Cell separation by centrifugation
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Transcript of Cell separation by centrifugation
Cell separation by Centrifugation
BY-Gautam ParmarIndustrial Biotechnology
Introduction
Principle
Types of Industrial Centrifuge
Merits Demerits
Applications
Stoke’s law
References
Preface
The various stages of processing that occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product.
Downstream process
Centrifugation is used to separate materials of different density when a force
greater than gravity is desired.
Centrifugation method is utilized to separate the cellular debris from the released
protein.
Example-a key role in many industrial processes, including the production of
insulin, is to separate liquid phases and solids from each other.
It depends on particles size, density difference between the cells and the broth and
broth viscosity.
It is based on the behavior of particles in an applied centrifugal field.
More dense components of the mixture move away from the axis of the centrifuge
while less dense components of the mixture move towards the axis
Introduction
5
Principle
Centrifugation is used to separate materials of different density when a
force greater than gravity is desired.
The particles will tend to sediment under the influence of gravity.
If the particles suspended in a liquid are so small or have a density so
close to that of the liquid, then the force of gravity fails to sediment the
particles into a separate layer.
So the basis of centrifugation techniques is to exert a larger force than
the gravitational force to enhance the effective sedimentation force for
the separating such particles from the liquid.
Particles which differ in density, shape or size can be separated since
they sediment at different rates in the centrifugal field, each particle
sedimenting at a rate which is proportional to the applied centrifugal
field.
The rate at which the sedimentation occurs in centrifugation is expressed in terms of sedimentation coefficient and is given by the formula:
Where, V = sedimentation of the molecules ω = Rotation of the rotor in radians/sec. (angular velocity) r = Distance in cm, from the centre of the rotor
S = V/ω2r
Bowl Basket Centrifuge- Useful for separating mould mycelia or crystalline
compounds.
Perforated & Lined with a filter bag of nylon, cotton.
Normally operated at speed of up to 4000 rpm for feed rate 50-300 dm³ min¯¹.
Continuous feed is used & when the basket is filled with the filter cake it is
possible to wash the cake before removing it.
It may be considered to be a centrifugal filter and holding capacity is 50-300 dm³
Blinding with soft biological materials so that high centrifugal forces cannot be
used.
Industrial Centrifuges
Disc-stack bowl centrifuge- This type is common in bioprocess. The developed forces is 5000-15000
G with minimal density difference between solid and liquid is 0.01-0.03 kg/m3.
Large particles have higher settling velocities than small particles
Cellular debris ends up at the outer edge of the bowl
Soluble intracellular material passes through with the clarified liquid
The minimum particle diameter is 5 µm.
The discs split the stream into a large number of very thin layers thereby improving separation.
Solids flow downwards on bottom face of disc.
Liquid flows upwards on top face of disc.
The close packing of the discs assists rapid sedimentation and the solids then slides to the edge of the
bowl.
Smaller in size compared with a bowl without discs for given throughput.
Requires in-situ steam sterilization and the discs arrangement makes this type of centrifuge laborious to
clean.
Feed rate 45-1800 dm³ min¯¹.
Tubular bowl centrifuge (Narrow tubular bowl centrifuge or ultracentrifuge, decanter
centrifuge)-Used for particle size ranging from 0.1-200 µm.
Simple and widely applied in food and pharmaceutical industry. Operates at 13000-16000 G, 105-
106 G for ultracentrifuge.
Feed containing solids, heavy liquid phases are introduced by nozzle and are kept separate in their
exit from the bowl by an adjustable ring.
High centrifugal force, good dewatering and ease of cleaning and plastic liners can be used in the
bowls to help improve batch cycle time.
It can be employed for Light phase/heavy phase liquid separation, Solid/light-liquid phase/heavy-
liquid phase separation.
Disadvantages re limited solids capacity, difficulties in the recovery of collected solids, gradual
loss in efficiency as the bowl fills, and foaming.
The clarification efficiency of centrifugation process is affected by harvest parameters such as centrifuge feed rate, G-force, bowl geometry, operating pressure, discharge frequency and ancillary equipment used in transfer of cell culture fluid.
Peak cell density, total cell density and culture viability during the culture process and harvest will also affect separation performance.
Particles of submicron size cannot be removed in the centrifuge, thus increases the burden on subsequent depth filtration.
Disk stack continuous centrifuge removes cell debris from viable cells and liquid phase but some cells gets disrupted during process especially feedstock with low viability culture fluid
Merits and demerits
Optimization of centrifugation process can be done at lab scale and pilot to select
the feed rate and bowl rotational speed using the scaling factors of feed rate (Q)
and equivalent settling area
As lower the value of sigma factor the better will be the clarification and it should
be govern that Q/ better will be the separation.
Feed rate containing fragile cells, viable cells are to be separated at lower speed
with constant value of sigma factor
Centrifuges are basically employed for separation of whole large cells from heterogeneous cell mixture.
Can be used to separate viable cells from cell debris using disc stack centrifuge. clarification (removal of solid impurities from milk prior to pasteurization) skimming (separation of cream from skim milk) standardizing whey separation (separation of whey cream (fat) from whey) bactofuge treatment (separation of bacteria from milk) quark separation (separation of quarg curd from whey) butter oil purification (separation of serum phase from anhydrous milk fat). Disc-stack centrifuges used by some companies in the oil sands industry to separate small
amounts of water and solids from bitumen. Large industrial centrifuges are commonly used in water and wastewater treatment to
dry sludges. The resulting dry product is often termed cake, and the water leaving a centrifuge after most of the solids have been removed is called centrate.
Large industrial centrifuges are also used in the oil industry to remove solids from the drilling fluid.
Centrifuge are also used in whole blood separation into different components.
Applications
Industrial centrifuges
Tubular bowl Chamber Disc
Tube◦ High centrifugal force◦ Good dewatering◦ Easy to clean
Chamber◦ Large solids capacity◦ Good dewatering◦ Bowl cooling possible
Disc type◦ Solids discharge◦ No foaming◦ Bowl cooling possible
◦ Limited solids capacity◦ Foams◦ Difficult to recover
protein
◦ No solids discharge◦ Cleaning difficult◦ Solids recovery difficult
◦ Poor dewatering◦ Difficult to clean
Properties of industrial centrifuges
Bacteria ◦ Small cell size◦ Resilient
Yeast cells◦ Large cells◦ Resilient
Filamentous fungi◦ Mycelial◦ Resilient
Cultured animal cells◦ Large cells◦ Very fragile
◦ High speed required◦ Low cell damage
◦ Lower speed required◦ Low cell damage
◦ Lower speed required◦ High water retention in
pellet
◦ Very susceptible to damage
Centrifugation properties of different cell types
16
gDu pfp
g2
18
rDu pfp
c22
18
The centrifugation theoryThe terminal velocity during gravity settling of a small spherical particle in dilute suspension is given by Stoke’s law:
Where ug is sedimentation velocity under gravity, ρp is particle density, ρf is liquid density, µ is liquid viscosity, Dp is diameter of the particle, and g is gravitational acceleration.In the centrifuge:
uc is particle velocity in the centrifuge, ω is angular velocity in rad/s, and r is radius of the centrifuge drum.
17
31
32
2
tan312 rr
gN
21
22
2
32
rrgb
The centrifugation theoryDisc-stack bowl centrifuge
N is number of disc, θ is half-cone angle of the disc. The r1 and r2 are inner and outer radius of the disc, respectively.
Tubular-bowl centrifuge
b is length of the bowl, r1 and r2 are inner and outer radius of the wall of the bowl.
Principle of Fermentation Technology by A. Whitaker, P.F Stanbury, S.J. Hall.
Wikipedia.org. Slideshare.com NCBI.com Hamel & Hunter- Modeling and Applications of
Downstream Processing. Downstream processing article- springer
References
THANX A LOT