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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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