CENTRIFUGATION

BY: Reddy Sailaja M (MSIWM031)

Centrifugation is one of the most extensively used technique in research and development fields of biochemistry, molecular biology, biochemistry and pharmaceutical industries for varied applications like isolation of cells, fractionation of sub cellular particles and other macromolecules for analytical and clinical applications.

Definition

Centrifugation is a process of separation (or concentration) of particles from suspended medium based on their size, shape, density, viscosity of the medium, rotor speed etc. Centrifugal force is a key in this technique to separate the particles in less time and it acts against gravitational force. Figure 1 gives overview of centrifugation process from muscle tissue.

Figure 1: Centrifugation overview

Principle:

In general, when a liquid suspension is placed idle for some time, particles of bigger size/density will start to settle at the bottom of the container because of gravitational force and so on. But, this is a slow process and can’t be applied practically. Centrifugation works on centrifugal force to separate the particles in a suspension in less time with more efficiency.

When a body with mass ‘m’ is rotating in a circle with radius ‘r’ and velocity ‘v’, the force acting on the body is measured using the following formula 1.

F = mv²/r

Where,

F = centrifugal force,

m = mass of body,

v = velocity of the body,

 r = radius of circle of rotation.

The gravitational force acting on the body ‘m’ is calculated using the formula 2: G = mg

Where,

G = gravitational force

g = acceleration due to gravity

U

The centrifugal force is further calculated using the formulae 1 and 2 as follows:

C = F/G = mv2/mgr = v2/gr

Since, v = 2π r n

Where,

 n = speed of rotation

C = F/G = (2π r n) 2/g r = 4π2 r2n2 = 2π2/g D n2 = kD n2

where,

k = 2π2/g = constant

D = maximum diameter of the centrifuge

D is able to measured either from centrifuge center to the free surface of the liquid or to the tip of the centrifuge tube.

From the equation C = kDn2 it was evident that,

Centrifugal effect ∝ diameter of centrifuge

Centrifugal effect ∝ (speed of rotation)².

When a liquid suspension containing container is rotated at a certain speed called revolutions per minute (RPM), particles will move at a certain speed away from the axis of rotation. The force that’s being generated on the particles to move away from the centre is called relative centrifugal force (RCF). RCF depends mainly on the rotational speed (measured in RPM) and the distance of the particles from the centre of the rotation (rotor).

RCF = 11.2 × r (RPM/1000)2

Where,

r – Distance in centimeters

More the density of the particles, faster is the settlement at the bottom of the tube, while less dense particles will be floating in the liquid. The rate of sedimentation depends on the size and density of the particles and can be explained by Stokes equation (explains movement of a sphere in a gravitational field).

Where,

V = viscosity of the medium

d = diameter of the sphere

p = particle density

L = medium density

n = viscosity of medium

g = gravitational force

Stokes equation explains behavior of particles based on the rate of particle sedimentation as follows:

• directly proportional to the size of the particle
• directly proportional to the difference between the particle and the medium densities
• zero when the particles and medium exhibits same density values
• decreases when the medium viscosity increases
• increases as the gravitational force increases

Table 1: Densities of cells and sub cellular fractions

The particles that gets settled at the bottom forms ‘pellet’ while the liquid suspension with lighter particles or no particles is called ‘supernatant’.  Therefore, centrifugation is a process that utilizes centrifugal force for the sedimentation of particles.

Figure 2: Densities and sedimentation coefficients of biomolecules, cell organelles and viruses

For example, ‘m’ is a particle in a centrifuge tube suspended in a liquid. During centrifugation process, the particle is influenced by three kinds of forces: F_C– the centrifugal force, F_B – the buoyant force and F_f – the frictional force between the particle and the liquid.

Figure 3: Centrifugal force

Centrifuge

Centrifuge is a tool designed to separate particles in the liquid suspension based on the centrifugation principle. It is operated using an electric motor that enables an object to move around in a fixed axis when a perpendicular force is applied to the axis.

Figure 4a: Front view of a typical centrifuge

Figure 4b: Rear view of a typical centrifuge

Centrifuge comprises of three major components:

1. Rotor – Holds containers (tubes/bottles etc) containing liquid suspension to be centrifuged. Rotors of different types and sizes are available
2. Fixed angle rotor – Requires short time to sediment particles as they need to travel only a little distance.

Figure 5a: Fixed angle rotor

• Swinging bucket rotor – Allows better separation of particles as the particles have to move long distance. Stronger pellet is formed and supernatant can be easily removed.

Figure 5b: Swinging bucket rotor

1. Drive shaft – Helps hold rotors which in turn connect to motor.
2. Motor – Helps to rotate the rotor based on the input speed by providing power.

All the major centrifuge components are surrounded by a protective cabinet with operating controls and indicator dials for speed and time mounted on it. Centrifuges come with brake system to control rotor and allow it to come to standstill when the centrifugation run gets completed. Refrigerated centrifuges allow option to control temperature so that the delicate biological samples won’t be degenerated during the process.

Types of centrifugation techniques

There are two major types of centrifugation techniques to separate particles.

1. Differential centrifugation
2. Density gradient centrifugation
3. Isopycnic centrifugation
4. Rate-zonal centrifugation
5. Differential centrifugation:

This is the simplest type of particle separation form, also called ‘pelleting’ down the particles. Sedimentation occurs at different rates based on the density of the particles. More dense particles will sediment fast and the lighter particles will be floating in the suspension. Sedimentation of the particles also depends on the centrifugal force applied. As the centrifugal force increased, pellet with decreased sedimentation rate will be formed and vice versa.

Differential centrifugation is applied during cell harvest or sub cellular fractionation from a tissue homogenate. When lower centrifugal force is applied, dense particles like nuclei, membrane vesicles etc gets pelleted first. To further pellet next order particles like mitochondria etc, more centrifugal force is applied. Greater than or equal to four differential centrifugation cycles are applied for sub cellular fractionation of the tissue homogenate. However, this process faces carry over contamination of the particles from previous fraction and not purity is less.

Figure 6: Differential centrifugation process overview

• Density gradient centrifugation: This method is mainly applicable to separate sub cellular particles and other macromolecules with more purity.

In this process, gradient media of different density are layered one above the other, more dense at the bottom of the tube and the lightest at the top. The cell fractionate that need to be separated is placed on the top of the gradient layer and the centrifugal force is applied.

Figure 7: Density gradient process overview

Density gradient method is further classified into two types as follows:

1. Isopycnic centrifugation:

This process is also known as buoyant or equilibrium separation. In this process, particles are separated base on density. Particle size plays a role when the density of the particles and the surrounding medium is same. When the centrifugal force is applied, initially the sample and gradient gets mix uniformly and the particles move through the gradient until the density of the particles and gradient medium becomes same. Now, the gradient is called as ‘isopycnic’ and the particles get separated based on their buoyancy. Therefore, it is important to make sure that the gradient medium is always dense than the particles to be separated. Particles get separated in the gradient medium in different layers, but never settle to the bottom of the tube. Gradient medium varies depending upon the kind of material being separated. Continuous gradient method is good for analytical separation while discontinuous gradient is more suitable for biological applications (E.g.: separation of lymphocytes from blood).

Table 2: Common density gradient media used for isopycnic centrifugation process

• Rate zonal centrifugation:

The carryover contamination of particles in differential centrifugation is prevented by implementing rate zonal centrifugation. In this process, sample was layered in a narrow zone on the top of the density gradient and the centrifugal force is applied. Particles segregate based on their size and mass rather than density, and also on the centrifugal force. As a result, narrow load zone prevents less sample volume (≤10%) that can be accommodated on the density gradient and it further stabilizes the bands and allows medium of increasing density and viscosity. Centrifugation is applied for a short time at a low speed. As the density of the particles is more than the density of the gradient, there is a chance that all the particles form pellet if the centrifugation continues for a long time.

Figure 8: Rate zonal and isopycnic centrifugation processes overview

Common applications of centrifugation

• Production of drugs and other biological products
• Separation of subcellular particles
• Separation of blood and urine components in forensic analysis
• Protein purification
• Clarification and stabilization of wine
• Fat removal from milk