Dr Gihan Gawish 1

Hydrodynamic methods

Common experimental manipulations in biochemistry

centrifugation, dialysis and filtration are strongly influenced by flow interaction arising from hydrodynamic properties of the sample.

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

Dr Gihan Gawish

Centrifugal force = buoyant force + frictional force

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1. The Centrifugal Force

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.

Dr Gihan Gawish

Fc = M * w * r0

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The Centrifugal Force

A more common expression is the relative centrifugal force (RCF):

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.

2

1000**18.11

rpm

rRCF

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2.The Buoyant Force

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. Dr Gihan Gawish

FB = Mw * w * r2

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The Frictional force

Frictional force (resistance of a molecule to movement)

v = velocity relative to the centrifuge tube,

f = frictional coefficient.

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Ff = f V

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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

sw in g in g b u ck e t ro to rs : * L o n g e r d is ta n c e o f tra v e l m a y a llo w b e tte r se p a ra tio n* E x c e lle n t fo r g ra d ie n t c e n tr ifu g a tio n * E a s ie r to w ith d ra w su p e rn a ta n t w ith o u t d is tu rb in g p e lle t

.

fix ed -an g le ro to rs :* S e d im e n tin g p a r tic le s h a v e o n ly sh o r t d is ta n c e to tra v e l b e fo re p e lle tin g* E x c e lle n t fo r fra c tio n a tio n p u rp o se s* T h e m o s t w id e ly u se d ro to r ty p e .

.

O th e r typ es in c lu d e v e rtica l ro to rs an d co n tin u o u s-flo w ro to rs

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2. Type of Centrifuge2-1.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. UltracentrifugeThe 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

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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 Dr Gihan Gawish 17

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

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.

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History of Ultracentrifuge1. Theodor Svedberg invented the analytical

ultracentrifuge in 1923, and won the Nobel Prize in Chemistry.

2. Edward Greydon Pickels invented the vacuum ultracentrifuge

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 for measuring temperature.

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

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as slide show

AnimationResearch work Related to Ultracentrifugation