Post on 08-Apr-2018
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Differential Centrifugation
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Differential Centrifugation
This is the most common method of
fractionating cells
Fractionation is the separation of the
different organelles within the cell
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Method:
1. Cut tissue in an icecold
isotonic buffer. It is cold to stop
Enzyme reactions, isotonic to
stop osmosis and a
buffer to stop pH changes. 2. Grind tissue in a
blender to break open
cells.
3. Filter to remove
insoluble tissue
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4. Centrifuge
filtrate at low
speeds. ( 1000 X g
or m ns This pellets the
nuclei as this is the
densest organelle.
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5. Centrifuge at
medium speeds( 10 000 x g for 30
This pellets
mitchondria which
are the second
densest organelle
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6. Centrifuge at
high speeds.( 100 000 x g for 30
mins)
This pellets ER,golgi apparatus
and other
membranefragments
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7. Centrifuge at
very high speeds
x g or3hrs)
This pellets
ribosomes
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Investigating Cell Function
Differential Centrifugation allows us to
look at each organelle within the cell
We can look at the individual organellesand study them in detail
s e ps o e erm ne eac organe esfunction within the cell
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The Electron Microscope
Microscopes allow us to see living organisms whichare too small to be seen by the naked eye
The electron microscope uses beams of electrons
rather than light to illuminate thespecimen
A eam of electrons has an effective wavelength of
less than 1 nm so it can be used to resolve small sub-cellular ultra-structure
The development of the electron microscope allowed
biologists to view the organelles within a cell for thefirst time
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There are two types of
electron microscope
The transmission
microscope. (TEM)
Works like a lightmicroscope, it transmits a
beam of electrons through
a thin s ecimen
The scanning electron
microscope (SEM)
This scans a fine beamof electron onto
specimen and collects
electrons scattered b
Then focussing theelectrons to form an image
on a screen
This is the most common
form of electron
microscope and gives
good resolution.
surface This has poor
resolution but gives
good 3-D images
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Disadvantages of the Electron Microscope
The specimens must be fixed and viewed in
dead.
Sometimes specimens can be damaged by
the electron beam and must bestained with an electron-dense chemical.
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Industrial centrifuges
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Tubular bowl centrifuge
Simplest type and can provide very high centrifugal force.
Centrifuge can be cooled and hence it is advantageous in
protein and other thermally labile bioproduct separation.
Mostly used in pilot plant level.
cons s s o a ong narrow cy n r ca ow suspen e rom
the top rotating at high speed, in an outer stationary casing.
Bowl dimensions range from 8 to 15 cm in diameter and upto
150 cm in height.
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The feed is introduced at the bottom of the
bowl and discharge of the supernatant occursthrough an annular opening at the top.
The feed liquid moves upward at a uniformvelocity carrying with it the solid particles.
The solid deposit on the bowl's inner wall as athick paste.
Bowl must be dismantled and cleaned, once theefficiency drops.
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Multiple chamber bowl
centrifuge
Contains a number of concentric tubes connected
in such a way that a zigzag flow of the feed
suspension through the chamber is achieved.
wall.
Solid discharge is done manually.
Employed in fractionation of human blood
plasma.
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Disc bowl centrifuge
Consists of shallow wide cylindrical bottomdriven bowl rotating at moderate speed in a
stationary casing.
Bowl is about 30 to 100cm in diametercontains a number of closely spaced metal
discs, located one above the other.
Disc have one or more set of matching
holes, which forms a channel through which
the feed material flows.
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Feed is introduced through a centrally located
feed pipe from above.
The clarified liquid flows out through an annular
slit near the neck of the bowl.
Under the influence of centrifugal force, the dense
phase of the feed travels towards the bowl wall.
While the lighter phase displaced towards the
centre.
The solids may be removed intermittently orcontinuously from the sides.
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roperties of industrial centrifuges
Tubular bowl
High centrifugal force
Good dewatering
Easy to clean
Chamber
Limited solids capacity
Foams
Difficult to recover protein
Good dewatering Bowl cooling possible
Disc type
Solids discharge No foaming
Bowl cooling possible
Cleaning difficult Solids recovery difficult
Poor dewatering Difficult to clean
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