Centrifugation Introduction and Types

Centrifugation Introduction

• Centrifugal force is the apparent force that draws a rotating body away from the center of rotation.
• Biological centrifugation is a process using centrifugal force to separate and purify biological particles in a liquid medium.
• It separates molecules based on their sedimentation rate under centrifugal force.
• It's a key technique for isolating and analyzing cells, subcellular fractions, and macromolecules like proteins and nucleic acids.
• Particles separate based on size, shape, density, medium viscosity, and rotor speed.

Types of Centrifuges

• Analytical centrifugation: Primarily focuses on studying purified macromolecules or supramolecular assemblies.
• Preparative centrifugation: Used for separating tissues, cells, subcellular structures, membrane vesicles, and other biochemically interesting particles.
• Clinical centrifuge: A type of centrifuge used in clinical settings.
• Small-scale laboratory microfuge: A small-scale, high-speed centrifuge.

Principle of Centrifugation

• Sedimentation is the movement of particles under gravitational force.
• Centrifugal force, applied by the centrifuge, causes particles to move faster.
• An example is adding sand to water in a bucket and swinging the bucket around in a circle, the water moves slower but the sand sediments faster.

Rate of Sedimentation

• A biological sample in a centrifuge experiences an outward centrifugal force.

• The rate of sedimentation depends on the radial distance of the particle from the axis of rotation and the square of the angular velocity of the rotor.

• G = ω²r ( where G is the centrifugal field, ω is the angular velocity, and r is the radial distance).

• The rate of sedimentation also depends on the particle's density, the surrounding medium's viscosity, and the particle's radius.

• ν = 2 r²(pp - pm)/9η × g ( where ν is the sedimentation rate, r is the radius of the particle, pp is the density of the particle, pm is the density of the medium, g is the acceleration due to gravity, and η is the viscosity of the medium).

Sedimentation Coefficient (s)

• The sedimentation rate or velocity is expressed as the sedimentation coefficient (s):
• s = ν/ω²r
• S20,w: The sedimentation coefficient corrected to standard conditions (water at 20°C).
• Svedberg units (S): A unit of measurement for sedimentation coefficients, where 1 S = 10⁻¹³ s.

Relative Centrifugal Force (RCF)

• RCF is a measurement of the force applied to a sample in a centrifuge.
• RCF is proportional to the radius of the rotor and the square of the rotor speed (RPM):
• RCF (g Force) = 1.118 × 10-5 × r × (RPM)2

Centrifugation Protocol Design Considerations

• Denser biological structures sediment faster in a centrifugal field.
• More massive particles have faster movement.
• Denser biological buffer systems cause slower movement of particles.
• Higher friction coefficients result in slower particle movement.
• Stronger centrifugal forces lead to faster sedimentation.
• Sedimentation rate is zero when the particle and medium densities are equal.

Types of Rotors

• Fixed-angle rotors: Tubes are held at an angle to the vertical axis, resulting in shorter run time.
• Vertical tube rotors: Rotors are held parallel to the vertical axis. The disadvantages include pellet falling back into solution at the end of centrifugation.
• Swinging-bucket rotors: Rotor swings to a horizontal position during acceleration. Advantageous for greater separation distance during isopycnic centrifugation.

Types of Centrifuges

• Large-capacity low-speed preparative centrifuges
• Refrigerated high-speed preparative centrifuges
• Analytical ultracentrifuges
• Preparative ultracentrifuges
• Large-scale clinical centrifuges
• Small-scale laboratory microfuges

Types of Centrifugation

• Low-speed centrifugation: Speeds 1-6000 rpm, used for separating large particles.
• High-speed centrifugation: Speeds 1000-25,000 rpm. Used for separating smaller particles.
• Ultracentrifugation: Very high speeds, 60,000 -150,000 rpm, used for separating very small particles and isolating organelles.

Preparative Centrifugation

• A procedure for separating samples into phases (pellet and supernatant) by placing the sample in the tube, inserting the tube in the fixed rotor, and spinning for a set time.
• Relatively heavy particles and precipitates settle in the pellet for further analysis.
• Light particles like ribosomes require ultracentrifugation.

Differential Centrifugation

• Separates components of a homogenate based on their sedimentation rates.
• Successive spins at increasing speeds lead to the separation and isolation of organelles.
• Example: Initially all particles of the homogenate are distributed evenly throughout the tube and the particles separate based on their sedimentation rates while moving down the tube.

Density Gradient Centrifugation

• Separates molecules based on differences in their densities.
• A density gradient is created in a medium in the centrifuge tube, similar to their own.
• Denser molecules shift to the bottom of the tube as they travel through the density gradient.
• Separates molecules and particles into layers that can be recovered for further analysis. Includes methods: a. Zonal centrifugation (size-based) b. Isopycnic centrifugation (density-based)

Analytical Centrifugation

• Used for accurate determination of molecular masses of solutes in their native states.
• Widely used for biological molecules with a range of molecular masses.
• The movement of particles during centrifugation, which are the rate of sedimentation can provide necessary information of the molecular mass.
• The sedimentation coefficient of a substance is a direct proportionality constant with the mass of a substance.

Sedimentation Coefficient

• Describes how fast particles experience movement during centrifugation.
• Conformational changes in biological macro-molecules can influence the rate at which it sediments through the solvent.
• The conformational change in biological macro- molecules can be exploited using sedimentation analysis.