Centrifugation

• Centrifugation is a technique used to separate components of a mixture based on their

size, shape, density, and the viscosity of the medium by applying centrifugal force. • The
process occurs inside a centrifuge, an instrument that spins samples at very high
speeds. This generates a force that acts outwardly from the axis of rotation, called
the centrifugal force.
• The centrifugal force acts on particles suspended in a liquid, causing denser particles to
move outward toward the bottom or side of the container (depending on
orientation), forming a pellet. Less dense particles remain in the supernatant, the
liquid above the pellet.
• Centrifugation works on the principle of sedimentation, where particles in suspension
move under the influence of gravity or, in a centrifuge, the much greater centrifugal
force.
• The sedimentation rate depends on:
• Particle size
• Particle shape
• Particle density relative to the surrounding medium
• Viscosity of the medium
• Speed of centrifugation (angular velocity)
Sedimentation Concepts
• When a particle sediment in a fluid, it experiences forces due to centrifugal
acceleration and buoyant force (displacement of fluid).
• The net centrifugal force driving sedimentation depends on the difference between the
particle's density and the density of the medium; this causes particles denser than
the medium to sediment.
• Particles that have higher density than the medium move outward (or downward) and
form a pellet.
• Particles less dense than the medium may float instead of sedimenting. • Sedimentation
rate increases with particle size, density difference, and applied centrifugal force.
Factors influencing sedimentation:
1. Particle mass and size
o Larger and heavier particles sediment faster.
2. Density difference between particle and medium
o Greater the difference, faster the sedimentation.
o If particle density = medium density → no sedimentation (isopycnic point).
3. Viscosity of the medium
o Higher viscosity slows down sedimentation.
4. Applied centrifugal force (RCF)
o Stronger forces increase sedimentation speed.
Sedimentation Coefficient (s-value):
 • Denoted as Svedberg unit (S).
• Defines the rate at which a particle sediment in a centrifugal field.
2
• s=vω r
v = sedimentation velocity (cm/s)
o
o ω = angular velocity (radians/s)
o r = radial distance from rotor axis (cm)
• Example: Ribosomes are classified as 70S (prokaryotes) or 80S (eukaryotes) based on
their sedimentation coefficients.

Relative Centrifugal Force (RCF)

• Also called g-force, RCF expresses the effective force acting on a particle during
centrifugation relative to the force of gravity (g).
• It depends on both the rotational speed and the distance of the particle from the
rotor axis.
Formula:
RCF = 1.118 × 10−5 × r × (RPM)2
Where:
• RCF = Relative Centrifugal Force (× g)
• r = radius of rotation (cm, distance from rotor axis to sample)
• RPM = revolutions per minute
Example:
• At 10,000 RPM with a rotor radius of 10 cm,
RCF = 1.118 × 10−5 × 10 × (10,000)2 = 11,180 g
Types of centrifuges: tabletop, ultracentrifuge, refrigerated, high-speed
1. Tabletop (Benchtop) Centrifuge
• Compact centrifuges designed to fit on laboratory benches, commonly used in clinical
and research settings.
• Suitable for small to moderate sample volumes (from under 1 mL to several liters
depending on model).
• Typically versatile with various rotors available, such as fixed-angle, swinging bucket,
and sometimes continuous flow types.
• Speeds can vary widely, with some models reaching up to 15,000 RPM or more. •
Applications:
• Routine sample separation (blood, urine).
• Pellet collection of cells, bacteria, yeast.
• Removing precipitates from solutions.
• Advantages: Small footprint, ease of use, moderate cost, flexible applications. •
Limitations: Generally lower maximum speed and capacity compared to floor model
centrifuges.
2. Ultracentrifuge
• Designed for extremely high speeds, capable of generating forces well beyond standard
centrifuges.
• Speeds can reach up to 100,000 RPM or more, producing centrifugal forces up to
1,000,000 x g.
• Used primarily for separating very small particles, such as viruses, sub-cellular
organelles, proteins, and nucleic acids.
• Requires specialized rotors and cooling systems to manage heat generated by high
speed operation.
• Applications:
• Separation of macromolecules (DNA, RNA, proteins).
• Study of ribosomes, viruses, lipoproteins.
• Determination of molecular weights and sedimentation coefficients. •
Advantages: High precision, extremely high force, efficient separation of very fine
particles.
• Limitations: High cost, complex operation and maintenance, requires trained
personnel.
3. Refrigerated Centrifuge
• Equipped with temperature control facilities to maintain sample integrity during
centrifugation.
• Used when samples are heat-sensitive, such as live cells, proteins, or enzymes that may
degrade at room temperature.
• Can be found as benchtop or floor models with moderate to high speed capabilities (up
to ~15,000 RPM).
• Temperature can typically be set and maintained down to 4°C or lower.
• Applications:
• Separation of macromolecules (DNA, RNA, proteins).
• Study of ribosomes, viruses, lipoproteins.
• Determination of molecular weights and sedimentation coefficients. •
Advantages: Protects temperature-sensitive samples, versatile for various sample sizes.
• Limitations: Higher cost and maintenance due to refrigeration components.
4. High-Speed Centrifuge
• Operates at speeds generally between 15,000 to 30,000 RPM.
• Often includes automated controls for speed and sometimes temperature. •
Can accommodate a variety of rotors and sample sizes.
• More advanced than low-speed centrifuges but less extreme than ultracentrifuges.

Applications:

• Isolation of cell organelles (mitochondria, nuclei, chloroplasts).

 • Harvesting microorganisms.
• Biochemical analysis requiring faster separation than normal centrifuges. •
Advantages: Fast and efficient, suitable for advanced biochemical applications. •
Limitations: Higher energy consumption and cost than lower-speed models.
Type Speed Max Sample Temperatu Typical
Range Force Volume re Control Use Cases
(RPM) (Approx.)

Tabletop Up to Moderate Small to Sometimes Clinical

(Benchtop) ~15,00 (~10,000 moderate (few models) labs,
0 x g) (mL) routine
separation
s

Ultracentrifuge Up to Very high Small Yes Molecular

100,00 (~1,000,0 (microvolumes) biology,
0+ 00 x g) virology

Refrigerated Up to Moderate Variable Yes Heat

~15,00 to high sensitive
0 biological
samples

Type Speed Max Sample Temperatu Typical

Range Force Volume re Control Use Cases
(RPM) (Approx.)

High-Speed 15,000- High Medium Often yes Protein,

30,000 (~50,000 DNA,
to organelle
100,000 x separation
g)

Centrifugation Accessories
• Rotors: The essential centrifuge accessory that holds tubes and spins samples. Main
types include:
• Fixed-angle rotor
• Tubes held at a fixed angle (usually 20–45°).
 • Particles sediment along the side wall and form a pellet at the bottom. •
Fast, high-speed separation.
• Used for pelleting cells, organelles, nucleic acids.
• Swing-bucket (horizontal) rotor
• Tubes swing out horizontally during centrifugation.
• Sediment forms a flat layer at the bottom.
• Suitable for density gradient centrifugation.
• Vertical rotor
• Tubes kept vertical.
• Short pathlength, rapid separation.
• Used in isopycnic centrifugation (e.g., plasmid DNA, CsCl gradients).

• Buckets and adapters: Used in swinging-bucket and some fixed-angle rotors to hold
tubes or bottles of varying sizes and shapes. Choosing the right adapter shape and
size is critical for sample containment and rotor balance.
• Tube holders and inserts: Facilitate compatibility with different tube sizes inside rotors
for versatile use.
• Lids and seals: Prevent sample spillage, evaporation, and contamination. Click-seal lids
may be used for biocontainment, especially with hazardous or radioactive samples. •
Buckets: Used in some rotors to hold tubes or bottles securely during centrifugation. •
Additional accessories: Some centrifuge systems have rolling cabinets for space
efficiency or specialized accessories for blood processing and workflow traceability.

Sample Types in Centrifugation

Different centrifuge applications depend on the sample type:
1. Biological Samples
o Blood: Separation of plasma, serum, buffy coat.
o Cells: Bacteria, yeast, mammalian cells for culture or analysis.
o Organelles: Mitochondria, nuclei, microsomes by differential centrifugation.
o Macromolecules: Proteins, nucleic acids, ribosomes.
o Viruses: Purification using ultracentrifugation.
2. Environmental Samples
o Soil and water sediments.
o Microorganism isolation.
3. Industrial/Chemical Samples
o Dairy (cream separation).
o Pharmaceutical formulations.
o Nanoparticles and colloid suspensions.

Tube Types and Characteristics

Choosing the correct tube is critical for sample integrity and safety.
Factors for Tube Selection
1. Material
o Plastic (Polypropylene, Polycarbonate):
▪ Lightweight, resistant to breakage.
▪ Disposable (reduces contamination).
▪ Polypropylene: chemical-resistant, opaque.
▪ Polycarbonate: clear, strong, but less chemical resistant.
o Glass:
▪ Clear, reusable.
▪ Risk of breakage at high speed.
▪ Usually used at low to medium speeds.
o Metal/Aluminum:
▪ For ultracentrifugation, reusable.
▪ Chemically inert, strong.

2. Tube Shape
o Round-bottom: Common, strong, good for pelleting.
o Conical-bottom: Concentrates pellet in a narrow tip → easy to recover.
o Thick-walled tubes: For ultracentrifugation (withstand high RCF).

3. Volume and Size

o Tubes range from 0.2 mL (microcentrifuge) to 500 mL (large-scale centrifuges).
o Must match rotor specifications.

4. Closure Type
o Open-top: Quick, but risk of spillage.
o Screw-cap / Snap-cap: For secure closure.
o Aerosol-tight caps: For infectious or hazardous samples.
Sedimentation Velocity (SV)
Principle
• Monitors the rate of sedimentation of particles under centrifugal force. • Particles move
through the solvent and form a moving boundary (sedimentation front), which can be
observed optically.
• The movement depends on molecular mass, size, shape, and solvent interactions.
Key Parameter: Sedimentation Coefficient (s)

Where:
 −13
• s = sedimentation coefficient (in Svedberg units, 1 S = 10 sec)
• v = sedimentation velocity
• ω = angular velocity
• r = radial distance from rotor axis
Applications
• Determining molecular size and shape of proteins, nucleic acids.
• Studying heterogeneity of a sample (monomers, dimers, aggregates). •
Analysing conformational changes in macromolecules.

Sedimentation Equilibrium (SE)

Principle
• At very high centrifugal force, sedimentation is balanced by diffusion (movement of
molecules back toward lower concentration).
• A stable concentration gradient is established without pellet formation. • This
equilibrium distribution is analyzed to determine molecular mass independent of shape.
Equation (Simplified)

Where:
• C = concentration at radius r
• M = molar mass

• = partial specific volume of solute

• ρ = solvent density
• R = gas constant
• T = absolute temperature
Applications
• Accurate determination of molecular weights of proteins, nucleic acids, and complexes.
• Studying protein–protein or protein–DNA interactions (stoichiometry, binding
constants).
• Determining oligomerization states (monomer, dimer, trimer, etc.).

3. Comparison of SV vs SE
Feature Sedimentation Velocity (SV) Sedimentation Equilibrium (SE) Purpose Measures
rate of sedimentation Measures equilibrium distribution
Absolute molecular weight, association
Data givesSize, shape, heterogeneity, constants
sedimentation coefficient
Time required Faster (few hours) Slower (can take 1–2 days) Sample use Small to medium
amounts Very small amounts shapeYes (shape affects s-value) No (shape-independent, only

Dependency on
molar mass)
Molecular mass and binding constants
OutputSedimentation coefficient distribution
Cell Fractionation and Organelle Isolation Methods

1. Introduction
• Cell fractionation = process of breaking open cells (cell lysis) and separating their
components (organelles, macromolecules) for biochemical analysis.
• Goal: to obtain pure, functional organelles for studying their structure, function, and
biochemical properties.
• Commonly achieved using centrifugation techniques after disrupting cells.
2. General Steps in Cell Fractionation
1. Extraction:
• Cells or tissues are first collected and suspended in an isotonic buffer (often
sucrose-based) to maintain organelle integrity and prevent osmotic damage. •
Kept at low temperatures to minimize enzymatic degradation.
2. Homogenization:
• The cells are disrupted (lysed) to release the internal organelles into a
homogenate.
• Methods include:
• Mechanical homogenization (blender, Potter-Elvehjem or Dounce
homogenizer)
• Sonication (ultrasound waves particularly for bacterial or small cells) •
Osmotic lysis (using hypotonic solutions for cells like red blood cells) •
High-pressure methods (French press or nitrogen bomb)
• Aim: Break cell membrane gently to preserve organelles intact.
3. Centrifugation:
• The homogenate is subjected to differential centrifugation:
• Low-speed spin (around 600-1,000 x g) pellets nuclei and unbroken cells.
• Medium-speed spin (around 10,000-15,000 x g) pellets mitochondria,
lysosomes, and peroxisomes.
• High-speed spin (100,000 x g) pellets microsomes and ribosomes.
• Each pellet fraction contains specific organelles based on size and density.
4. Density Gradient Centrifugation:
• Further purification using gradients (e.g., sucrose or Percoll) based on buoyant
density.
• Organelles float to their specific density layers allowing finer separation. • Useful
 to separate similar-sized organelles (e.g., lysosomes from peroxisomes).

Organelle Isolation Techniques:

A. Differential Centrifugation
• Principle: Larger and denser components sediment faster at lower centrifugal forces;
smaller organelles require higher speeds.
• Procedure:
• Low-speed spin → removes unbroken cells, nuclei (largest, densest). •
Medium-speed spin → sediments mitochondria, chloroplasts, lysosomes. •
High-speed spin → sediments microsomes (ER fragments), small vesicles. •
Ultracentrifugation → isolates ribosomes, viruses, macromolecules. • Application:
Rough separation of organelles, commonly used in basic labs.

B. Density Gradient Centrifugation

Two types:
1. Rate-zonal centrifugation (velocity-based)
o Separation based on size and mass.
o Organelles are layered on top of a density gradient medium (e.g., sucrose,
Percoll).
o After centrifugation → organelles move as zones according to sedimentation
rate.
o Used for ribosomes, RNA, protein complexes.
2. Isopycnic (equilibrium) centrifugation
o Separation based on buoyant density.
o Each organelle moves until it reaches a position in the gradient where its
density equals the medium’s density (isopycnic point).
o Used for mitochondria, nuclei, plasmids, and viral particles.

Organelle Isolation – Typical Fractionation Order

From animal cells (using differential centrifugation):
1. Nuclei → low-speed centrifugation (~600 × g).
2. Mitochondria, Lysosomes, Peroxisomes → medium speed (~10,000 × g).
3. Microsomes (ER fragments), Golgi → high-speed (~100,000 × g).
4. Ribosomes, Viruses, Macromolecules → ultracentrifugation (>100,000 × g).

Applications of Organelle Isolation

• Study of organelle-specific functions (e.g., mitochondrial respiration, lysosomal
enzymes).
• Purification of biomolecules (DNA, RNA, proteins, lipids).
• Clinical and diagnostic use (e.g., plasma fractionation, virus isolation). •
Biotechnology: production of organelle-derived vesicles or enzymes.