2. 2
Principles and methods of
Differential, Density Gradient
Centrifugation, and Ultracentrifugation
Definition of Centrifugation
Centrifugation is a process that involves the use of a centrifuge to separate particles from
a solution based on their size, shape, density, or other physical properties. A centrifuge is
a device that rotates a sample at high speeds to generate a centripetal force, which causes
the heavier particles to sediment to the bottom of a tube or rotor while the lighter particles
remain suspended.
There are several types of centrifuges, including high-speed centrifuges, low-speed centrifuges,
and ultracentrifuges. High-speed centrifuges are used to separate cells and other biological
materials, while low-speed centrifuges are used to separate non-biological materials such as
suspensions of crystals or inks. Ultracentrifuges are used to separate very small particles or to
measure the size, shape, and density of particles.
Centrifugation is a widely used technique in research, industry, and clinical laboratories, and it
has many applications, including the purification of biological samples, the separation of cells
and organelles, the analysis of proteins and nucleic acids, and the preparation of diagnostic
specimens.
Centrifuge definition
A centrifuge is a device that uses centrifugal force to separate particles from a solution
based on their size, shape, density, or other physical properties. It consists of a motor, a
rotating head or rotor, and a series of tubes or cups that hold the sample. When the rotor
is rotated at high speeds, the centripetal force generated causes the heavier particles to
sediment to the bottom of the tubes or cups while the lighter particles remain suspended.
There are several types of centrifuges, including high-speed centrifuges, low-speed centrifuges,
and ultracentrifuges. High-speed centrifuges are used to separate cells and other biological
3. 3
materials, while low-speed centrifuges are used to separate non-biological materials such as
suspensions of crystals or inks. Ultracentrifuges are used to separate very small particles or to
measure the size, shape, and density of particles.
Centrifuges are widely used in research, industry, and clinical laboratories, and they have many
applications, including the purification of biological samples, the separation of cells and
organelles, the analysis of proteins and nucleic acids, and the preparation of diagnostic
specimens.
What is Relative Centrifugal Force (RCF)?
Relative centrifugal force is a measurement for the rate of strength in rotors that are of
different sizes and types.
The force is that is exerted on the inside of the rotor by the force of the rotor’s rotation.
RCF is the force perpendicular to the surface applied to the sample, which is always in
relation to the gravity of the earth.
The RCF of different centrifuges is a good tool for analysis of rotors and allowing to select
the best centrifuge to fulfil a specific task.
The formula used to calculate the force of centrifugal relative (RCF) could be written as
follows:
RCF (g Force) = 1.118 × 10-5 × r × (RPM) ^2
Centrifugation Principle/how does centrifugation work?
Centrifugation takes advantage of these density differences to separate various particles in
a solution. Instead of relying on gravity, which may be relatively weak, centrifugation
employs the much stronger centrifugal force generated by a centrifuge. A centrifuge is a
specialized piece of equipment designed to rotate an object around a fixed axis, applying a
powerful outward force perpendicular to the axis of spin.
The centrifuge operates based on the sedimentation principle, wherein the centripetal
acceleration caused by the rotation of the centrifuge causes denser substances and particles
to move outward in the radial direction. Simultaneously, objects that are less dense are
displaced and move towards the centre.
4. 4
In a laboratory centrifuge equipped with sample tubes, the radial acceleration exerted on
the solution causes denser particles to settle at the bottom of the tube, forming a pellet,
while substances with lower density rise to the top. This separation allows researchers to
isolate and collect different components of the solution based on their relative densities.
By using centrifugation, scientists and researchers can separate and purify a wide range of
materials, including cells, organelles, proteins, nucleic acids, and other particles. It is a
fundamental technique employed in various fields such as biology, biochemistry,
chemistry, and clinical diagnostics, enabling efficient and precise separation and analysis
of complex mixtures.
Fig 1 Centrifugation process
A. Differential centrifugation
Differential centrifugation is a commonly used technique in cell biology and biochemistry to
separate cellular components based on their size, shape, and density. The principle behind
differential centrifugation involves subjecting a sample to a series of increasing centrifugal
forces, allowing different components to settle at different rates.
Principle of Differential centrifugation
Differential centrifugation operates based on the principle of differences in sedimentation rate,
size, and density of biological particles. Here are the key principles involved in the process:
5. 5
1. Sedimentation Rate: Differential centrifugation takes advantage of the fact that particles
of different sizes and densities settle at different rates under the influence of centrifugal
force. Larger and denser particles tend to settle more rapidly, while smaller and lighter
particles take longer to sediment.
2. Centrifugal Force: By applying increasing centrifugal forces during the centrifugation
process, particles experience greater sedimentation forces. The centrifugal force causes
particles to move outward from the centre of rotation, and the settling rate depends on the
force applied.
3. Sedimentation Steps: In differential centrifugation, a series of centrifugation steps is
employed, with each step involving different centrifugal forces and durations. Initially, at
lower forces, the larger and denser particles settle down, forming a pellet at the bottom of
the centrifuge tube. As the centrifugal force is increased, smaller particles with slower
sedimentation rates settle progressively.
4. Size and Density Differences: The settling behaviour of particles in differential
centrifugation depends on their size and density relative to the surrounding medium. Larger
and denser particles sediment more quickly and at lower centrifugal forces, while smaller
and less dense particles take longer and require higher forces to settle.
5. Pellet Formation: The differential sedimentation rates lead to the formation of distinct
pellets or sediment fractions at the bottom of the centrifuge tube. The pellet typically
contains the largest and densest particles, while the smaller-sized structures and lighter
particles remain in the supernatant.
6. Floating Particles: In some cases, particles that are less dense than the surrounding
medium may float instead of settling to the bottom. This can occur when the buoyant force
acting on the particles is greater than the sedimentation force. These floating particles can
be collected from the upper layers of the centrifuge tube.
By exploiting the differential sedimentation rates based on size and density, differential
centrifugation enables the separation and fractionation of biological particles into distinct
fractions. This technique has been widely used to isolate and study specific components, such
6. 6
as organelles, subcellular structures, and various cellular particles, contributing to our
understanding of cell biology and enabling further analysis and characterization of these
components
.
Fig 2 Differential Centrifugation Principles involved
Steps of Differential centrifugation
Differential centrifugation involves a series of steps to separate particles based on their size
and density differences. Here is an outline of the steps involved in the process:
1. Sample Homogenization: The sample solution, which may contain cells, organelles, or
other biological particles, is homogenized in a buffer medium. The buffer helps maintain
the stability and integrity of the sample during centrifugation.
2. Initial Centrifugation: The homogenized sample is placed in a centrifuge tube and
subjected to centrifugation at a specific centrifugal force, duration, and temperature. This
initial centrifugation step is designed to separate the largest and densest particles from the
rest of the sample.
3. Pellet Formation: As a result of the initial centrifugation, the larger and denser particles
sediment and form a pellet at the bottom of the centrifuge tube. The pellet contains the
separated particles, while the supernatant, which is the liquid portion above the pellet,
contains the remaining particles.
7. 7
4. Supernatant Transfer: The supernatant, which contains smaller particles and
components, is carefully transferred to a new centrifuge tube. This allows for further
separation and isolation of specific particles in subsequent steps.
5. Repeat Centrifugation: The transferred supernatant is subjected to another round of
centrifugation, but at a different centrifugal force, duration, and temperature. This step is
aimed at separating the next fraction of particles based on their size and density differences.
6. Pellet Collection: After each centrifugation step, a new pellet forms at the bottom of the
tube, and the supernatant is once again collected. This process is repeated multiple times,
with each subsequent centrifugation step separating particles of decreasing size and
density.
7. Particle Identification: Once all the desired separations have been performed, the
collected pellets and supernatant fractions can be further analysed. The separated particles
can be identified through various methods, such as biochemical assays, microscopy, or
specific staining techniques that target unique indicators or markers of the specific particles
of interest.
By systematically adjusting the centrifugal force, duration, and temperature in each step,
differential centrifugation allows for the separation and isolation of particles based on their size
and density. This technique has been widely used in biological research and diagnostics to
study organelles, subcellular components, and various cellular particles, providing valuable
insights into their structure, function, and composition.
Uses of Differential centrifugation
Differential centrifugation finds several applications in biological research and laboratory
settings. Here are some key uses of this technique:
1. Organelle and Membrane Separation: One of the primary applications of differential
centrifugation is the separation of cell organelles and cellular membranes. By adjusting the
centrifugal force and duration, different organelles such as mitochondria, lysosomes,
peroxisomes, and endoplasmic reticulum can be isolated. This allows for the study of their
structure, function, and biochemical properties.
8. 8
2. Nucleus Separation: While not providing high-resolution separation, differential
centrifugation can be employed for the low-resolution separation of the nucleus. This
allows for the isolation of nuclear components and the study of nuclear processes.
3. Purification of Extracts: Differential centrifugation is useful for the purification of
biological extracts containing larger-sized impurities. By removing the larger particles or
debris through successive centrifugation steps, the target molecules or components of
interest can be obtained in a more purified form.
4. Subcellular Fractionation: The technique enables the fractionation of complex mixtures
into distinct subcellular fractions based on their size and density differences. This facilitates
the study of specific cellular compartments and their associated functions.
5. Enzyme and Protein Studies: Differential centrifugation can be applied to separate enzymes
and proteins based on their subcellular localization. By isolating specific organelles or
subcellular fractions, researchers can investigate the distribution, activity, and regulation
of enzymes and proteins within different cellular compartments.
6. Diagnostic Applications: In clinical laboratories, differential centrifugation is used for the
separation and isolation of specific components in bodily fluids, such as blood or urine.
This enables the detection and analysis of disease-related markers, cell types, or infectious
agents.
B. Density gradient centrifugation
Density gradient centrifugation is a powerful technique used for the separation and
purification of molecules based on their density. It takes advantage of the principle that
molecules will migrate through a density gradient when subjected to centrifugal force, with
the rate of migration determined by their density.
The process of density gradient centrifugation involves creating a density gradient within
a centrifuge tube. This gradient is achieved by layering solutions of different densities,
typically in the form of a sucrose or caesium chloride gradient. The sample containing the
molecules to be separated is carefully layered on top of the density gradient.
9. 9
When the tube is placed in the centrifuge and spun at high speeds, the molecules in the
sample experience a centrifugal force that drives them through the density gradient. As the
molecules migrate, they encounter regions of the gradient with increasing or decreasing
density, depending on their own density. Consequently, molecules of different densities
will reach different positions in the tube and form distinct bands or zones.
The separation is based on the principle that molecules with higher density will sediment
or move towards the bottom of the tube faster than molecules with lower density. As a
result, molecules of different densities will eventually form separate bands or layers along
the length of the gradient.
After centrifugation, the tube is carefully removed from the centrifuge, and the distinct
bands or layers are visualized and collected. Each band represents a specific population of
molecules with similar densities. This technique allows for the purification and isolation
of molecules with specific density characteristics.
Density gradient centrifugation has a wide range of applications in various fields of
research. It can be used to separate and purify different biomolecules, such as proteins,
nucleic acids, lipids, and subcellular organelles, based on their densities. It is also utilized
for virus purification and isolation, as viruses often have distinct densities that can be
exploited for separation.
Furthermore, density gradient centrifugation is employed in studies of molecular biology,
biochemistry, and biophysics to investigate the structure and composition of
macromolecular complexes. By analysing the distribution of molecules along the density
gradient, researchers can gain insights into their composition, interactions, and molecular
characteristics.
In summary, density gradient centrifugation is a technique that exploits the density
differences of molecules to achieve their separation. By creating a density gradient and
subjecting the sample to centrifugal force, molecules migrate through the gradient and form
distinct bands or layers based on their densities. This technique is widely used for
10. 10
purification, isolation, and characterization of biomolecules, as well as in the study of
molecular complexes and virus particles.
Fig 3 Schematic representative of gradient density ultracentrifugation-based exosome
isolation
Principle of Density gradient centrifugation
The principle of density gradient centrifugation is based on the behaviour of molecules under
the influence of centrifugal force and their interaction with a density gradient medium. The key
aspects of this principle are as follows:
1. Sedimentation under Centrifugal Force: When a sample containing molecules is
subjected to centrifugal force in a centrifuge, the molecules experience a sedimentation
force that causes them to move through the sample solution. This sedimentation occurs as
the molecules attempt to reach a region with a density matching their own.
2. Density Gradient Medium: To facilitate separation based on density, a density gradient
medium is employed. This medium can have either decreasing or increasing density along
its length. Typically, sucrose or caesium chloride solutions are used to create the density
gradient. The gradient is formed by layering solutions of different densities, and the sample
is placed on top of the gradient.
3. Migration through the Density Gradient: As the sample is subjected to centrifugal force,
the molecules within it start migrating through the density gradient. The rate of migration
11. 11
is determined by the density of the molecules relative to the density of the gradient at each
point.
4. Separation based on Density: Molecules with higher density than the surrounding
medium will gradually settle towards the bottom of the tube or container as they move
through the density gradient. As they reach a point where their density matches that of the
surrounding medium, they become suspended or evenly distributed at that position.
Molecules with lower density will remain suspended at higher levels within the density
gradient.
5. Layer Formation and Recovery: The separation process results in the formation of
distinct layers or bands along the length of the density gradient. Each layer contains
molecules with a similar density. These layers can be carefully recovered by various
methods such as fraction collection or gradient fractionation techniques.
Density gradient centrifugation allows for the separation and purification of molecules based
on their density. By exploiting the principle that molecules will settle to a region with a
matching density, different molecules can be separated into distinct layers or bands within the
density gradient. This technique is widely used in various fields, including biochemistry,
molecular biology, and cell biology, for the isolation of biomolecules, purification of
subcellular components, and characterization of macromolecular complexes.
Steps of Density gradient centrifugation
Density gradient centrifugation involves several steps to separate particles based on their
density. Here are the key steps involved in density gradient centrifugation:
1. Preparation of Density Gradient: A density gradient is created by layering a medium
with varying concentrations of a solute in a centrifuge tube. The lower concentration
solution is gently layered over the higher concentration solution to form a gradient.
Common density gradient media include sucrose or caesium chloride solutions.
2. Sample Application: The sample containing the particles to be separated is carefully
applied on top of the density gradient. This can be done using a pipette or other suitable
methods to ensure the sample is layered without disturbing the gradient.
12. 12
3. Ultracentrifugation: The tubes containing the density gradient and sample are placed in
an ultracentrifuge. The centrifuge is operated at high speeds to generate a strong centrifugal
force. This force causes the particles in the sample to migrate through the density gradient.
4. Particle Migration: As the centrifuge spins, the particles in the sample begin to migrate
through the density gradient. The rate of migration is determined by the relative density of
the particles compared to the surrounding medium. Heavier particles will settle towards the
bottom of the tube, while lighter particles will remain suspended at higher levels within the
gradient.
5. Equilibrium and Fractionation: Over time, the particles reach a point in the density
gradient where their density matches that of the surrounding medium. At this point, they
become evenly distributed or suspended within the gradient. This state of equilibrium
allows for the separation of particles based on their density.
6. Fraction Collection: After the equilibrium is reached, the centrifuge is stopped, and the
tubes are carefully removed. The tubes are then fractionated by collecting fractions from
different levels of the density gradient. Each fraction contains particles with a specific
density.
7. Particle Isolation: The collected fractions are processed further to isolate the particles of
interest. Depending on the nature of the particles and the experimental requirements,
various techniques such as centrifugation, filtration, or chromatography can be employed
to isolate the particles as individual units.
By following these steps, density gradient centrifugation enables the separation and isolation
of particles based on their density. This technique is widely used in various fields, including
biochemistry, cell biology, and molecular biology, for the purification of biomolecules,
separation of subcellular components, and isolation of specific particles from complex
mixtures.
13. 13
Uses of Density gradient centrifugation
Density gradient centrifugation is a versatile technique with a wide range of applications in
various fields of research. Here are some key uses of density gradient centrifugation:
1. Purification of Biomolecules: Density gradient centrifugation is commonly employed for
the purification of biomolecules, such as proteins, nucleic acids, and subcellular organelles.
By exploiting the differences in density, the technique enables the separation of these
biomolecules from impurities or contaminants. It is particularly useful for purifying large
volumes of biomolecules efficiently and effectively.
2. Virus Purification and Study: Density gradient centrifugation is extensively used in
virology for the purification of different types of viruses. Viruses often have specific
densities, and by using density gradient centrifugation, researchers can separate and isolate
viruses from other components in a sample. This purification step is crucial for further
studies on virus structure, composition, and function.
3. Separation of Particles: Density gradient centrifugation serves as a powerful separation
technique for particles with different densities. It enables the separation of particles based
on their density differences, allowing for the isolation of specific particles from complex
mixtures. This can be applied to separate various types of particles, such as cellular
components, subcellular organelles, and biological macromolecules.
4. Determination of Particle Densities: In addition to its separation capabilities, density
gradient centrifugation can also be used to determine the densities of particles. By creating
a density gradient and analysing the positions at which particles equilibrate within the
gradient, their density can be estimated.
This information is valuable for characterizing particles, assessing their composition, and
understanding their behaviour in different environments.
5. Study of Particle Interactions: Density gradient centrifugation can be utilized to
investigate particle interactions. By subjecting particles to density gradient centrifugation
under different conditions or in the presence of specific ligands or substrates, researchers
14. 14
can observe changes in their distribution along the gradient. This provides insights into the
nature of particle interactions, including molecular recognition, complex formation, and
binding affinities.
Examples of Density gradient centrifugation
Density gradient centrifugation has been employed in several notable experiments and
applications. Here are a couple of examples:
1. Meselson-Stahl Experiment on DNA Replication: Density gradient centrifugation
played a crucial role in the landmark Meselson-Stahl experiment, which provided evidence
for the semi-conservative nature of DNA replication. In this experiment, different isotopes
of nitrogen were used to label DNA. By subjecting the DNA samples to density gradient
centrifugation, it was possible to separate DNA molecules based on their density. The
experiment demonstrated that after one round of DNA replication in the presence of heavy
nitrogen (15N), the DNA molecules formed a hybrid band with intermediate density. This
result supported the semi-conservative model, where each newly synthesized DNA strand
contains one original strand and one newly synthesized strand.
2. Isolation of Microsomal Fraction and Membrane Vesicles: Density gradient
centrifugation is widely used in cell biology to isolate specific cellular components and
organelles. One example is the isolation of the microsomal fraction from muscle
homogenates. Microsomes are small vesicles derived from the endoplasmic reticulum (ER)
and contain membrane-bound proteins and other ER components. By subjecting the muscle
homogenate to density gradient centrifugation, the microsomes can be separated based on
their density. Subsequently, the membrane vesicles within the microsomal fraction can be
further separated based on their differing densities, allowing for the isolation of specific
membrane components for further analysis and study.
Advantages Density gradient centrifugation
1. High resolution: Density gradient centrifugation allows for the separation of molecules
with a high degree of resolution, making it useful for the purification of specific molecules
or organelles.
15. 15
2. High efficiency: Density gradient centrifugation is highly efficient at separating molecules
based on their size, shape, and density, allowing for the separation of large quantities of
sample in a short amount of time.
3. Gentle on samples: Density gradient centrifugation is relatively gentle on samples, as it
does not rely on physical forces such as filtration or sedimentation.
4. Versatility: Density gradient centrifugation can be used to separate a wide range of
molecules, including proteins, nucleic acids, cells, and organelles.
Disadvantages Density gradient centrifugation
1. Complexity: Density gradient centrifugation requires the preparation of a gradient, which
can be time-consuming and requires specialized equipment and expertise.
2. Limited to separating molecules based on density: Density gradient centrifugation is
limited to separating molecules based on their density, size, and shape, and it may not be
suitable for separating molecules based on other physical properties.
3. Limited to separating molecules with a narrow size range: Density gradient
centrifugation is most effective at separating molecules with a narrow size range, and it
may not be suitable for separating molecules with a wide size range.
4. Expense: Density gradient centrifugation requires specialized equipment and
consumables, which can be expensive.
16. 16
C. Ultracentrifugation
Ultracentrifugation is a specialized technique used to spin samples at exceptionally high
speeds. Current ultracentrifuges can spin to as much as 150 000 rotations per minute (rpm)
(equivalent to 1 000 000 g). However, extreme centrifugal forces may cause overheating,
so to avoid sample damage, ultracentrifuges are equipped with vacuum systems that keep
a constant temperature in the centrifuge’s rotor. Centrifugation, and ultracentrifugation, is
nowadays, at the core of the laboratory routine. Bench top centrifuges are essential devices
in any biology or chemistry laboratory, and they are used on a day-to-day basis in a wide
range of experimental protocols, from concentrating solutions to isolating cells and sub
cellular components. Ultracentrifugation widened the applications of bench top
centrifugation, allowing the isolation of smaller sized particles, and the study of purified
molecules and molecular complexes. In biology, the development of ultracentrifugation in
the early 1900s, widened the possibilities of scientific research to the subcellular level,
allowing for the differential separation of cellular components, such as organelles, lipid
membranes, and even to purify proteins and ribonucleic acids (DNA and RNA).
Principle of Ultracentrifugation
The basis of ultracentrifugation is the same as normal centrifugation: to separate the
components of a solution based on their size and density, and the density (viscosity) of the
medium (solvent). A general principle, (ultra)centrifugation abides by the following rules:
The denser a biological structure is, the faster it sediments in a centrifugal field.
The more massive a biological particle is, the faster it moves in a centrifugal field.
17. 17
The denser the biological buffer system is, the slower the particle moves in a
centrifugal field.
The greater the frictional coefficient (i.e., the friction between the component and
the neighbouring environment) is, the slower a particle moves
The greater the centrifugal force is, the faster the particle sediments
The sedimentation rate of a given particle will be zero when the density of the
particle and the surrounding medium is equal.
Types of Ultracentrifugation
Analytical ultracentrifuges: are equipped with optical detection systems that allow the
researcher to follow the centrifugation process in real-time. These systems may use
ultraviolet (UV) light absorption or refracting index interference (RII) optical detection
systems (ultracentrifuges may be equipped with one or both types of optical systems).
While UV detection directly measures the absorbance (abs) of a substance at a specific
wavelength, RII measures changes in the refraction index (radiation direction) of a given
substance, compared to the solvent it is dissolved in. The purpose of analytical
centrifugation is different from other types of centrifugation. Although component
isolation is possible with analytical centrifugation, the goal of this technique is to obtain
data to characterize the sample that is spun (sedimentation velocity, viscosity,
concentration, etc.). With analytical centrifugation, it is possible to follow the variations
in sample concentration as a function of the applied centrifugal force. This technique is
used in two main experimental settings: sedimentation velocity and sedimentation
equilibrium studies, which are key in macromolecular characterization.
Preparative ultracentrifuges: are mostly used to process biological samples for further
analysis. The most common application of preparative ultracentrifugation is in tissue and
sub cellular fractionation, to isolate increasingly smaller components of the biological
samples. For that, two main centrifugation methods are used: differential and density-
gradient centrifugation
