CENTRIFUGATION

Centrifugation is a commonly used method in downstream processing for separating particles

based on their size and density. It works by applying centrifugal force, which is a force that
pushes objects outward from the centre of rotation. This force causes denser particles to settle
at the bottom of the centrifuge tube, while less dense particles remain suspended in the liquid
phase.

Centrifugation is a separation technique that uses centrifugal force to settle particles of

different sizes and densities at the bottom of a tube. It is a versatile technique that is used in a
wide variety of applications, including biotechnology, pharmaceuticals, and food science.

Density difference between the particles and the surrounding medium is a key factor in
centrifugation. Denser particles will settle faster than less dense particles. Particle size also
affects the rate of sedimentation. Larger particles will settle faster than smaller particles.
Liquid viscosity also affects the rate of sedimentation. More viscous liquids will slow down
the sedimentation of particles.

There are two main types of centrifugation: preparative centrifugation and analytical
centrifugation. Preparative centrifugation is used to isolate or purify a particular component
from a mixture. Analytical centrifugation is used to measure the size or density of particles in
a mixture.

Steps in centrifugation:

 Sample preparation: The sample is prepared by mixing it with a solvent that will not
interact with the particles to be separated.
 Loading the centrifuge: The sample is loaded into the centrifuge tube and the rotor is
balanced.
 Centrifugation: The rotor is spun at a high speed, creating centrifugal force. This
force causes the denser particles to settle at the bottom of the tube.
 Collection: The supernatant, which is the liquid phase containing the less dense
particles, is carefully removed. The pellet, which is the solid phase containing the
denser particles, is collected for further analysis or purification.
 DOWNSTREAM CENTRIFUGATION
Downstream processing is the series of steps following cell cultivation and harvesting to
isolate and purify the desired product, which could be proteins, biomolecules, or even cells
themselves. It involves a multitude of techniques, including cell harvesting, cell debris
removal, product purification, and clarification, each playing a crucial role in achieving the
final goal.

Cell Harvesting:

Purpose: To isolate and collect the desired cells from the culture medium.

Importance: Ensures a starting material for further downstream processing steps.

Steps:

Preparation: Monitoring cell culture, selecting harvesting media, and preparing equipment.

Detachment: Enzymatic or non-enzymatic methods for adherent cells, simple collection for
suspension cells.

Centrifugation: Pelleting cells by centrifugation.

Cell washing: Removing residual media components by washing with buffer.

Cell counting and viability assessment: Determining cell yield and viability.

Cell resuspension: Suspending cells in suitable buffer for downstream processing.

Cryopreservation (optional): Long-term storage of cells for future use.

Cell Debris Removal:

Purpose: To eliminate unwanted dead cells, cellular components, and other contaminants
from the harvested cell suspension.

Importance: Improves product purity and prevents unwanted reactions during downstream
processing.

Methods:

Centrifugation: Separating cell debris based on their different sedimentation rates.

Filtration: Removing debris using filters with appropriate pore size.

Density gradient centrifugation: Separating cells and debris based on their densities.

Immunomagnetic separation: Removing debris using magnetic beads coated with specific
antibodies.
Product Purification:

Purpose: To isolate and concentrate the desired product, such as proteins or biomolecules,
from the remaining components of the cell lysate or fermentation broth.

Importance: Ensures high purity of the final product for its intended application.

Methods:

Precipitation: Utilizing changes in temperature, pH, or precipitation agents to induce product

precipitation.

Chromatography: Separating molecules based on their size, charge, affinity, or other

properties using various techniques like affinity chromatography or ion-exchange
chromatography.

Extraction: Using solvents to selectively extract the desired product from the mixture.

Dialysis: Removing unwanted small molecules from the product solution using a semi-
permeable membrane.

Clarification:

Purpose: To remove insoluble particles, such as cell debris, protein aggregates, and other
contaminants, from the product solution.

Importance: Improves product clarity and reduces potential issues during further downstream
processing or final product formulation.

Methods:

Centrifugation: Pelleting insoluble particles by high-speed centrifugation.

Filtration: Using filters with appropriate pore sizes to remove particles.

Depth filtration: Utilizing deep filter beds to remove particles and clarify the solution.

Flocculation and sedimentation: Adding flocculating agents to aggregate particles, followed

by sedimentation and removal.

It's important to note that the specific methods used for each step, particularly in cell debris
removal, product purification, and clarification, will vary depending on the type of product
being isolated, its properties, and the overall downstream processing strategy. The chosen
methods should ensure high yield, purity, and recovery of the desired product while
maintaining its integrity and functionality.
 WORKINGPRINCIPLE

The working principle of centrifugation is based on the difference in sedimentation rate of

particles with different sizes and densities. When a mixture is placed in a centrifuge tube and
the rotor is spun at high speed, centrifugal force is generated. This force pushes the denser
particles outward from the centre of the rotor, causing them to settle at the bottom of the tube.
Less dense particles, on the other hand, remain suspended in the liquid phase.
The strength of this centrifugal force is measured in relative centrifugal force (RCF), which is
a multiple of Earth's gravitational force (g). Higher RCF values translate to faster
sedimentation and more efficient separation of particles. In simpler terms, spinning the
sample creates a force that pushes denser particles towards the bottom of the container,
allowing them to be separated from the less dense liquid and other particles.

Types of Centrifuges

Centrifuges are categorized into two main types based on their design and operation:

Discrete centrifuges: These centrifuges have a fixed bowl that is filled with the sample
liquid. The bowl is then spun at a high speed, causing the particles to settle to the bottom.
Discrete centrifuges are typically used for batch processing and are well-suited for separating
larger particles.

Continuous centrifuges: These centrifuges have a rotating bowl that continuously feeds the
liquid mixture into the bowl and discharges the separated solids. Continuous centrifuges are
more efficient for processing large volumes of liquid and are well-suited for separating
smaller particles.
 TUBULAR BOWL CENTRIFUGE

The tubular bowl centrifuge is a high-speed centrifuge characterized by its elongated bowl
with a high length-to-diameter ratio (typically around 4.8). This design allows it to achieve
high rotational speeds, often reaching 15,000 rpm, leading to efficient separation of particles
from liquids.

Here are some features and applications of the tubular bowl centrifuge:

High Speed: As mentioned, the high rotational speed generates a powerful centrifugal force,
effectively separating particles.

Long Residence Time: The elongated bowl provides a longer path for particles to sediment,
further enhancing separation efficiency.

Continuous or Batch Operation: Tubular bowl centrifuges can be operated in both batch
and continuous modes, offering flexibility for different processing needs.

Compact Design: The compact design makes them suitable for space-constrained
environments.

Applications:

Emulsion Separation: The high centrifugal force and long residence time make tubular bowl
centrifuges ideal for separating emulsions, such as oil-water mixtures.

Solid-Liquid Separation: They are effective in separating solids from liquids, even when the
solid content is small.

Clarification: Tubular bowl centrifuges can clarify liquids by removing suspended particles
and impurities.

Concentration: They can be used to concentrate solids from liquids, such as in protein
purification processes.

High Separation Efficiency: Tubular bowl centrifuges provide high purity and yield due to
their efficient separation capabilities.

Low Shear Stress: The design minimizes shear stress on the separated components, which is
crucial for sensitive materials like biological products.

Ease of Operation: They are relatively easy to operate and maintain, making them suitable for
various industrial applications.
 MULTI CHAMBER CENTRIFUGE

The multi-chamber centrifuge is a modification of the tubular bowl centrifuge designed for
enhanced separation performance. It consists of several chambers arranged in a way that
directs the feed flow in a zigzag fashion, maximizing the separation efficiency.

Multiple chambers are arranged in series within an elongated bowl.

Each chamber is separated by a weir or dam that controls the flow of the feed.

The feed enters the inner chamber and progresses through the chambers in a zigzag pattern,
increasing the path length and residence time.

Separation Mechanism:

The centrifugal force varies within the different chambers due to their varying radii. The
outer chambers experience the highest centrifugal force, while the inner chambers experience
the lowest force. This creates a gradient of centrifugal force that effectively separates
particles based on their size and density. Larger and denser particles experience a greater
force and settle down faster, accumulating in the outer chambers. Smaller and less dense
particles remain suspended longer and exit the centrifuge in the later chambers.

Particle Size Range:

Multi-chamber centrifuges are suitable for separating particles in the range of 0.1 to 200
micrometres in diameter.

This makes them ideal for a wide range of applications, including cell separation, protein
purification, and wastewater treatment.

Advantages:

Flexibility: The ability to customize the number and size of chambers allows for tailoring the
centrifuge to specific separation requirements.

Reduced Shear Stress: The zigzag flow path minimizes shear forces on the separated
components, making it suitable for sensitive materials.

Applications:

Cell Harvesting and Separation: Isolating specific cell populations from fermentation broths
or cell cultures.

Protein Purification: Removing impurities and contaminants from protein solutions.

Wastewater Treatment: Separating suspended solids and clarifying water.

 DISC CENTRIFUGE

The disc centrifuge is another powerful separation tool that utilizes a unique design for
efficient particle separation. Here's a detailed breakdown of its structure and operation:

Structure:

The core component is a stack of conical metal sheets called discs, arranged with small
clearances between them. The size of each disc is typically around 0.3mm, creating numerous
thin layers within the centrifuge bowl. The bowl itself is also conical in shape and rotates at
high speeds.

Operation:

Feed Introduction: The slurry or liquid mixture is fed into the centrifuge through a central
tube.

Centrifugal Force: As the bowl rotates, a powerful centrifugal force is generated.

Thin Layer Formation: The force distributes the feed into thin layers between the discs,
maximizing the surface area exposed to centrifugal force.

Separation: Denser particles, such as solids, sediment towards the lower surface of the discs
due to the centrifugal force.

Clarified Liquid: The less dense liquid, known as the clarified liquid, flows upwards between
the discs and exits through an outlet at the top of the bowl.

Solids Collection: The accumulated solids are discharged periodically through a separate
outlet at the bottom of the bowl.

Advantages:

High Separation Efficiency: The thin layer formation and large surface area exposed to
centrifugal force maximize separation efficiency, leading to high purity and yield.

Wide Particle Size Range: They can handle a wide range of particle sizes, from submicron
particles to large solids.

Continuous Operation: Disc centrifuges operate in a continuous mode, allowing for

continuous processing of large volumes of material.

Self-Cleaning: The design allows for a certain degree of self-cleaning, as the solids are
continuously discharged from the bottom of the bowl, minimizing downtime for cleaning.
 SCROLL CENTRIFUGE OR DECANTER

The scroll centrifuge, also known as a decanter, is a versatile and efficient centrifuge
designed for separating liquids and solids, particularly when dealing with high solid
concentrations. Here's a detailed explanation of its features and operation:

Horizontal Bowl: The centrifuge consists of a horizontal bowl that rotates at high speed. The
bowl is typically cylindrical but tapers at one end.

Conveyor Scroll: A screw-shaped conveyor, known as the scroll, runs along the inner wall of
the bowl.

Feeding and Discharge Ports: The feed mixture enters the bowl through a central inlet, while
the separated liquids and solids are discharged through separate outlets.

Operation:

Feed Introduction: The feed mixture, containing both liquid and solid components, is fed into
the rotating bowl through the central inlet.

Centrifugal Force: As the bowl rotates, the centrifugal force throws the heavier solids towards
the inner wall of the bowl.

Solids Deposition: The deposited solids are continuously pushed towards the tapered end of
the bowl by the rotating scroll.

Liquid Separation: The lighter liquid phase, known as the clarified liquid, flows towards the
center of the bowl and exits through an outlet near the feed inlet.

Solids Discharge: The solids are conveyed by the scroll to the tapered end of the bowl and
discharged through a separate outlet.

Advantages:

High Solid Concentration: Scroll centrifuges are particularly efficient in handling slurries and
mixtures with high solid concentrations.

Continuous Operation: They operate continuously, allowing for efficient processing of large
volumes of material.

Adjustable Scroll Speed: The speed of the scroll can be adjusted to optimize the separation
process for different types of feed mixtures.

Wide Range of Applications: Scroll centrifuges are versatile and can be used in various
applications.

Self-Cleaning: The continuous discharge of solids minimizes the need for frequent cleaning.