Dr. Tarek Elbashiti Assoc. Prof. of Biotechnology Bioseparation
Techniques Centrifugation
Slide 2
A centrifuge is a device that separates particles from
suspensions or even macromolecules from solutions according to
their size, shape and density (weight per unit volume) by
subjecting these dispersed systems to artificially induced
gravitational fields. Centrifugation can only be used when the
isolated material is denser than the medium in which they are
dispersed.
Slide 3
Table 6.1 lists the densities of different biological
substances that are usually separated by centrifugation.
Slide 4
Based on data shown in Table 6.1 one may wrongly assume that
proteins and nucleic acids would settle faster than cells and
organelles. Biological macromolecules in aqueous solution exist in
an extensively hydrated form i.e. in association with a large
number of water molecules. Hence the effective densities of these
substances in solution are only slightly higher than that of water.
Also, these macromolecules are significantly smaller than
cells.
Slide 5
The substances listed in Table 6.1 would settle at extremely
low velocities under gravity and hence separation would not be
feasible. In a centrifugation process, these settling rates are
amplified using an artificially induced gravitational field.
Slide 6
Process Functions There are several process functions using
centrifuges in biotech separation. These are listed below. 1.
Separation (solid/liquid, solid/liquid/liquid and
solid/solid/liquid separation) Centrifugation can be used for
solid- liquid separation provided the solids are heavier than the
liquid.
Slide 7
Centrifuge can also be used to separate a heavy phase, and two
lighter liquid phases, with one of the lighter phases being lighter
than the other. Solids can be lighter than liquid and separation is
by flotation of the dispersed solid phase.
Slide 8
2. Clarification- minimal solids in liquid product Centrifuge
can be used to clarify the discharge separated lighter liquid
phase. The objective is to minimize the discrete suspended solids
in the light continuous phase. Usually, only fine submicron
biosolids are left uncaptured by centrifugation and they escape
with the discharged light phase.
Slide 9
3. Classification -sort by size and density Centrifuge is used
to classify solids of different sizes. One of the several possible
applications is to classify crystals of different size range, with
the finer submicron sizes leaving with the light phase and
retaining only the larger sizes in the separated heavy phase.
Either of the separated solids can be the product.
Slide 10
For example, the larger crystals can be the product crystals
while the finer crystals are returned to the crystallizer to grow
to larger crystals. Another similar application is to classify
smaller size cell debris in the light liquid phase from the heavier
products after homogenizing cells.
Slide 11
4. Degritting- remove oversized and foreign particles
Degritting is similar to classification where unwanted particles,
larger or denser, are rejected in the sediment, with product
(smaller or less dense) overflowing in the lighter liquid phase.
Another situation is where smaller unwanted particles are rejected
in the light liquid phase, and valuable heavier solids are settled
with the heavier phase.
Slide 12
5. Thickening or concentration- remove liquid, concentrate
solids Centrifuge is frequently used to concentrate the solid phase
by sedimentation and compaction, removing the excess liquid phase
in the overflow or centrate. This reduces the volume of the product
in downstream processing.
Slide 13
6. Separation and repulping - remove impurities by washing or
diluting With a concentrated suspension containing contaminants
such as salts and ions, it is diluted and washed so that the
contaminants are dissolved in the wash liquid. Then, the suspension
is sent for centrifugation to remove the spent wash liquid with
dissolved contaminants or finely suspended solids. Subsequently,
the product can be further concentrated by centrifugation.
Slide 14
The abovementioned processes can be combined to achieve several
objectives concurrently or in series. Cells, sub-cellular
components, virus particles and precipitated forms of proteins and
nucleic acids are easy to separate by centrifugation. When
macromolecules such as proteins, nucleic acids and carbohydrates
need to be separated, normal centrifuges cannot be used and special
devices called ultracentrifuges which generate very strong
artificial gravitational fields are used.
Slide 15
Slide 16
Centrifuges are classified into two categories: I. Laboratory
centrifuges II. Preparative centrifuges I. Laboratory centrifuge
Laboratory centrifuges are used for small-scale separation and
clarification (i.e.removal of particles from liquids). Typical
liquid volumes handled by such devices are in the range of 1-5000
ml.
Slide 17
Slide 18
The principle of separation by centrifugation is shown in Fig.
6.1. There are two types of rotors: fixed angle rotors and swing
out rotors. A fixed angle rotor holds the centrifuge in a fixed
manner at particular angle to the axis of rotation. Swing out
rotors hold the tubes parallel to the axis of rotation while the
rotor is stationary but when the rotor is in motion, the tubes
swing out such that they are aligned perpendicular to the axis of
rotation.
Slide 19
Slide 20
When the centrifuge tubes are spun, the centrifugal action
creates an induced gravitational field in an outward direction
relative to the axis of rotation and this drives the particles or
precipitated matter towards the bottom of the tube. Typical
rotation speeds of laboratory centrifuges range from 1,000-15,000
rpm.
Slide 21
The magnitude of the induced gravitational field is measured in
terms of the G value: a G value of 1000 refers to an induced field
that is thousand time stronger than that to gravity. The G value
which is also referred to as the RCF (relative centrifugal force)
value depends on the rotation speed as well as the manner in which
the centrifuge tubes are held by the rotor:
Slide 22
Slide 23
It is virtually impossible to make very exact calculations for
a laboratory centrifugation process. This is due to the fact that
it usually takes a certain amount of time after start-up for the
rotation speed of the centrifuge to reach the operating value.
Similarly it takes a certain amount of time for the rotation speed
to decrease from the operating speed to zero at the end of the
process.
Slide 24
The settling particles go through different G value zones while
moving toward the bottom of the centrifuge tube.
Slide 25
II. Preparative centrifuge Preparative centrifuges can handle
significantly larger liquid volumes than laboratory centrifuges,
typically ranging from 1 litre to several thousand litres.
Preparative centrifuges come in a range of designs, the common
feature in these being a tubular rotating chamber. 1.Tubular Bowl
Centrifuge For particle size ranges of 0.1 to 200 micrometer and up
to 10% solids in the in-going slurry.
Slide 26
A simple diagram of the most common type of preparative
centrifuge (the tubular bowl centrifuge)
Slide 27
The suspension to be centrifuged is fed into such a device from
one end while the supernatant and precipitate are collected from
the other end of the device in a continuous or semi- continuous
manner. Typical rotating speeds for preparative centrifuges range
from 500 - 2000 rpm. The motion of a particle at any point within a
tubular centrifuge in the radial direction is governed by the
following force balance equation:
Slide 28
The force used on a particle = frictional force beared by the
particle The particle would continue to settle due to centrifugal
acceleration till the two forces are balanced. Thus the centrifuge
may be altered to use for: a. Light-phase/heavy-phase liquid
separation. b. Solids/light-liquid phase/heavy-liquid phase
separation. c. Solids/liquid separation
Slide 29
Slide 30
2. Disk Stack or Multichamber Centrifuge For slurry of up to 5%
solids of particle size 0.1 to 200 micrometer diameter. A disc
stack centrifuge is a special type of preparative centrifuge which
is compact in design and gives better solid-liquid separation than
the standard tubular bowl centrifuge. Fig. 6.6 shows the working
principle of a disc stack centrifuge. The feed enters from the top
of the device and is distributed at the bottom of the disk bowl
through a hollow drive shaft.
Slide 31
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Slide 33
The particles are thrown outer and these come into contact with
the angled disc stack. Once this happens they slide down the disc,
are collected at the periphery of the bowl and discharged from the
device in the form of a slurry. The liquid flows up the device
along the central regions and is discharged from the top. The
smaller particles collect in the outer chambers where they are
subjected to greater centrifugal forces (the greater the radial
position of a particle, the greater the rate of
sedimentation).
Slide 34
Although these vessels can have a greater solids capacity than
tubular bowls and there is no loss of efficiency as the chamber
fills with solids, their mechanical strength and design limits
their speed to a maximum of 6500 rpm for a rotor 46- cm diamter
with a holding capacity of up to 76 cubic dm.
Slide 35
Slide 36
3. Ultracentrifuge An ultracentrifuge is a special type of
centrifuge in which the rotor rotates at a much higher speed than a
standard centrifuge. Typical rotation speeds in ultracentrifuges
range from 30000 rpm-50000 rpm. An ultracentrifuge is usually used
for separating macromolecules from solvents or for fractionating
mixtures of macromolecules. Ultracentrifuges are used for
analytical as well as for preparative applications.
Slide 37
An analytical ultracentrifuge (AUC) is mainly used for studying
the properties of macromolecules as well as for analyzing complex
mixtures of macromolecules. Preparative ultracentrifuges are used
to purify macromolecules such as proteins and nucleic acids based
on their physical properties such as size, molecular weight,
density and mobility.
Slide 38
The high rotating speeds used in ultracentrifuges can generate
considerable amount of heat. Therefore cooling arrangements are
required in these devices. An ultracentrifuge is also an angled
spin tube, and with a titanium rotor that provides mechanical
integrity, i.e. for high shear and yield strengths.
Slide 39
It can go up to 500,000-1,000,000 g for separating very small
particles, particles and liquid with a small density difference,
and/or separation in a viscous liquid phase. Theodore Svedberg
invented the analytical ultracentrifuge in 1923, and won the Nobel
Prize in Chemistry in 1926 for his research on colloids and
proteins using the ultracentrifuge.
Slide 40
4. Decanter Centrifuge The figure below shows the
countercurrent flow decanter, or solid-bowl centrifuge.
Slide 41
After accelerating in the rotating feed compartment or
accelerator, feed slurry is introduced to the annular pool. Under
high centrifugal force, the heavier solids migrate radially
outwards towards the bowl, displacing the lighter liquid to the
pool surface at a smaller radius.
Slide 42
Decanter centrifuge is most often used for clarification of
liquid containing high concentrations of solids, removing solids
from liquid. A decanter centrifuge is a sedimentation centrifuge
for separation of suspended solids from one liquid. The
characteristic which distinguishes a decanter centrifuge from other
types of centrifuges such as disc stack separators is that it has a
cono-cylindrical rotor equipped with a conveyor for continuous
unloading of sedimented solids.
Slide 43
Decanter centrifuges are used in a wide range of applications
where their ability to achieve both good clarity and low moisture
in the discharged solids is appreciated. They are very versatile as
they can handle both large and small particles as well as a wide
variety of solids concentrations. The performance of the centrifuge
depends on various operating variables, such as the feed rate, pool
depth, rotation speed or G- force, and they should be optimized for
a given process.