centrifugation Protocol

    • Density of Structure: Denser items sediment faster.

    • Mass of Particles: More massive particles move quicker in centrifuge.

    • Density of Medium: Denser buffers slow particle movement.

    • Frictional Coefficient: Higher coefficients result in slower particle speed.

    • Applied Force: Greater centrifugal forces lead to faster sedimentation.

    • Equilibrium: Sedimentation rate becomes zero when particle density equals that of the surrounding medium.

Page 6: Frictional Drag in Sedimentation

  • Frictional Drag

    • Particles moving through a viscous medium experience drag, counteracting sedimentation.

    • Drag force = velocity × frictional coefficient; the coefficient is influenced by particle size and shape.

  • Velocity Increase

    • As particles descend, their velocity increases due to radial distance, incurs drag which slows down sedimentation.

Page 7: Sedimentation Rate Equation

  • Factors in Equation

    • Sedimentation rate (v) depends on centrifugal field (G; measured in cm/s²), radial distance (r), and angular velocity (ω).

  • Essential Formula

    • G = ω² × r

Page 8: Angular Velocity

  • Definition

    • Angular velocity of a rigid body is angular displacement over time interval.

  • Radian Measurement

    • 1 radian subtends arc length equal to the radius of a circle; 360° = 2π radians.

  • Conversion to RPM

    • ω (rad/s) relates to rotor speed (RPM) as: ω = 2π rad × RPM.

  • Centrifugal Field Representation

    • G = 4π² × r × RPM²

Page 9: Calculating Centrifugal Field

  • Variable Components

    • RPM: rotor speed (revolutions/min)

    • r: radial distance from rotation center

  • RCF Calculation

    • RCF relates centrifugal acceleration at a specific radius to standard gravitational force: RCF = G/g = 4π² × r × RPM²/g.

Page 10: RCF Units

  • Dimensionless Nature

    • RCF units are dimensionless, representing multiples of gravitational constant.

  • Numerical Convenience

    • Constants grouped for easier calculations.

Page 11: Centrifugation Manuals

  • Nomograph Utility

    • Centrifugation manuals often include nomographs for converting between RCF and centrifuge speed at varying radii.

  • Three Columns in Nomographs

    • Represent radial distance, RCF, rotor speed to facilitate conversions using a straight edge for intersection readings.

Page 12: Nomograph Example

  • Visualization

    • Three columns illustrate relationship for determining RCF from rotor speed; example calculation given for 5 cm rotor at 40,000 RPM yielding ~90,000×g.

Page 13: Stokes’ Law

  • Sedimentation Dependencies

    • Rate of sedimentation (ν) is influenced by particle density (ρp), hydrodynamic radius (R_hydro), medium density (ρm), and viscosity (η).

  • Stokes’ Law Formula

    • ν = (ρp - ρm) * g * R_hydro² / (η)

Page 14: Sedimentation Time Calculation

  • Sedimentation Time

    • Given as distance/velocity (ν): t = distance/ν

  • Particle Separation

    • Spherical particles can be separated based on size/density in a centrifugal field.

Page 15: Sedimentation Coefficient

  • Definition

    • Sedimentation rate expressed in coefficient units (s), indicating time taken for sedimentation.

Page 16: Normalization of Sedimentation Coefficients

  • Standard Conditions

    • Experimental values often corrected for comparison to standard density and viscosity (water at 20°C).

  • Svedberg Units

    • Sedimentation coefficients denoted in Svedberg units (S), where 1 S = 10⁻¹³ s.

Page 17: Basic Components of a Centrifuge

  • Essential Parts

    • Metal rotor (accommodates vessels) and motor (for spinning).

    • Additional parts support operation/environment management during centrifugation.

Page 18: Types of Centrifuges

  • Centrifuge Categories

    • Microcentrifuge

    • Benchtop/Tabletop centrifuges

    • Ultracentrifuges

Page 19: Desk Top Centrifuges

  • Characteristics

    • Small, simple, least expensive model.

    • Used for rapidly sedimenting substances (e.g., RBCs, yeast cells).

    • Max speed ~3000 RPM, no temperature regulation.

Page 20: Capacity of Desk Top Centrifuges

  • Capacities for separation range from 10, 50, or 100 cm³ tubes; larger versions available (4-6 dm³).

Page 21: Balancing Centrifuge Tubes

  • Loading Protocol

    • Tubes must be balanced accurately with even numbers for stability.

    • Partially loaded rotors should have opposing loading to balance forces.

Page 22: High-Speed Centrifuges

  • Performance

    • Maximum speeds up to 25,000 RPM (~90,000 x g).

    • Refrigeration systems manage heat from friction; maintain 0-4°C.

  • Typical Applications

    • Used to collect microorganisms, cell organelles, etc.

Page 23: Limitations of High-Speed Centrifuges

  • Organelles

    • Effective for larger organelles but not suitable for smaller ones like ribosomes or microsomes.

Page 24: Ultracentrifuge Preparative Function

  • Capabilities

    • Operates up to 75,000 RPM (>500,000 x g); significantly generates heat requiring evacuated rotor chambers for cooling.

  • Structural Features

    • Small diameter drive shaft accommodates rotor imbalances; constructed with high-strength materials to resist high forces.

Page 25: Safety Precautions in Ultracentrifuges

  • Overspeed Device

    • Prevents excessive speeds; potential rotor explosion hazard.

  • Enclosure

    • All chambers protected by heavy armor plating.

Page 26: Resolution Power of Ultracentrifuges

  • Applications

    • Can distinguish between DNA molecules differing in isotopic composition, essential for structural-function studies of organelles and macromolecules.

  • Methods

    • Used for isolation, purification, or characterization of subcellular components.

Page 27: Ultracentrifuge Types

  • Categories

    • Preparatory ultracentrifuge for isolation/purification.

    • Analytical ultracentrifuge for characterization studies.

Page 28: Types of Rotors

  • Rotor Varieties

    • Fixed-angle rotors

    • Vertical tube rotors

    • Swinging bucket rotors

    • Zonal rotors

Page 29: Fixed-Angle Rotors

  • Design Features

    • Holes at angles (14°-40°) allow particles to slide down, forming pellets at tube outer walls.

  • Advantage

    • Quick sedimentation due to wall effects.

  • Disadvantage

    • Limited resolution for particles with similar sedimentation characteristics.

Page 30: Vertical-Tube Rotors

  • Operating Mechanism

    • Holes parallel to rotor shaft; reorientation of solution during rotation leads to quicker sedimentation across tube diameter.

  • Pellet Formation

    • Pellet deposited along outer wall; potential for back-dissolution after centrifugation.

Page 31: Swinging Bucket Rotors

  • Functional Movement

    • Buckets swing to a horizontal position under centrifugal acceleration; similar sedimentation mechanics as fixed-angle rotors, but can be used for density-gradient centrifugation.

Page 32: Disadvantages of Swinging Bucket Rotors

  • Wall Effects

    • Particles still experience wall impacts and sliding, but less pronounced due to density gradients.

Page 33: Zonal Rotors

  • Advantages

    • Allow particle movement without wall contact; essential for limiting sample volumes in density gradient experiments.

  • Operational Method

    • Gradients loaded dynamically as rotor is spinning, enhancing efficiency.

Page 34: Zonal Rotor Mechanism

  • Sedimentation Process

    • Zonal rotors allow for controlled gradient additions, providing optimized conditions for sedimentation within sectors.

Page 35: Sample Loading in Zonal Rotors

  • Dynamic Loading

    • Samples introduced during rotor operation for effective sedimentation near rotational center.

Page 36: Final Steps in Zonal Centrifugation

  • Gradient Management

    • After centrifugation, dense solutions are pumped to collect separated layers effectively.

Page 40: Conclusion

  • Endnote

    • THANK YOU!