CENTRIFUGATION
Centrifugation
is a method for separating biomolecules from each other in a solution. This is done by subjecting a
biological sample to an intense force by spinning the sample at high speed. The
application of this technique range from collection & separation of cells,
organelles & molecules to the study of molecular weights of macromolecules.
Centrifugation
is a technique, in which the gravitational force on a particle is increased to
effect its sedimentation . sedimentation of particle depends on, the density of
the particle, the size of the particles & the viscosity of the medium. The
gravitational force under normal condition is 980 cm/sec2 i.e.,
equal to one ‘g’ unit.
Heavier
particles sedimented quickly due to greater gravitational pull on them. But
lighter particles that do not sediment easily can also be sediment quickly by
subjecting them to higher gravitational force. Centrifuges are used to increase
the gravitational forces on particles.
CENTRIFUGAL FORCE & PRINCIPLE OF SEDIMENTATIONS
Centrifugal force, as defined by Isaac
Newton, is the tendency of any rotating object to move away from its center of
rotation. The opposing force is known as centripetal force, which is directed
towards the centre of rotation.
Therefore , the
centrifugal force on an object mainly depends on the mass of the object & the
velocity or speed of rotation.
In vacuum,
centrifugal for F=ω2r
ω = its angular velocity
r =
radius of rotation
in any medium
other than vacuum, the centrifugal force is opposed by two opposing forces:
Buoyant force – Tendency of particles to
float
Force of viscous drag – Friction
developed by the viscosity of the medium
Types of Rotors
The rotors are the important
components of the centrifuges. Three types of centrifuges are used in the
centrifuges.
i.
Fixed
angle rotors
ii.
Vertical
tube rotors
iii.
Swinging
bucket rotors
Since
the centrifuge rotors have to withstand enormous amount of force during the
spin, the rotors are robustly built to withstand tremendous strains. Inspite of
their heavy construction, high speed rotors show signs of metal fatigue on
continues use. To avoid catastrophic accidents, it is necessary to debate their
safe maximum speed on a scheduled basis, usually 10% of initial maximum speed
per 1000 hours use.
Fixed angle rotors
The
centrifuge tubes containing the sample are kept at an angle 30° to the
horizontal and the particles are subjected to a lateral force. When the
particles strike the side of the tube, they are driven downwards and sediments.
Vertical tube rotors
It
is also called as zero angle rotors, the tubes are placed vertically, (i.e)
parallel to the axis of rotation. In these rotors, the particles are separated
as vertical zones. The diameter of the
tube is shorter than the length of tube, there is a shorter distance for the
components in the tubes to travel and hence separation requires lesser time.
When the centrifuge is turned off, the separated zones reorient themselves to
form horizontal zones. The vertical rotors are widely used especially for
purification of DNAs using cesium chloride density gradients.
Swinging bucket rotors
The
tubes spin horizontally and hence the particle is subjected to a constant
force. The path length for the particle to travel is increased which is equal to depth of the suspension.
Therefore, the sedimentation times is increased accordingly in this type of
rotors.
TYPES OF CENTRIFUGATION
The
basic centrifuge consists of two components, an electric motor with derive
shaft to spin the sample and a rotor to hold tubes or other containers of the
sample. Many centrifuges of different designs are available in the market.
However all centrifuges can be roughly categorized into different types on the
basis on their operating speed. They are
i.
Low
speed centrifuges
ii.
High
speed centrifuges
iii.
Medium
speed centrifuges
iv.
Ultra
centrifuges
i.
Low speed
centrifuges
Most
laboratories have a strande low speed centrifuge used for routine sedimentation
of relatively heavy particles. The common centifuge has a maximum speed in the
range of 4000 – 5000 rpm, with RCF values up nto 3000 x g. These instruments
usually operate at room temperature with no mass of temperature control of the
samples. Two types of rotors, fixed angle and swinging bucket may be used in
the instrument. Centrifuge tubes or bottles that contain 12 or 50 ml of sample
are commonly used. Low speed centifuges are especially useful for the
sedimentation of coarse precipitates or red blood cells.
The
sample is centrifuges until the particles are tightly packed into a pellet at
the bottom of the tube. The upper liquid portion, the supernatant is then
separated by decantation. This is also called as desktop centrifuges. In all
these centrifuges the rotors are mounted on a rigid shaft. It is therefore very
important that the contents of the centrifuge tubes are balanced accurately and
that they are never loaded with an odd number of tubes. If the rotor is only
partially loaded, the tubes have to be placed diametrically opposite to each
other to disperse the load evenly. These precautions are true for all types of
centifuges.
ii.
High
speed centrifuges
High
speed centifuges can operate with maximum speed up to 25,000 rpm providing
about 90,000 g centrifugal force in a process. They are usually equiped with
refrigeration equipment to remove heat generated due to fiction between the air
and the spinning rotor. The temperature can easily be maintained in the range
0-4°C by means of a thermocouple. The highest carrying capacity may be 1.5dm3.
Three types of rotors are available for high speed centrifugation, the fixed
angle, the swing bucket and the vertical rotor. Rotors were constructed from
metals such as aluminium and titanium. Although metal rotor have great
strength. But they are very have to handle, they are not corrosion resistant,
and they become fatigued with use. Rotors are now available that are fabricated
from carbon fiber composite materials.
These
new rotors are 60% lighter than comparable aluminium and titanium rotors.
Because of the lighter weight, acceleration and deceleration times are reduced,
thus centrifuge run times are shorter. This also results in lower service and
maintenance costs. Instruments are equipped with a brake to slow the rotor
rapidly after centrifugation. These instruments are routinely used to collect
microorganisms, cell debris, cells, large cellular organelles, precipitates of
chemical reactions and immunoprecipitates. Although these centrifuges are
usefull in isolating sub cellular organelles such as the nuclei, mitochondria,
lysosomes etc.
iii.
Medium speed
centrifuges
It
is also called as microfuge. These instruments, which are designed for the
bench top, are used for rapid pelleting of small samples. Fixed angle rotors
are available to hold up to eighteen 1.5 og 0.5 tubes. The maximum speed
of most commercial microfuges is between
12,000 and 15,000 rpm, which delivers a force of 11,000 – 12,000 x g. Some
instruments can accelerate to full speed in 6 seconds and deaccelerate within
18 seconds. Most instruments have a variable speed control and a momentary
pulse buttons for minispins.
iv.
Ultra centrifuges
The
most sophisticated of the centrifuges are the ultracentrifuges. Because of the
high speed attainable, intense heat is generated in the rotor. So the spin
chamber must be refrigerated and placed under a high vacuum to reduce fiction.
The sample in a cell or tube is placed in a rotor, which is then driven by an
electric motor. Although it is rare, metal rotors when placed under high stress
sometimes break into fragments.
The
rotor chamber on all ultracentrifuges is covered with protective steel armor
plate. The drive shaft of the ultracentrifuge is constructed of a flexiable
material to accommodate any “Wobble” of the motor due to imbalance of the
samples. The low, medium and high speeds are of value only for preparative
work, that is, for the isolation and separation of precipitates in biological
samples.
Ultracentrifuges
can be used both for preparative work and for analytical measurements. The
shaft is made up of aluminum or titanium alloy of high tensile strength to with
stand the great forces generated during centrifugation. Ultrcentrifuge can be
carried out to obtain certain biological material in isolation from the
components (macromolecule or sub-cellular particle) with respect to its
molecular weight or sedimentation coefficient. This centrifuge can operate at
speeds upto75,000 rpm providing centrifugal force in excess of 5,00,000 x g.
Centrifugation
for isolation and purification of components is known as preparatory
centrifugation, while that carried out with a desire for characterization is
known as analytical centrifugation. One of the most versatile model is the
Beckman TLX, a microprocessor-controlled table top ultracentrifuge. With a
typical fixed angle rotor, which holds six, 0.2-2.2 ml samples, the instruments
can generate 1,00,000 rpm and an RCF of 540,000 x g.
Analytical ultracentrifuge
Analytical ultra centrifuge is the most
commonly used centrifuge. These instruments can operate at 70,000 rev min-1.
And the relative centrifugal field is about 500000 g.
Instrumentation
It consists of a motor, rotor in a
protective armored chamber. This chamber is refrigerated and evacuated. And
there is an optical system to observe the sedimenting material throughout the
process.
Optical system
·
Three types of optical systems are available in
the analytical centrifuge.
·
Light absorption system
·
Schlieren system
·
Rayleigh interferometric system.
Rotor
·
The rotor is solid, with holes to hold the test
tube.
·
This rotor is connected with a wire coming from
the drive shaft of a high speed motor.
·
The tip of the rotor contains a thermistor for
measuring the temperature.
·
Several types of rotors are available.
Analytical cell & counter poise cell
§
A wide variety of cells is available and has a
capacity of between 0.4 and 1.0 cm3.
§
Analytical cells are used with the ultra violet
light absorption optical system.
§
This is used to prevent the convection and
usually have a 12mm optical path length centerpiece.
§
Double sector cells are used to absorbing
components in the solvent and to correct the redistribution of solvent
components.
§
Double sector cells also facilitate the
measurements of differences in sedimentation co-efficient.
Lens
·
The rotor chamber contains an upper condensing
lens & lower collimating lens. The condensing lens with a camera lens
focuses light on to a photographic plate. The collimating lens collimates the
light, so the sample cell is illuminated by parallel light. In more advanced
instruments the photographic detector system is replaced by an electronic
scanning system.
Procedure
§
In the ultra violet absorption system, suitable
wavelength of light is passed through the moving analytical cell.
§
Analytical cell contains the solution under
analysis(protein/nucleic acid)
§
The intensity of the transmitted light is
recorded on a photographic plate/scanning system.
§
The scanning system allowing direct
visualization of the results during the experiment.
§
Different wavelengths of light can be selected.
§
The optical system records the change in
refractive index of a solution.
§
In the schlieren optical system, the deviated
light is passed through a schlieren diaphragm and a cylindrical lens.
§
The result is production of peak in the final
image on the photographic plate, which is used as the detector system.
§
After a period of sedimentation, the peak height
is diminished and the width increases.
§
But the disadvantage is schlieren system is not
sufficiently sensitive to detect small concentration differences.
§
Hence more sensitive Rayleigh interference
optical system is used.
§
Thus the analytical ultra centrifuge plays a
crucial role in the techniques.
Applications:
·
It has many applications in biology, especially
in protein chemistry, nucleic acid chemistry etc.
Determination of relative molecular mass:
·
To determine the relative molecular mass of a
macro molecule based on sedimentation velocity and sedimentation equilibrium.
In the sedimentation velocity method, the sedimentation co efficient of the
molecule is initially determined either by boundary sedimentation or band
sedimentation. In this method, the particles are uniformly distributed through
the cell. The relative molecular mass of the particle may then determined by
using the Svedberg equation.
RTs
D (1-
V P)
Mr = relative molecular mass
R = molar gas constant
T = absolute temperature
D = diffusion co efficient
V = partial specific volume
P = density of the solvent at 20° C.
·
This technique is extremely useful for the
determination of the relative molecular mass of proteins.
Estimation of purity of macromolecules:
§
This is extensively used in the estimation of
the purity of DNA preparations, viruses and proteins.
§
This is done by, sedimenting boundary using the
technique of sedimentation velocity.
§
Homogeneity is recognized by a single sharp
symmetrical sedimenting boundary.
§
Impurities in the preparation are displayed as
additional peaks.
§
Detection of conformational changes in
macromolecules.
§
It is successfully applied to the detection of
conformational changes in macromolecules.
§
For e.g. DNA may exist as single or double
stands, either linear or circular in nature.
§
DNA will undergo a number of conformational
changes, if it is exposed to organic solvent or elevated temperature.
§
The conformation changes may be ascertained by
examining differences in the sedimentation velocity of the sample.
§
The more compact molecules having a lower
frictional force and more disordered molecules have a greater frictional force
and sedimentation occurs more slowly.
§
Conformational changes therefore detected by
differences in sedimentation rates of the sample.
Thus it has a number of important applications and
therefore plays an important role in biological world.
TYPES OF CENTRIFUGATION
i.
Differential
centrifugation ( makes use of different speeds of effect sedimentation of
different particles, step & )
ii.
Density
gradient centrifugation ( makes use of different densities of the particles at
same speed)
DIFFERENTIAL CENTRIFUGATION
1. During centrifugation, particles
sediment through the medium in which they are suspended at rates related to their
size, shape & density. Differences in the sedimentation coefficients of the
various subcellular particles provide the means for their effective separation.
2. Differential
centrifugation, a technique introduced to cellular research in the early 1940s by
the noted biologist & Nobel laureate
Albert claude is one of the classic procedures for isolating subcellular
particles & involves the stepwise removal of classes of particles by
successive centrifugation at increasing RCF.
3. The most
common & simple method is the differential centrifugation pelleting of
samples. In this method, the centrifuge tube is filled with a homogenous
solution & then centrifuged at required rpm for required time &
temperature. It results in two fractions, a pellet at the bottom of the tube
containing the sedimented material at that speed & a supernatant solution
containing the unsedimented material. ( any particular component of the
homogenous solution, that may end up in the supernatant (or) in the pellet (or)
if may be distributed in both the fraction dependeds upon its size/or the speed
& length of centrifugation).
4. The process
of separation of cell organelles is known as subcellular fraction. To isolate a
specific organelle are homogenized in a suitable homogenizing medium at 4°c.
the resulting suspension containing many subcellular organization.
This type of
separation is commonly used in simple pelleting & in obtaining partially
pure-preparation of subcellular organelles & macromolecules.
For the study of
subcellular organelles, tissue or cells are first disrupted to release their
internal contents. This crude disrupted cell mixture is referred to as a
homogenate. During centrifugation of a cell homogenate, larger particles
sediment faster than smaller ones & this provides the basis for obtaining
crude organelle fractions by differential centrifugation. A cell homogenate can
be centrifuged at a series of progressively higher g-forces & times to
generate pellets of partially purified organelles.
5. This method
uses a series of 4 different centrifugation steps. Each step yields a pellet
& supernatant. The supernatant is subjected to centrifugation next step.
6. The two
fractions are recovered by decanting the supernatant solution into a fresh tube
& leaving the pellet in the original tube. The supernatant can now be
recentrifuged at a higher speed to effect further sedimentation of lighter
particles with formation of a new pellet & supernatant, the pellet can also
be recentrifuged after resuspending in a suitable media. Thus, by increasing
the speed (rpm) & hence the g value step by step, one can pellet down
different components of the homogenate at each step. This type of
centrifugation, is known as differential centrifugation.
7. Differential
centrifugation is used to fractionate different organelles of the cells. For
examples in, a typical procedure, the cells are homogenized in 10 volumes of
buffer containing 0.25 sucrose ( to maintain isotonic condition). The
homogenate is centrifuged at 1,000 x g for 20 minutes to pellet the nuclei. The
supernatant solution is recentrifuged again at 10,000x g for 20 minutes to
pellet the mitochondria, lysosomes & peroxisomes. The supernatant solution
is again centrifuged at 30,000xg for 30 minutes to pellet the microsomes.
The
supernatant is further centrifuged at 1,05,555xg for 60 minutes to pellet
ribosomes. The final supernatant solution is the cytosolic fraction, which
contains tRNAs, soluble enzymes & precursors for macromolecules. Thus,
different organelles of the cells can be fractionated by differential
centrifugation.
B.DENSITY GRADIENT CENTRIFUGATION
Density
gradient centrifugation employs medium which has gradients. The separation
under the centrifugal field is therefore dependent upon the buoyant densities
of the particles. The gradients, apart from existing their separating effect,
eliminate mixing of separated components due convection & mechanical
vibrations.
This method
gives a much better separation than differential centrifugation.
This
technique was introduced to cellular research in the late 1940’s & early
1950’s by N.G. Anderson & M.K.Brakke & is an exceedingly powerful
preparative tool.
Types of density gradient centrifugation
The density
gradient centrifugation is of two types
1.Rate-zonal centrifugation
using a preformed gradient of different densities (linear or step gradients
using glycerol or sucrose solution.)
2. Isopycnic
centrifugation using a self-forming gradient during the centrifugation (e.g.
cesium choride gradient).
Characteristics of Good Gradient Materials
§ It should not
affect the biological activity of the sample.
§ It should be
non-corrosive to the rotor.
§ It should be easily sterilizable.
§ It should be
cheap & readily available.
§ It should not
interfere with the easy technique.
§ It should be
easily removable from the purified product.
§ It should not
get absorbed in the ultraviolet range.
Examples
Sucrose, caesium
Chloride, Ficoll and Polyvinyl pyrrolidone (Percoll)
Sucrose
It
is a low cost material and its modest viscosity makes it the most commonly used
gradient solute. However, a maximum density of about 1.29 g/cm3 only
can beobtained with sucrose. It cannot be used in certain applications because
it has an osmotic effect due to its ability to penetrate through some cell membranes.
Thus it may cause shrinkage of some organelles by osmotic withdrawl of water
from them. Since ordinary contains a high concentration of calcium, it cannot
be used for making gradients. A specially purified reagent grade sucrose should
be used for analytical purpose. Sucrose solution should be prepared freshly.
Solutions that are slightly turbid or cloudy should be discarded promptly. Low
concetrations of sucrose solution (0.25M) support microbial growth.
Caesium Chloride
Since,
it s freshly soluble in water, solutions of high density and low viscosity can
be prepared. However, the ionic nature of the saltand its low molecular cause
severe osmotic effects when cells or organelles are suspendedin the medium.
Therefore caesium chloride is used extensively for the separation of RNA and
DNA only. The solutions of ceasium chloride produce gradients from 1.099 to 1.9
cm-3 by simple centifugation. The major disadvantage is the high cost of the
salt.
Ficoll
The
molecular weight of ficoll is about4,00,000. It is a polymer of sucrose and
epichlorohydrin. It is originally developed as a plasma substitute for
transfusion purposes. It is stable non ionic polymer. But it is contaminated with small ionic materials
and therefore ficoll solutions are dialyzed extensively before use. It has
become a useful gradient solute because of its low osmotic effects. Ficoll
solutions are highly viscous in nature and it is more difficult to dissolve
them in high concentration then sucrose. It is also quite expensive. Therefore
it is used only in essential cases.
Percoll
It
is polyvinyl pyrolidone coated colloidal silica. Using percoll gradients with
density ranges from 1.0 to 1.3 g / cm3 can be formed. It is non
toxic to cell and has a very low viscosity. Its osmotic effects also very low.
Percoll suspensions produce self generating gradients by simple centrifugation.
An initially uniform suspension of percoll when centrifuged at forces >
10,000 x g redistribution itself to form a gradients. The steepness of the
gradient charges with time of centrifugation, the geometry of the rotor and the
initial concentration of the suspension.
Principles of rate zonal centrifugation
Particle
separation by the zonal technique is based upon differences in the size, shape
and the density of the particles, the density and viscous of the medium and the
applied centrifugal field. However, subcellular organelles such as
mitochondria, lysosomes and peroxisomes which have different densities but are
similar in size cannot be separated efficiently using this method.
The
bottom of the centrifugation medium contains the highest density. However, its
density does not exceed that of the densest particles to be separated. The
sample is carefully layered on top of a pre-formed liquid density gradient. The
density of the gradient increases very slow from the top to bottom. The sample
is then centrifuged until the particles travel through the gradient to form
discrete zones. Centrifugation must be terminated before any one of the
separated zones reaches the bottom of the tube.
Principles of rate isopycnic centrifugation
Isopycnic
centrifugation depends only upon the buoyant density of the particle. Shape and
size of the particle do not have any effect on separation. In isopynic
centrifugation, the maximum density of the gradient always exceeds the
densityof the most dense particles. During centrifugation, sedimentation of the
particles occurs until the buoyant density of the particle and the density of
the gradient are equal. At this point of isodensity no further sedimentation
occurs, irrespective of howlong centrifugation continues.
This
technique is used to separate particles of similar size but of differing
density. Subcellular organelles such as golgi apparatus (r = 1.11 g/cm3),
mitochondria (r = 1.19 g/cm3), and peroxisomes (r = 1.23 g/cm3)
can be effectively separated by this method.
The
sedimentation rate of rigid spherical particle is calculated using the equation
V = 2/9
x r2p (ρp-ρm)/η x g
In
the above equation,the density ot the sedimentation particle(ρp)
becomes equivalent to the density of the medium (ρm) then V will
become zero. A density gradient is prepared in a tube in such a manner that the
density goes on increasing toward the bottom of the tube. A solution of
different particles differing in their buoyant densities is centrifuged in this
medium. Different particles will travel different length and become stationary
at a region where the density of the layer below them is greater than their own
buoyant density. This is the basis of isopycnic centrifugation technique. This
is also known as sedimentation equilibrium centrifugation.
Commonly use
gradient materials and their densities at 20°C
Material
|
Maximum
density (g/cm3)
|
Sucrose
(66%)
|
1.33
|
Glycerol
|
1.26
|
RbBr
|
1.63
|
CsCl
|
1.91
|
Cesium
formate
|
2.11
|
Ficol
|
1.18
|
Preparation of Density Gradients
Two types of
density gradients may be prepared for centrifugation purposes. They are
·
Discontinuous
density gradient
·
Continuous
density gradient
Discontinuous density gradient
In
Discontinuous density gradient, density increases abruptly from one layer to
another. It is prepared by carefully layering solutions of continuously
decreasing densities over each other. The highest density solution is placed at
the bottom and the lightest density solution is at the top. The sample is then
layered at the top layer.
Continuous density gradient
Continuous
density gradient is prepared using a special device which has two chambers. One
chamber contains high density solution and the other chamber contains a very
less dense solution.
The chamber that contains a high dense
solution has an outlet and a stirrer. When the tap of the first chamber is
opened, the dense solution drips into the centrifuge tube. The levels of
solution are the first chamber thus decreases and is compensated by the less
dense solution from the second chamber.
Using the stirrer, the solution is
mixed well. The density of the solution in the first chamber is slightly less
than that of original solution. The tap is agin opened to fill up a particular
length of the centrifuge tube and then closed. By repeating this, a continuous
linear density gradient is formed.
Sample Application
The volume of the sample that can be
applied to the centrifuge tube depends upon the cross sectional area of the
gradient prepared in the centrifuge tube. Thus a volume of 1ml can be applied
to centrifuge tubes having a inner diameter of 2.5 cm. the concentration of the
medium in which the sample is prepared should be about 10 times less than the
concentration of the strating gradient. The sample can be applied through the
syringe which is held about 2 to 3 mm above the gradient inclined at an angle
of 45°C touching the wall of the centrifuge tube. For fragile solution like
DNA, a pipette is used.
Recovery of the sample
The first method used in the recovery
of samples involves puncturing of the centrifugation tube at the bottom with
the help of a needle and collecting the dripping medium in separate tubes.
The second method is called the
displacement technique. In this a cover with an outlet is placed at the top of
the gradient. A very dense solution is pumped through this tube. The dense
solution displaces the gradient layers which strat coming out from the outlet
provided in the cover and are collected in separate tubes.
Applications
i.
Rate
– zonal centrifugation method is useful for separation of RNA-DNA hybrids and
ribosomal subunits.
ii.
Isopycnic
centrifugation method is suitable for separation of intracellular organelles
such as mitochondria, lysosomes and peroxisomes.
iii.
It
is also suitable for nucleic acid fractionation.
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