# centrifugation

Views:

Category: Entertainment

## Presentation Description

No description available.

By: monapharma89 (122 month(s) ago)

By: mehras007 (128 month(s) ago)

By: amgchira (129 month(s) ago)

good ppt

By: khawish (129 month(s) ago)

## Presentation Transcript

### Centrifugation Theory and Practice :

Centrifugation Theory and Practice Routine centrifuge rotors Calculation of g-force Differential centrifugation Density gradient theory

### Centrifuge rotors :

Centrifuge rotors

### Geometry of rotors :

Geometry of rotors

### k’-factor of rotors :

k’-factor of rotors The k’-factor is a measure of the time taken for a particle to sediment through a sucrose gradient The most efficient rotors which operate at a high RCF and have a low sedimentation path length therefore have the lowest k’-factors The centrifugation times (t) and k’-factors for two different rotors (1 and 2) are related by:

### Calculation of RCF and Q :

Calculation of RCF and Q RCF = Relative Centrifugal Force (g-force) Q = rpm; r = radius in cm

### RCF in swinging-bucket and fixed-angle rotors at 40,000 rpm :

RCF in swinging-bucket and fixed-angle rotors at 40,000 rpm Beckman SW41 swinging-bucket (13 ml) gmin = 119,850g; gav = 196,770g; gmax = 273,690g Beckman 70.1Ti fixed-angle rotor (13 ml) gmin = 72,450g; gav = 109,120g; gmax = 146,680g

### Slide 7:

Velocity of sedimentation of a particle

### Differential centrifugation :

Differential centrifugation Density of liquid is uniform Density of liquid << Density of particles Viscosity of the liquid is low Consequence: Rate of particle sedimentation depends mainly on its size and the applied g-force.

### Size of major cell organelles :

Size of major cell organelles Nucleus 4-12 m Plasma membrane sheets 3-20 m Golgi tubules 1-2 m Mitochondria 0.4-2.5 m Lysosomes/peroxisomes 0.4-0.8 m Microsomal vesicles 0.05-0.3m

### Differential centrifugation of a tissue homogenate (I) :

Differential centrifugation of a tissue homogenate (I)

### Differential centrifugation of a tissue homogenate (II) :

Differential centrifugation of a tissue homogenate (II) Homogenate – 1000g for 10 min Supernatant from 1 – 3000g for 10 min Supernatant from 2 – 15,000g for 15 min Supernatant from 3 – 100,000g for 45 min Pellet 1 – nuclear Pellet 2 – “heavy” mitochondrial Pellet 3 – “light” mitochondrial Pellet 4 – microsomal

### Differential centrifugation (III)Expected content of pellets :

Differential centrifugation (III)Expected content of pellets 1000g pellet: nuclei, plasma membrane sheets 3000g pellet: large mitochondria, Golgi tubules 15,000g pellet: small mitochondria, lysosomes, peroxisomes 100,000g pellet: microsomes

### Differential centrifugation (IV) :

Differential centrifugation (IV) Poor resolution and recovery because of: Particle size heterogeneity Particles starting out at rmin have furthest to travel but initially experience lowest RCF Smaller particles close to rmax have only a short distance to travel and experience the highest RCF

### Differential centrifugation (V) :

Differential centrifugation (V)

### Differential centrifugation (VI) :

Differential centrifugation (VI) Rate of sedimentation can be modulated by particle density Nuclei have an unusually rapid sedimentation rate because of their size AND high density Golgi tubules do not sediment at 3000g, in spite of their size: they have an unusually low sedimentation rate because of their very low density: (p - l) becomes rate limiting.

### How does a gradient separate different particles? :

How does a gradient separate different particles?

### Predictions from equation (I) :

When p > l : v is +ve When p = l : v is 0 Predictions from equation (I)

### Predictions from equation (II) :

When p < l : v is -ve Predictions from equation (II)

### Summary of previous slides :

Summary of previous slides A particle will sediment through a solution if particle density > solution density If particle density < solution density, particle will float through solution When particle density = solution density the particle stop sedimenting or floating

### Buoyant density banding Equilibrium density bandingIsopycnic banding :

Buoyant density banding Equilibrium density bandingIsopycnic banding

### 3 Formats for separation of particles accordingto their density :

3 Formats for separation of particles accordingto their density When density of particle < density of liquid V is -ve