The Basic Separation Technique

CENTRIFUGATION

Centrifugation is a basic separation technique. A centrifuge is a device for separating particles in an applied centrifugal field in a solution.

There are two different forces act on an object moving in a circular motion.

Centrifugal force:

Force directed outward from the center. E.g. While turning a bus in twist way, the passengers strike on the bus wall is due to centrifugal force.

Centripetal force:

The force exerted towards the center is now as centripetal force. E.g. the force acts on passengers by the turning car.

Now, suppose a particle is exerted to sediment by centrifugal force, then

The rate or velocity at which it sediments is proportional to the force applied

• Sedimentation is more rapid when the force applied is greater than the gravitational force of the Earth
• Basis of separation is to exert a larger force than does the Earth’s gravitational force.

Basic Principle of Sedimentation

The particles to be separated are suspended in a specific liquid media, held in tubes or bottles which are located in rotor in centrifuge machine, positioned centrally to the drive shaft. These particles are differing in size, shape and density.

As we have already mentioned that,

The rate of sedimentation is dependent upon the applied centrifugal field (G)

G          = W²R …………………………equ (i)

Where

W:        Angular velocity of revolving particle (Remember: one revolution of the rotor is equal to 2 radians)

R:         Radial distance from axis of rotation

In terms of revolution per minute, we have W= 2p rev min^-1/ 60

Therefore:

G          = W²R

           

It is expressed as a multiple of the earth’s gravitational field (g=981 cm s^-2).

Hence

RCF, Relative Centrifugal Field

= G / g

=

RCF      = 1.119 x 10^-5(rev min^-1)² R …………………………….equ (ii)

= x g unit (number times g)

It means, RCF is the ratio of the weight of the particle in the applied centrifugal field to the weight of the same particle when acted by gravity alone. Therefore the rotor speed, radial dimensions and time of the rotor must be quoted during the centrifugation.

However:

This is not the only case in Biochemical experiments as biological samples are always found in dissolved or suspended form in a solution. Thus, the rate of sedimentation not only depends on the centrifugal field but also on

1. Mass of particle

2. Density of particle

3. Density and viscosity of the medium used

4. The extent to which its shape deviates from spherical

Now according to Newton’s Second law of Motion, the centrifugal force (F) exerted on particle is

= M. a

= M. W²R ……………………………….equ (iii)

Where:

M: mass of particle

a: acceleration while in angular motion= W²R

Increasing the sharpness of a turn, w and r decreases. Since r is linear, w has greater effect on the particle.

It causes the molecules to sediment down the centrifuge tube. They start to move downward to sediment; however they encounter opposing force, a frictional resistance in their movement.

Frictional force          = f

                             = 6p. h. R_p. ) ……………………………equ (iv)

Where:

f:          Frictional force

dr/dt: Rate of sedimentation expressed as the change in radius with time (velocity v)

h:         Viscosity coefficient of medium

R_p:        Radius of sedimenting particle

The sedimenting molecule must also displace the solvent into which it sediments and give rise to a buoyant force

Buoyant force            = mass x a

= V. d_m W²R ……………………..equ (v)

Where:

V:         Specific volume of the molecule

d_m:       Density of the medium

While sedimenting, the velocity of the particle increases until it equals the frictional force resisting its motion through the medium. This is an equilibrium state when the particles stop to move or sediment. From equations iii, iv and v.

Centrifugal force       = Frictional force + Buoyant force

M. W²R           = 6p. h. R_p. ) + V. d_m W²R

  pR_p³ d_p W²R             = 6p. h. R_p. ) +   pR_p³ d_m W²R   

  pR_p^3 (d_p - d_m) W²R       = 6p. h. R_p. )

                  =  h R_p^{2  (}d_p - d_m) W²R 

v          =  h R_p^{2  (}d_p - d_m) W²R  ……………………………..equ (vi)

Where:

dr/dt:   v, is the velocity of the sedimenting particle

Mass:   Density x Volume

d_p:        Density of particle

d_m:       Density of medium

From above equation, it seems clear that velocity is proportional to its size, to the differences in density between the particle and medium and to the applied centrifugal field. It is zero when the density of the particle and medium are equal. It decreases when the viscosity of the medium increases.

Since the R_p is in square form, the size of particle has greater influence on velocity.

For a particle, h, R_p^, d_p^, d_m and W all are constants

                        t           =   In

Where

t:          The sedimentation time in seconds

R_t:        Radial distance from the axis of rotation to liquid meniscus

R_b:        Radial distance from the axis of rotation to bottom of tube

It is now clear that a mixture of heterogeneous approximately spherical particles can be separated by centrifugation on the basis of their densities, their sizes and etc.

t           µ          

It means, higher the size particles, faster is the sedimentation (Short time for sedimentation) of it and smaller the size slower is the sedimentation (takes longer time).

CENTRIFUGATION: RCF CALCULATION

The relative centrifugal force (RCF) can be calculated from the following equation:

RCF = (1.119 x 10-5) (rpm)²(r)

Where rpm is the speed of rotation expressed in revolutions per minute and r (radius) is the distance from the axis expressed in cm. The RCF units are "x g" where g represents the force of gravity. RCF can also be determined from the NOMOGRAPH below. Place a straight edge to intersect the radius and the desired RCF to calculate the needed rpm. Alternatively place the straight edge on the radius and the rpm to calculate the g-force. For example, spinning a sample at 2500 rpm in a rotor with a 7.7 cm radius results in a RCF of 550 x g.

 

Figure 1: Nomograph showing relationship between RCF, RPM and Radius
Centrifuges and their uses

1.      Low  Speed Centrifuge

·         Least expensive and simplest in many design

·         Maximum rotor speed of 4000-6000rpm (3000-7000 X g)

a) Small bench centrifuges

·         To collect small amounts of materials (250mm³⁾ that is rapidly sediment (1-2 min)

·         No special cooling system

·         Ambient air flows around the rotor to cool the system

·         Use to rapid sedimentation of blood samples

b) Large capacity refrigerated centrifuges

·         Refrigerated rotor chambers for cooling the sample

·         Large volumes 10, 50 and 100 cm³ processing depending upon the rotors and tubes

·         Maximum capacity of 1.25 dm³

·         Rotors are mounted on a rigid suspension

·         Erythrocytes, coarse or bulky precipitates, yeast cells, nuclei and chloroplasts

2.      Microcentrifuge

·         Maximum rotor speed of 12000rpm with RCF of 10000g

·         Have total capacity of 1.5ml over very short time (0.05-5 min)

·         Use to sediment large particles like cell ppt 

3.      High speed refrigerated centrifuge

·         Maximum rotor speed of 25000rpm with RCF of 60000g

·         Have total capacity of 1.25 dm³

·         Interchangeable fixed angle and swinging buckets rotors

·         Use to collect microorganisms, cellular debris, larger cellular organelles and proteins precipitates by ammonium sulphate

·         Not use for viruses and smaller organelles like ribosome

4.      Continuous flow centrifuge

·         Relatively simple and high speed centrifuge

·         Special design rotor (long and tubular) with non interchangeable system

·         Have total capacity of 1-1.25 dm³/min with continuous flow

·         Particles sediment at wall and excess clarified medium overflows through an outlet port

·         Use to collect bacterial and yeast cells from their mass culture of about 100-500 dm³

5.      Ultracentrifuge

·         Powerful with speed

·         2 types

a) Preparative ultracentrifuge

• Maximum rotor speed of 30000-80000 rpm with RCF of 600000 x g
• Highly sophisticated with refrigerated, sealed and evacuated to minimize excess heat generate
• More sophisticated temperature monitoring system employing an infrared temperature sensor
• Overspeed control system to prevent operation of rotor above its max rated speed
• Vibration minimize system (a flexible drive shaft system) during unequal loading of the centrifuge tubes
• Enclosed in heavy armour plating
• Airfuse for some biochemical applications requiring high centrifugal force
• Use for sediment macromolecule/ligand binding kinetic studies, steroid hormone receptor assays, separation of major lipoprotein from plasma and deproteinisation of physiological fluids for amino acid analysis

b) Analytical ultracentrifuge

• Maximum rotor speed of 70000 rpm with RCF of 500000 x g
• Highly protective chambers with refrigerated and evacuated system also have an optical system to enable the sedimenting material to be observed throughout the process.
• Three types of optical system, a light absorption system, alternative Schlieren system and Rayleigh interferometric system (both measures refractive index of solution)

Design and types of Preparative Rotors

• These are rotating instruments in Centrifuges
• During rotation at high speed, higher stress forces generated
• Made up of aluminum alloy and titanium alloy which don’t rust-brass, steel or Perspex
• Can tolerate nearly twice the centrifugal force of rotors
• Protective coating to the metal surface by anodizing or by applying black epoxy paint
• Various types of rotors

a)      Swing bucket rotors

·         Common in low speed centrifuges

·         Also high speed, ultracentrifuges

·          Tubes accommodated in a pivoted bucket which rotates from a vertical to a horizontal position during acceleration

·         Bucket returns to vertical as centrifuge decelerates

·         Meniscus of sample always remains at right angles to axis of tube

·         Six-place rotor (6 buckets) most useful – can spin 2,3,4 or 6 samples (or sets of samples)

·         Pelleted material symetrically distributed in a hemisperical section at bottom of tube

·         Only particles in bottom of tube which move directly to bottom

·         Other particles move first to wall of tube, then towards bottom

 

Figure 2: Swinging-bucket rotor and its spinning

b)     Fixed-Angle Rotors

·         Tubes in pocket at fixed angle in rotor

·         Angle 10 to 50 degrees from vertical – at rest and during spin

·         Use up to 600,000 x g

·         Particles migrate to wall before moving towards bottom

·         Pellets always asymetrically distributed toward the outer aspect of the bottom of the tube

V

Figure 3: Fixed-angle rotor

c)      Vertical Rotors

·         First introduced in 1970’s – high-speed and ultracentrifuges

·         Solution re-orientates below 800 rpm, no disruption to gradient

·         Good for isopycnic and rate-zonal centrifugation

·         Not used for pelleting – pellet would be along length of tube and would fall off as liquid decanted

·         Also – “near-vertical” rotors – tube angle = 8 degrees

Figure 4: Vertical or near vertical rotor

Figure 5: Axis of rotation 

SEPARATION METHODS IN PREPARATIVE ULTRACENTRIFUGATION

1. Differential Centrifugation: The process of differential centrifugation is based on the fact that organelles have differences in size, shape and density. As a result, the effect of gravity on each is different. We can use this principle to separate an organelle from a homogenous solution of particles by artificially controlling the gravity of a solution. This is done by putting the solution in a variable speed centrifuge and rotating them at a high rate of speed. This creates a force that can be much greater than the force of gravity, and particles that would normally stay in solution will fall out and form a pellet at the bottom of the tube.

Differential centrifugation schemes involve stepwise increases in the speed of centrifugation. At each step, more dense particles are separated from less dense particles, and the successive speed of centrifugation is increased until the target particle is pelleted out. The final supernatant is removed, the pellet is resuspended and further study or purification can be done on it. The fractionation of rat liver is an example of how this process works:

 

Figure 6: Separation of cell fractionate by Differential Centrifugation

Figure 7: Separation of cell organelles from rat liver fractionate by Differential Centrifugation

2. Density Gradient Centrifugation

Density gradient centrifugation is a technique that allows the separation of cells, organelles and macromolecules, depending on their size, shape and density.

A density gradient is created in a centrifuge tube by layering solutions of varying densities with the dense end at the bottom of the tube. Cells and large molecules are usually separated on a shallow gradient of sucrose or other inert carbohydrates even at relatively low centrifugation speeds, while macromolecules such as proteins and nucleic acids are separated at higher centrifugation using ultracentrifuges.

Criteria for an ideal density gradient centrifugation medium are:

• the additive must form a solution within the required density range
• the additive must not interfere with, or damage, the sample
• the solvent must be compatible with the sample
• the solution must have a refractive index within the practical range, as well as a low viscosity
• The additive must be easily removable from the sample.

The additives for density gradient centrifugation can be divided into four main categories:

1.      Salts of Alkali Metals

These solutions fulfill most of the above requirements. However, due to the high ionic strength, hydrogen bonding within biological macromolecules (protein, nucleic acid - protein complexes) is impaired by a chaotropic effect. Therefore these salts are mainly used for DNA and RNA separations. Cesium chloride is used most frequently. Other useful salts include sodium iodide, sodium bromide, cesium sulfate and cesium acetate. Potassium tartrate has been used to separate viruses from host cells.

It should be kept in mind that the density of the sample is highly dependent on the hydration of the macromolecule, which in turn depends to a large extent on the dehydration power of the salt solution.

2.      Neutral, Water-Soluble Molecules

In this class of compounds sucrose is most widely used. It has a useful density range of up to 1.29. This range can be increased to 1.37 by addition of glucose or by dissolving sucrose in D₂O. Sucrose has very little effect on macromolecules, but affects enzyme activity. Due to its high osmotic pressure, sucrose solution dehydrates cells and their organellae very efficiently. Glycerol solutions are the preferred media for the separation of enzymes because they do not affect enzyme activity. They exhibit a high viscosity, requiring prolonged centrifugation times. More importantly however, glycerol penetrates biological membranes.

3.      Hydrophilic Macromolecules

Dextran gradients have been used for the separation of microsomes. Separations achieved with dextrans show similar results to those obtained by using synthetic sucrose/ epichlorohydrin co-polymers. In some cases bovine serum albumin has been applied, but the preparation of an appropriate solution is very difficult.

4.      Synthetic Molecules

These additives are the sodium or methyl glucamine salt of triiodobenzoic acid and of metrizoic acid. It should be kept in mind that the parent acid of these salts may precipitate on adjusting the pH to acidic values. Metrizamide, a covalently bonded compound of glycosamine and metrizoic acid is most widely used. This additive forms solutions of relatively low viscosity. These solutions are stable over a wide range of pH and ionic strength, and show practically no interference with the analytes.

Density gradient centrifugation methods are of two types, the rate zonal technique and the isopycnic (isodensity or equal density) technique.

Rate Zonal Technique: When mixtures of cellular extracts are layered on top of a density gradient in a tube and subjected to centrifugation, the various components move through the gradient at different rates that are dependent on their sizes and shapes. These different components appear as distinct bands or zones in the gradient with large components migrating farthest in the tube in a given period of time. The rate with which a fraction moves the fixed distance in the gradient tube is dependant of its sedimentation value (S) that, in turn is determined by the size and shape of that fraction. By comparing the different position of the components in the gradient, it is possible to make an approximate measurement of their molecular weight. It is, however, difficult to precisely determine these molecular weights, as this requires knowledge about the shape of these molecules, which is hard to determine with accuracy. This density gradient separation technique is called rate zonal centrifugation and is usually performed with a shallow sucrose gradient. The different components being separated by this technique are denser than any of the sucrose concentrations used in the gradient. Samples are, therefore, centrifuged just long enough to separate the components of interest. Longer centrifugation than necessary would allow all components to form a pellet at the bottom of the tube. One of the most important applications of this technique over the past decades was the separation of transfer RNA (4S) from ribosomal RNA that forms three different classes with distinct sedimentation values 23S, 16S and 5S. This helped to facilitate the characterization of the protein synthesizing system.

                                            

             

Figure 8: Rate zonal Density Gradient Centrifugation                                                                    

Isopycnic Technique: A second density gradient technique, called equilibrium density-gradient centrifugation is used to separate cellular components on the basis of their buoyant density. In this case the cellular mixture is centrifuged through a steep density gradient that contains a high concentration of sucrose, or more often, cesium chloride (CsCl). In these gradients, the molecules being studied have a density somewhere in between the highest and lowest densities of sucrose or CsCl generated in the gradient. The components of a sample begin to move down this gradient in the same way as they do in a rate-zonal density gradient. When a component of the mixture reaches a point where the density of the solution is equal to its own density, it stops moving further and forms a distinct band. The position of the band in the tube is characteristic of the buoyancy of that component. Buoyancy or buoyant density of a substance is its tendency to float in a medium, which in this case is the density gradient. Hence soluble proteins which have similar density (p=1.3 g cm^-3 in sucrose solution) cannot usually be separated by this method, whereas subcellular organelles (e.g. Golgi apparatus p=1.11 g cm^-3, mitochondria p=1.19 g cm^-3 and peroxisomes p=1.23 g cm^-3in sucrose solution can be effectively separated.

Figure: Isopycnic Density Gradient Centrifugation 

Equilibrium density gradient centrifugation using CsCl was for decades the method of choice in the purification of highly pure plasmid DNA. Meselson and Stahl, who developed this technique, were the first to use it in an experiment that provided evidence for the semi-conservative replication of DNA and confirmed the double helix structure of DNA proposed by Crick and Watson.

Application of Centrifugation

—Basic separation of Biomolecules

—Purification of mammalian cells

—Fractionation of subcellular organelles (including

—membranes / membrane fractions)

—Fractionation of membrane vesicles

—Identification of molecules

Extensive tool in molecular biology