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 Dr Gihan Gawish 2

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. Dr Gihan Gawish 3

Sedimentation Theory Dr Gihan Gawish Centrifugal force = buoyant force + frictional force 4

1. The Centrifugal Force  M o 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 * r 0 2 5

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. Dr Gihan Gawish 6

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 * r 2 7

The Frictional force Frictional force (resistance of a molecule to movement)  v = velocity relative to the centrifuge tube,  f = frictional coefficient. Dr Gihan Gawish F f = f V 8

 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. Dr Gihan Gawish 9

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 principlesedimentation principle  Theory: The amount of acceleration to be applied to the sample, rather than specifying a rotational speed such as revolutions per minute. Dr Gihan Gawish 10

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 Dr Gihan Gawish 11

1. Types of Rotors Dr Gihan Gawish 12

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. Dr Gihan Gawish 13

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 Dr Gihan Gawish 14

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 Dr Gihan Gawish Analytical Ultracentrifuge 15

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 Dr Gihan Gawish 16

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 Dr Gihan Gawish 18

Dr Gihan Gawish 19 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. Dr Gihan Gawish 20

History of Ultracentrifuge 1. Theodor Svedberg invented the analytical ultracentrifuge in 1923, and won the Nobel Prize in Chemistry.Nobel Prize in Chemistry 2. Edward Greydon Pickels invented the vacuum ultracentrifuge  Vacuum allowed a reduction in friction generated at high speeds. Vacuum  Vacuum systems also enabled the maintenance of constant temperature.temperature Dr Gihan Gawish 21

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.???? Dr Gihan Gawish 22

Dr Gihan Gawish 23 Research work Related to Gradient Centrifugation Click down as slide show

Animation Research work Related to Ultracentrifugation 2- Meselson and Stahl’s experiment