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Questions and Answers
What does the sedimentation coefficient (s) represent in sedimentation principles?
What does the sedimentation coefficient (s) represent in sedimentation principles?
Which factor is NOT included in the calculation of Relative Centrifugal Force (RCF)?
Which factor is NOT included in the calculation of Relative Centrifugal Force (RCF)?
In Stokes' Law, which parameter affects the sedimentation rate of a rigid spherical particle?
In Stokes' Law, which parameter affects the sedimentation rate of a rigid spherical particle?
What is the primary use of a nomograph in centrifugation?
What is the primary use of a nomograph in centrifugation?
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How is the sedimentation rate affected when the density of the particle increases?
How is the sedimentation rate affected when the density of the particle increases?
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What is the defining characteristic of Svedberg units when discussing sedimentation coefficients?
What is the defining characteristic of Svedberg units when discussing sedimentation coefficients?
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What role does gravitational acceleration play in calculating sedimentation rates?
What role does gravitational acceleration play in calculating sedimentation rates?
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What is typically found in a centrifugation manual to assist with RCF calculations?
What is typically found in a centrifugation manual to assist with RCF calculations?
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What is the maximum Relative Centrifugal Force (RCF) achievable in low-speed centrifugation?
What is the maximum Relative Centrifugal Force (RCF) achievable in low-speed centrifugation?
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Which type of centrifugation is designed to operate with a rotor chamber maintained at low temperatures?
Which type of centrifugation is designed to operate with a rotor chamber maintained at low temperatures?
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Which of the following types of rotors is NOT typically used in low-speed centrifugation?
Which of the following types of rotors is NOT typically used in low-speed centrifugation?
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In which centrifugation type is the speed typically in the range of 60,000 to 150,000 rpm?
In which centrifugation type is the speed typically in the range of 60,000 to 150,000 rpm?
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Which method is primarily used when maximum resolution of banding zones is required?
Which method is primarily used when maximum resolution of banding zones is required?
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What is the typical purpose of high-speed centrifugation?
What is the typical purpose of high-speed centrifugation?
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What fundamental principle is used in sedimentation to separate particles during centrifugation?
What fundamental principle is used in sedimentation to separate particles during centrifugation?
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Which of these centrifuge types is primarily used for large-scale clinical applications?
Which of these centrifuge types is primarily used for large-scale clinical applications?
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How is the relative centrifugal force (G) calculated in a centrifugation process?
How is the relative centrifugal force (G) calculated in a centrifugation process?
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Which type of centrifuge is primarily used for the separation of pure macromolecules?
Which type of centrifuge is primarily used for the separation of pure macromolecules?
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What factors influence the rate of sedimentation of particles in a centrifuge?
What factors influence the rate of sedimentation of particles in a centrifuge?
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Which factor is NOT involved in the sedimentation process when using centrifugation?
Which factor is NOT involved in the sedimentation process when using centrifugation?
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In centrifugation, what is the main purpose of using a preparative centrifuge?
In centrifugation, what is the main purpose of using a preparative centrifuge?
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What is the significance of the radial distance in the context of centrifugation?
What is the significance of the radial distance in the context of centrifugation?
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Which of the following is true regarding the use of a nomograph in centrifugation?
Which of the following is true regarding the use of a nomograph in centrifugation?
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What commonly happens to particles when subjected to centrifugal force in a centrifuge?
What commonly happens to particles when subjected to centrifugal force in a centrifuge?
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Study Notes
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
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A biological sample in a centrifuge experiences an outward centrifugal force.
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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.
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G = ω²r ( where G is the centrifugal field, ω is the angular velocity, and r is the radial distance).
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The rate of sedimentation also depends on the particle's density, the surrounding medium's viscosity, and the particle's radius.
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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.
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Description
This quiz covers the fundamentals of centrifugation, focusing on the principles of centrifugal force and its applications in biological settings. Explore various types of centrifuges, including analytical, preparative, and clinical centrifuges, and their specific uses in separating biological particles. Test your knowledge of how these processes aid in isolating and analyzing cellular components.
