Microbiology -10:

Bioinstrumentation Techniques

Dr. Neha Jangbari

Assistant Professor
Department of Microbiology
Christ College, Rajkot
 Unit – 4: Centrifugation

1. Centrifugation techniques- Basic principles,

Types of rotors.
2. Preparative and analytical centrifugation:
Instrumentation and application.
3. Ultracentrifugation methods.
4. Density gradient centrifugation.
 INTRODUCTION

• Centrifugation is a process that uses

centrifugal force to separate particles from a
solution based on their size, shape, density,
and viscosity of the medium.
• It is widely used in biological, chemical, and
industrial applications.
 Basic Principles of Centrifugation

1. Centrifugal Force:
• Centrifugation relies on centrifugal force, which
is generated by the rapid rotation of a sample
around a fixed axis.
• The force pushes heavier particles outward,
causing them to sediment at different rates based
on their mass and density.
Relative Centrifugal Force (RCF)
• RCF (or g-force) determines the
effectiveness of centrifugation.
• It is calculated using the formula:
RCF=1.118×10−5× r × (RPM)2
where:
• r = radius of rotation (cm)
• RPM = revolutions per minute
2. Sedimentation Rate
• The speed at which particles settle depends on
several factors:
1. Particle Size & Shape – Larger and denser
particles sediment faster.
2. Density Difference – The greater the density
difference between the particle and the medium,
the faster it sediments.
3. Viscosity of the Medium – Higher viscosity
slows down sedimentation.
3. Stokes’ Law (Sedimentation Velocity)
• The motion of a particle in a liquid medium under centrifugal force
follows Stokes’ Law:
v=2r2 (ρp−ρm)g / 9 η
where: v = sedimentation velocity
r = particle radius
ρp = particle density
ρm = medium density
g = gravitational acceleration
η = viscosity of the medium
• This equation shows that larger and denser particles settle faster,
while smaller particles require higher speeds for separation.
4. Types of Centrifugation Based on Principle

A. Differential Centrifugation
• Relies on size and density differences to
separate components.
• Used for pelleting cells, nuclei, organelles (e.g.,
mitochondria, ribosomes).
• Higher speeds separate smaller components.
B. Density Gradient Centrifugation
• Uses a density gradient medium (e.g., sucrose, cesium
chloride).
• Particles separate based on buoyant density rather than
size.
• Used in DNA, RNA, virus, and lipoprotein separation.
• Further divided into:
• Rate-Zonal Centrifugation (separates based on size
and mass).
• Isopycnic Centrifugation (separates based on density
equilibrium).
5. Sedimentation Coefficient (Svedberg Unit - S)
• The sedimentation coefficient (S) is a measure of
how fast a particle sediments.
• It is expressed in Svedberg units (S):
S=v/ω2r
• Where: v is velocity, ω is angular velocity, and r is
radius.
• Smaller values indicate slower sedimentation (e.g.,
ribosome subunits: 30S, 50S).
• Centrifugation is based on the principles of
centrifugal force, sedimentation rate,
Stokes' Law, and density gradients.
• The technique is widely used in
biochemistry, molecular biology, and
clinical diagnostics for the separation of
biological macromolecules and organelles.
 Types of Rotors in Centrifugation

• Centrifuge rotors are essential components that hold

the sample tubes and determine the efficiency and
type of separation.
• They come in different designs, each suited for
specific applications.
• The three main types of rotors are fixed-angle
rotors, swinging bucket rotors, and vertical
rotors.
1. Fixed-Angle Rotor

• In a fixed-angle rotor, the tubes are held at a constant

angle (typically between 15° and 45°) relative to the axis
of rotation.
• Characteristics:
✓ Tubes remain at a fixed angle throughout the run.
✓ Particles move radially outward and pellet against the
tube wall.
✓ Sedimentation occurs in a shorter distance, making it
faster than other rotor types.
Applications:
• Used for pelleting cells, organelles, and precipitates (e.g.,
mitochondria, nuclei).
• Suitable for differential centrifugation to separate cellular components.
• Ideal for protein and nucleic acid precipitation.
Advantages:
• High-speed operation (can reach up to 150,000 × g in ultracentrifuges).
• Efficient for rapid pelleting.
Disadvantages:
• Pellets form along the side of the tube, which can make resuspension
difficult.
• Not suitable for density gradient separation due to uneven sedimentation.
 2. Swinging Bucket Rotor

• In a swinging bucket rotor, the sample buckets

hang vertically when at rest and swing outward to
90° during centrifugation.
• Characteristics:
✓ Provides horizontal sedimentation, making
separation more uniform.
✓ Ideal for gradient-based separations since particles
separate in a straight path rather than at an angle.
Applications:
• Density gradient centrifugation (e.g., separating DNA, viruses,
ribosomes).
Isolation of blood components (plasma, serum, RBCs, WBCs).
Suitable for cell culture harvesting.
Advantages:
• Pellets form at the bottom of the tube, making resuspension easier.
• Excellent for isopycnic and rate-zonal centrifugation.
Disadvantages:
• Lower speed capability than fixed-angle rotors (max ~50,000 × g).
Longer run time due to increased sedimentation distance.
 3. Vertical Rotor

• A vertical rotor holds tubes parallel to the axis of

rotation. Unlike fixed-angle and swinging bucket rotors,
particles do not have to travel far before reaching their
isopycnic position.
• Characteristics:
✓ Tubes remain vertical throughout centrifugation.
✓ Particles move directly outward and separate quickly.
✓ Short sedimentation path improves resolution.
Applications:
• Ultracentrifugation of macromolecules (DNA, RNA,
lipoproteins).
• Used for isopycnic separations (e.g., cesium chloride gradients
for DNA isolation).
Advantages:
• Fastest separation among all rotors.
• Reduces run time due to short sedimentation path.
Disadvantages:
• Pellets may be difficult to recover as they form along the tube
walls.
• Limited to gradient-based separations, not suitable for pelleting
applications.
4. Zonal Rotor (Continuous Flow Rotor)
• A zonal rotor is designed for high-throughput
applications, where a large volume of sample
needs to be processed.
• Characteristics:
✓ A large cylindrical chamber replaces
individual tubes.
✓ Sample and gradient are loaded into the
chamber before spinning.
Applications:
• Large-scale virus purification.
• Used for mammalian cell fractionation.
• Ideal for industrial or research-scale protein purification.
Advantages:
• Handles large sample volumes efficiently.
• Reduces tube-based limitations in fixed-angle and swinging bucket
rotors.
Disadvantages:
• Requires specialized equipment.
• Not as commonly used in routine laboratory applications.
 Preparative Centrifugation
• Preparative centrifugation is primarily used for
isolating and purifying particles from complex
mixtures.

• It enables the separation of components based

on their size and density, allowing researchers
to obtain purified fractions for further analysis.
Instrumentation:

1. Centrifuge Machine:

o A device that generates centrifugal force by

spinning samples at high speeds.

o Equipped with a motor, temperature control

system, vacuum system (for ultracentrifuges),
and safety features to prevent rotor
imbalance.
2. Rotors:
o Fixed-angle rotors – Used for pelleting particles, such
as cells and organelles.
o Swinging bucket rotors – Used for density gradient
centrifugation to separate different layers of biological
samples.
o Continuous flow rotors – Used in large-scale
separation applications.
3. Tubes & Bottles:
o Made of durable materials like polycarbonate,
polypropylene, or stainless steel to withstand high
centrifugal forces.
o Designed with caps and sealants to prevent leakage.
Types of Preparative Centrifugation
1. Differential Centrifugation:
o Separation based on sedimentation rates.
o Larger, denser particles sediment first, while smaller
particles remain in the supernatant.
o Used for cell fractionation, organelle isolation (e.g.,
nuclei, mitochondria, lysosomes).
2. Density Gradient Centrifugation:
o Utilizes a density gradient medium (e.g., sucrose or
cesium chloride) to separate particles based on their
buoyant density.
 Applications of Preparative
Centrifugation
• Cell Fractionation:
o Separation of different organelles (nuclei, mitochondria,
ribosomes).
• Protein Purification:
o Isolation of specific proteins using ultracentrifugation and density
gradient methods.
• Virus and Bacteria Isolation:
o Concentration of viruses for vaccine production.
• Blood Component Separation:
o Used in blood banks to separate plasma, platelets, and red blood
cells.
• Industrial & Pharmaceutical Applications:
o Purification of antibiotics, enzymes, and vaccines.
 Analytical Centrifugation

• Analytical centrifugation is used to study the

properties of macromolecules and particles, such
as size, shape, density, and molecular
interactions.

• It provides quantitative data that help in

understanding the behaviour of biological
macromolecules.
Instrumentation:
• Analytical Ultracentrifuge (AUC):

o A specialized centrifuge with high-speed

capabilities (up to 500,000g) and an optical
detection system to monitor sedimentation in real
time.

o Equipped with a vacuum system to reduce air

resistance and improve rotor stability.
Instrumentation:

• Optical Detection Systems:

o Absorption Optics – Measures UV-visible light absorption of

biomolecules like proteins and nucleic acids.

o Interference Optics – Measures changes in refractive index

due to particle sedimentation.

o Fluorescence Optics – Used for highly sensitive detection of

fluorescently labelled macromolecules.

• Sample Cells:

o Made of quartz or sapphire to allow light transmission for

optical measurements.
 Types of Analytical Centrifugation

1. Sedimentation Velocity (SV) Analysis:

o Measures how fast a molecule moves under
centrifugal force.
o Provides information about molecular shape, size, and
interaction dynamics.
2. Sedimentation Equilibrium (SE) Analysis:
o Measures the equilibrium distribution of molecules at
different radial positions.
o Used to determine molecular weight and self-
association of proteins.
Applications of Analytical Centrifugation:
• Determination of Molecular Weight:
o Used to study protein oligomerization and complex
formation.
• Protein-Protein and Protein-DNA Interactions:
o Provides insights into macromolecular binding and
association.
• Characterization of Nanoparticles & Polymers:
o Used in material science to determine nanoparticle
stability and size distribution.
• Pharmaceutical Research:
o Studies drug-protein binding and aggregation.
 Differences Between Preparative
and Analytical Centrifugation
Preparative Analytical
Feature
Centrifugation Centrifugation

Studying Physical
Purpose Separation & Purification
Properties
Low to High (up to Very High (up to
Speed
100,000g) 500,000g)
Fixed-angle, Swinging Analytical Rotor with
Rotor Type
bucket, Continuous Optical Detection
No direct optical UV, Interference,
Detection Method
detection Fluorescence
Output Isolated Fractions Sedimentation Data
Cell biology, Biophysics, Molecular
Applications
Biochemistry, Medicine Biology, Polymer Science
• Centrifugation is a critical technique in research and
industry.

• Preparative centrifugation is essential for separating and

purifying biomolecules, while analytical centrifugation
provides in-depth analysis of molecular properties.

• The choice between the two depends on the research

objective whether isolating biological components or
analysing their physical characteristics.

• As technology advances, new methods such as ultra-fast

centrifugation and automated data analysis continue to
enhance the accuracy and efficiency of centrifugation
techniques.
 Ultracentrifugation
• Ultracentrifugation is a high-speed centrifugation
technique used for the separation and analysis of
biomolecules, subcellular organelles, viruses, and
nanoparticles.

• It operates at very high centrifugal forces (up to

1,000,000g) and is commonly used in molecular
biology, biochemistry, and nanotechnology.
Principle of Ultracentrifugation:

• Relies on sedimentation of particles under

centrifugal force.

• The rate of sedimentation depends on particle

size, shape, density, and medium viscosity.

• It allows both preparative (separation and

purification) and analytical (molecular
characterization) applications.
 Types of Ultracentrifugation
Ultracentrifugation is categorized into Preparative
Ultracentrifugation and Analytical Ultracentrifugation based
on the purpose of the study.
A. Preparative Ultracentrifugation
Used for isolating and purifying biological molecules or
organelles from complex mixtures.
Methods of Preparative Ultracentrifugation:
1. Differential Ultracentrifugation
2. Density Gradient Ultracentrifugation
o Rate-Zonal Centrifugation
o Isopycnic (Equilibrium) Centrifugation
 Differential Ultracentrifugation

Principle:

• Particles are separated based on differences in

sedimentation rate under high-speed
centrifugation.

• Larger and denser particles sediment faster,

forming a pellet at the bottom.
Process:
1. A sample is placed in a centrifuge tube and
subjected to increasing centrifugal forces.
2. The centrifugation steps occur in a sequential
manner:
o Low speed: Removes large debris (e.g., whole
cells, nuclei).
o Medium speed: Separates mitochondria,
lysosomes.
o High speed: Pellets ribosomes, microsomes.
The final supernatant contains the smallest
particles (e.g., soluble proteins, viruses).
Applications:

• Isolation of subcellular organelles (nucleus, mitochondria,

ribosomes).

• Separation of different cell components for biochemical

analysis.

• Purification of viruses and bacterial components.

Limitations:

• Less precise as it does not separate particles of similar size

and density.

• Requires multiple centrifugation steps for complete

fractionation.
Density Gradient Ultracentrifugation

This method uses a density gradient medium to

separate particles based on their size, shape, and
buoyant density.

Types of Density Gradient Ultracentrifugation:

1. Rate-Zonal (Velocity) Centrifugation

2. Isopycnic (Equilibrium) Centrifugation

Rate-Zonal Centrifugation (Velocity Sedimentation)

Principle:

• Separation is based on particle size and mass in a

density gradient.

• Heavier particles sediment faster than lighter

ones.

• The gradient prevents convective mixing and

provides stability.
Process:

1. A pre-formed gradient (e.g., sucrose) is prepared in

the centrifuge tube.

2. The sample is layered on top of the gradient.

3. Centrifugation is performed at moderate speeds for

a specific time.

4. Particles separate into distinct bands based on their

sedimentation rate.

5. Fractions are collected from the bottom or by

puncturing the tube.
Applications:

• Separation of macromolecules like proteins,

ribosomes, and viruses.

• Isolation of different species of RNA and DNA.

• Purification of organelles from cell lysates.

Limitations:

• Requires careful gradient preparation.

• Overcentrifugation can lead to sample

contamination or mixing.
Isopycnic (Equilibrium) Centrifugation

Principle:

• Separation occurs based on buoyant density, not

size.

• Particles migrate until they reach a position where

their density equals that of the surrounding
gradient.
Process:

1. A self-forming gradient (e.g., cesium chloride or

Percoll) is created in the tube.

2. The sample is mixed with the gradient solution.

3. Centrifugation is conducted at high speeds for

extended periods (e.g., 24 hours).

4. Particles move through the gradient until they reach

their equilibrium position.
Applications:

• Separation of DNA isoforms (e.g., supercoiled vs.

linear DNA).

• Purification of viruses and subcellular organelles.

• Density-based separation of cell membranes.

Limitations:

• Time-consuming (long ultracentrifugation runs).

• Requires careful selection of gradient material.

Analytical Ultracentrifugation (AUC)

Used for quantitative analysis of macromolecules like

proteins, nucleic acids, and polymers.

Principle:

• Uses high-speed centrifugation with real-time

optical detection.

• Measures sedimentation velocity and equilibrium

properties to analyse molecular interactions and
size.
Instrumentation:

• Ultracentrifuge with optical detection

system (UV absorbance, interference
optics).

• Sample cells made of quartz or sapphire.

• Rotors designed for small-volume

samples.
Methods of Analytical Ultracentrifugation:

1. Sedimentation Velocity (SV) Analysis

o Determines molecular shape, size, and interactions.

o Measures how fast a molecule sediments under

centrifugal force.

2. Sedimentation Equilibrium (SE) Analysis

o Determines molecular weight without needing

external standards.

o Used for studying protein self-association and

macromolecular interactions.
Applications:

• Determination of molecular weight and shape

of proteins and nucleic acids.

• Analysis of protein-protein and protein-DNA

interactions.

• Characterization of nanoparticles, liposomes,

and synthetic polymers.
 Comparison of Ultracentrifugation
Methods
Feature Differential Rate-Zonal Isopycnic Analytical
Separation Buoyant Sedimentation
Size & DensitySize & Mass
Based On Density Behavior
Gradient
None Pre-formed Self-forming No Gradient
Used
Speed Range Low to High Moderate Very High Very High
Cell Proteins, DNA, Molecular
Application
Organelles Viruses Membranes Analysis
Precision Moderate High Very High Extremely High
Advantages:

• High-resolution separation of macromolecules.

• Allows both preparative (isolation) and analytical (study

of properties) applications.

• Essential for molecular biology, virology, and structural

biochemistry.

Limitations:

• Expensive equipment and maintenance.

• Requires careful handling and calibration.

• Long centrifugation times in some methods.

• Ultracentrifugation is a powerful technique for
separating and analysing biomolecules.

• Preparative ultracentrifugation is widely used in

cell fractionation and molecular purification, while
analytical ultracentrifugation provides detailed
insights into molecular weight and interactions.

• Advances in automation and real-time monitoring

continue to enhance the efficiency and accuracy of
ultracentrifugation methods in modern research.
THANK YOU