Download 2-Hydrodynamic Methods (Sedimentation, Centrifugation and

Dr Gihan Gawish
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Hydrodynamic methods
 Common experimental manipulations in biochemistry
 centrifugation, dialysis and filtration are strongly
influenced by flow interaction arising
hydrodynamic properties of the sample.
from
 These properties arise from physical interaction between
molecules or particles and aqueous solvent.
 They are even more important in large scale industrial
situations in countered in biotechnology such as down
stream processing of protein products
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1- Sedimentation
 Sedimentation describes the motion of
molecules in solutions or particles in suspensions
in response to an external force such as gravity,
centrifugal force or electric force.
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Sedimentation Theory
solvent
FC: The Centrifugal Force
solvent
=M0* 2* r
FB: the Buoyant Force (Archimedes)
=Mw* 2* r
Ff: the Frictional Force
=f*v
Centrifugal force = buoyant force + frictional force
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1. The Centrifugal Force
2
Fc = M 0* w * r
 Mo is the particle weight, or molecular weight
  (omega)= angular velocity (radians/sec)
 r is the radius of rotation
This equation says that the larger the molecule, or the
faster the centrifugation, or the longer the axis of
rotation, the greater the centrifugal force and the
rate of sedimentation.
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The Centrifugal Force

A more common expression is the relative centrifugal force (RCF):
 rpm 
RCF  11.18 * r * 

1000


2
 r = Radial distance of particle from axis of rotation
 rpm = Revolutions per minute
 RCF reports centrifugal force relative to earth’s gravitational
force; commonly refer to as “number times g.”
 A sample rotating at 20, 000 rpm with r = 7 cm will experience RCF=
33,000 x g.
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2.The Buoyant Force
FB = Mw * w * r
2
 The buoyant force opposes the centrifugal force.
 where Mw is the mass of the solvent displaced by the
particle.
The net force= (Fc-FB) will determine whether a particle
floats or sediments
 Particles with higher density will experience
smaller buoyant force, and thus, sediment
faster.
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Dr Gihan Gawish
The Frictional force
Ff = f V
Frictional force (resistance of a molecule to
movement)
 v = velocity relative to the centrifuge tube,
 f = frictional coefficient.
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 The frictional coefficient depends
upon:
1. the size
2. shape of the molecule,
3. the viscosity of the gradient material.
 The frictional coefficient f of a
compact particle is smaller than
that of an extended particle of the
same mass.
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Centrifuge
 Centrifuge is a piece of equipment, generally
driven by a motor, that puts an object in rotation
around a fixed axis, applying a force
perpendicular to the axis. The centrifuge works
using the sedimentation principle
 Theory: The amount of acceleration to be
applied to the sample, rather than specifying a
rotational speed such as revolutions per
minute.
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Applied Centrifugation
 Parameters you need to know:
1. Type of rotor:
 fixed angle, swinging bucket, vertical
2. Type of centrifuge:
 Low speed , high Speed, ultracentrifuge
3. Type of centrifugation
 Differential, preparative, or analytical
 Also, the Speed and duration of centrifugation
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1. Types of Rotors
swinging bucket rotors:
* Longer distance of travel may allow
better separation
* Excellent for gradient centrifugation
.* Easier to withdraw supernatant without
disturbing pellet
fixed-angle rotors:
* Sedimenting particles have only short
distance to travel before pelleting.
* Excellent for fractionation purposes
* The most widely used rotor type.
Other types include vertical rotors and continuous-flow rotors
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2. Type of Centrifuge
2-1.Low-speed centrifuges
 Also called: microfuge, Clinical, Table top or
bench top centrifuges
 Max speed ~ 20,000 rpm
 Operate at room temperature
 Fixed angle or swinging bucket can be used
 Commonly used for rapid separation of coarse
particles
 E.g. RBC from blood, DNA from proteins, etc.
 The sample is centrifuged until the particles
are tightly packed into pellet at the bottom of
the tube. Liquid portion, supernatant, is
decanted.
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2-2. High-speed Centrifuges
Preparative centrifuges.
 Max speed ~ 80,000 rpm
 Often refrigerated, and
requires
vacuum to operate
 Fixed angle or swinging bucket can
be used
 Generally
used
to
separate
macromolecules (proteins or nucleic
acids)
during
purification
or
preparative work.
 Can be used to estimate
sedimentation coefficient and MW,
 No optical read-out
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2-3. Ultracentrifuge
The most advanced form of
centrifuges: (specialized and
expensive)
 Used to precisely determine
sedimentation coefficient and
MW of molecules, Molecular
shape,
Protein-protein
interactions
 Uses very high speed and/or
RCF
 Uses small sample size (< 1 ml)
 Uses relatively pure sample
 Built in optical system to
analyze
movements
of
molecules during centrifugation
Analytical Ultracentrifuge
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3. Types of Centrifugation
There are basically three modes of
centrifugation
3-1.Differential or pelleting
 Cellular fractionation and/or separating coarse
suspension
 removal of precipitates
 crude purification step
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3-2. Preparative or Density gradient
centrifugation:
 Separation of complex mixtures
 Finer fractionation of cellular components
 Purification of proteins, nucleic acids, plasmids
 Characterization of molecular interactions
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3-3. Analytical
 determining
hydrodynamic or thermodynamic
properties of biomolecules in the absence of solid
supports (vs. electrophoresis, chromatography)
 Relative MW
 Molecular shape
 Aggregation behavior
 Protein-protein interactions
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Ultracentrifugation
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Ultracentrifuge
 The ultracentrifuge is a centrifuge optimized for
spinning a rotor at very high speeds, capable of
generating acceleration as high as 1,000,000 g
(9,800 km/s²).
 There are two kinds of ultracentrifuges, the
preparative and the analytical ultracentrifuge.
 Both classes of instruments are used in molecular
biology, biochemistry and polymer science.
Dr Gihan Gawish
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History of Ultracentrifuge
1. Theodor
Svedberg
invented
the
analytical
ultracentrifuge in 1923, and won the Nobel Prize in
Chemistry.
2. Edward
Greydon
ultracentrifuge
Pickels
invented
the
vacuum

Vacuum allowed a reduction in friction generated at high
speeds.

Vacuum systems also enabled the maintenance of
constant temperature.
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3. Beckman
Instruments
 The rotor is an evacuated and
cooled the chamber and is
suspended on a wire coming from
the drive shift of the motor.
 The tip of the rotor contains a
thermistor
temperature.
for
measuring
 Electrical contact of the thermistor
to the control circuit is by means of
a pool of mercury which the rotor
tip touches.
 The rotor chamber content an
upper and a lower lens.????
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Research work Related to Gradient Centrifugation
Click
down as
slide
show
1-THE GRADIENT
CENTRIFUGATION TECHNIQUES
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Research work Related to Ultracentrifugation
2- Meselson and Stahl’s experiment
Animation