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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 TheorySvedberg equations

so lv e n t

so lv e n t

F f: th e F r ic tio n a l F o rce= f * v

F C : T h e C en tr ifu g a l F o rce

= M 0* 2* r F B : th e B u o ya n t F o rce (A rch im ed es)

= M w * 2* r

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Centrifugal force = buoyant force + frictional force

1. The Centrifugal Force

Mo is the particle weight, or molecular

weight w (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.4

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 ( round) per minute RCF reports centrifugal force relative to earth’s

gravitational force; commonly refer to as “number of times or g.” A sample rotating at 20, 000 rpm with r = 7 cm will experience

RCF= 33,000 x g.

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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.

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FB = Mw * w * r2

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

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

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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 Rotorssw 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 er 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

Dr Gihan Gawish11

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

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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 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

3. Types of Centrifugation

There are basically three modes of centrifugation

3-1.Differential or pelletingCellular fractionation and/or separating

coarse suspension removal of precipitatescrude 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. AnalyticalDetermining hydrodynamic or thermodynamic

properties of bio molecules

Relative MW Molecular shapeAggregation behavior Protein-protein interactions

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