Centrifugation/Cell-disruption techniques

❖Based upon behaviour of particles in an applied centrifugal field
❖Particles are suspended in specific liquid medium, held in tubes or bottles, which
are located in a rotor
❖Rotor is positioned centrally on the drive shaft of the centrifuge.
❖Particles which differ in density, shape or size can be separated since they sediment
at different rates in the centrifugal field, each particle sedimenting at a rate which is
proportional to the centrifugal field.

▪ Preparative: actual separation, isolation and purification of various cellular
components for subsequent biochemical investigations.
▪ Analytical: study of pure or virtually pure macromolecules or particles.

When a body travels a circular path, the angle subtended by an arc when its size is
equal to radius is called one radian i.e. a central angle to a circle has a size of one
radian if it subtends an arc length on the circle equal in length to the radius.
During one revolution, the distance covered is 2𝜋𝑟
and the radians subtended = 2𝜋𝑟 /r = 2𝜋 radians

The rate of sedimentation is dependent upon the applied centrifugal field (G) being
directed radially outwards, which is determined by the square of the angular velocity of
the rotor (𝜔, in radians per second) and the radial distance r, in cm, of the particle
from the axis of rotation, according to the equation:
𝐺 = 𝜔2
𝑟
One revolution of rotor= 2𝜋 radians, its angular velocity, in radians per
second, can be expressed in terms of revolutions per minute (rpm), the
common way of expressing rotor speed being:
𝜔 =
2𝜋𝑟
60

The centrifugal field (G) in terms of rpm is then:
𝐺 =
4𝜋2𝑟𝑝𝑚2𝑟
3600
and is expressed as a multiple of earth’s gravitational field g= 980cm2
i.e. the ratio of the weight of the particle in the centrifugal field to the weight of the
same particle when acted on by gravity alone, and is then referred to as the relative
centrifugal field (RCF).
𝐺
𝑔
=
4𝜋2
𝑟𝑝𝑚2
𝑟
3600 × 960
= 𝑅𝐶𝐹 = 1.11×10−5rpm2r

For spherical particle of known volume and density,
F=
4
3
𝜋𝑟𝑝
3
(𝜌𝑝− 𝜌𝑚)𝜔2
𝑟
Frictional force as stoke’s law:
𝑓𝑜 = 6𝜋𝑛𝑟𝑝𝑣

Velocity or sedimentation rate, 𝑣 =
2
9
𝑟𝑝
2(𝜌𝑝−𝜌𝑚)𝜔2𝑟
𝑛
Proportional to size, difference in density between particle and medium
Rate will be zero when the density of particle and medium are equal
Rate decreases when viscosity of medium increases
Rate increases as the CF increases

For assymetric as in rod-like molecules such as DNA and proteins like myosin, the
frictional coefficient of molecule can be increased by ten times that of frictional
coefficient of a sphere.
This results in sedimentation at a slower rate.

▪ As per the rotor design, radial dimension of a given particle changes
▪ r (min.) and r (max) will vary
▪ Rotors: fixed and swinging
In fixed rotor, as 𝐺 = 𝜔2𝑟, particle will experience greater field further away
from axis of rotation.The operative centrifugal field, in a fixed angle rotor for
example, differ by a factor of two between the top and bottom of the centrifuge tube.
Thus, the sedimentation rate of particles at the bottom of the tube will be twice that
of identical particles near the top of the tube.

Amount of solution in centrifuge tube:
If sample tube is only partly filled, then, in case of fixed angle and swinging bucket
Rotors, the r (min.) is effectively increased and the particles will therefore start to
Sediment in a higher gravitational field and have a reduced path length to travel.
Thus, the sedimentation will be quicker.
Apparatus Manual: maximum speed of rotor, maximum relative centrifugal field
generated

The sedimentation rate or velocity (v) of a particle can also be expressed in terms
of sedimentation rate per unit of centrifugal field, commonly referred to as its
sedimentation coefficient, s.
𝑣 = 𝑠𝜔2
𝑟
Since there are wide variety of solvent-solute systems involved in sedimentation
studies, the value of sedimentation coefficient varies according to temperature,
solution viscosity, and density, is often corrected to a value that would be obtained
in a medium with a density and viscosity of water at 20°C =
Standard sedimentation coefficient or 𝑠20𝑤

One Svedberg Unit (S):
The sedimentation coefficients of most biological particles are very small, and for
convenience its basic unit is taken as 10-13 seconds
For example, a ribosomal RNA molecule possessing a sedimentation coefficient of
5×10-13 seconds is said to have a value of 5S.

Sedimentation coefficient is influenced by
Size
Shape
Density of particle
# larger the molecule or particle, the larger its svedberg unit, and faster its
sedimentation rate.

▪ Small benchtop:
Least expensive,
Collect small amounts of material that rapidly sediments (yeast cells, erythrocytes,
coarse precipitates)
4000-6000 rpm
CF= 3000-7000 g
Ambient temperature or refrigerated
#small microfuges provide instant acceleration to maximum speed of 8000-13000
rpm developing fields of approx. 10,000 g.

▪ Large capacity Refrigerated centrifuges:
These have a maximum speed of 6000 rpm and produce a maximum relative
centrifugal field of approx. 6500 g.
Have refrigerated rotor chambers and vary only in their carrying capacity
Interchangeable swinging bucket and fixed angle rotors enabling use of 10, 50 & 100
cm3 tubes
# rotors must never be loaded with odd number of tubes
# In case of partially loaded tubes, these must be diagonally opposite to each other
so that load is evenly distributed around the rotor axis

▪ Separation and fractionation of macromolecules by subjecting them to a strong
centrifugal force originated with T. Svedberg and co-workers, who in the early
1920s invented and developed the instrument called the ultracentrifuge.
▪ centrifugal forces that can be attained in uItracentrifugation are in the order of
500,000g
▪ The high forces can be used for the determination of molecular weights of
macromolecules or for preparative fractionations

▪ Analytical ultracentrifuge:
study of the behaviour of macromolecules
in solution under the influence of a strong
gravitational force
provide a photographic record
▪ automatic temperature and speed controls for the rotor,
▪ a high vacuum chamber to reduce friction,
▪ an optical system for measuring the rate at which individual
peaks (representing different proteins) move towards the
bottom of the cell,
▪ an automatic photographic system for recording changes
in concentration at specified intervals, and special cells

▪ Optical systems are available: Schlieren, interference, and absorption
▪ most commonly used system is the astigmatic Schlieren optics.
▪ The photographic record of the sedimentation pattern using Schlieren optics
gives the concentration gradient in the cell in terms of the refractive index
gradient.
Molecular weight of proteins:
a) Sedimentation equilibrium:

in which v is the partial specific volume of the protein (increase in volume when
1 g of dry protein is added to a large volume of liquid), can be assumed to be
0.75.
The angular velocity can be expressed as
where V is the velocity of the centrifuged solution and x the distance from the
center of the rotor. If the number of revolutions per second is z, then

It is customary to give the velocity in rpm; since Z = rpm/60, then
▪ # main disadvantage of the sedimentation equilibrium method is that it requires
several days for a determination

b) sedimentation velocity:
high speeds at which the protein particles sediment at a fast rate are used. If the
molecular weight of the particles is high, their rate of diffusion can be
neglected.
▪ The sedimentation velocity depends on the shape and the hydration of the protein
molecules
▪ The rate of sedimentation is usually expressed in terms of the sedimentation constant
s, the velocity per
unit centrifugal field force
▪ A sedimentation constant of 1 x 10-13 is called a Svedberg unit (S), and
sedimentation constants generally are given in Svedberg units.

Major areas of application of analytical ultracentrifuge:
(1) the study of reversibly se1f-associating systems, leading to the identification
of specific oligomers and
(2) the analysis of stable or transient heterogeneous associations between
membrane proteins

Density Gradient:
▪ Size of the separated molecules in a mixture
▪ can be carried out with low concentrations of solute
▪ Permits separation on a preparative scale

▪ Each substance sedimenting at its own rate forms a band or zone in the fluid
column
▪ The solute zones will be separated from one another by distances related to
their sedimentation rates
▪ After centrifugation, each substance can be drawn off separately for the
determination of its sedimentation rate and for further analysis
▪ Limitations:
#only small amounts of material can be separated at high speeds,
#and the separation is incomplete due to the wall effect (from some particles being reflected
from the tube walls back into the solution, sticking to the walls, or clumping).

Isopycnic gradient centrifugation:
▪ Separation is based on differences in density of the macromolecules in a sample solution that is usually
distributed evenly throughout the gradient column before ultracentrifugation
• Gradient is steep, gradient density is maximum greater than that of the most dense sedimenting species
• Centrifugation is carried out at high speed for long time
• Applications: DNA, plasma, lysosomes, mitochondria, peroxisomes

▪ Isopycnic density gradient ultracentrifugation is an equilibrium (static) method
that depends on the buoyant density of the macromolecules.
▪ The gradient is self-generating in the centrifugal field, and its density range is
so adjusted as to be denser at the bottom of the tube and less dense near the
meniscus than the macromolecules in the column of solution.
▪ Thus, the macromolecules, most frequently nucleic acid or viruses, form a
definite band at a definite level in the column and remain there irrespective of
the length of centrifugation

▪ Materials used to form gradients should be chemically inert to the studied system,
nontoxic, and soluble in water and salt solutions; they also should have a high density, high
molecular weight, and low viscosity
▪ For separation and analysis of proteins, the gradient material should contain no nitrogen.
▪ High-density materials are required to form a steep gradient;
▪ Low viscosity permits easy handling during gradient formation, and rapid sedimentation
and fractionation.
▪ Sucrose is the most widely used material for the gradient. Its main disadvantage is high
viscosity
▪ Ficoll is a commercially available, water-soluble, neutral colloid with properties similar to those of
a polysaccharide.
▪ Its average molecular weight is about 50,000, and it is stable in nonoxidizing neutral or alkaline
solutions.
▪ Its viscosity and density are lower than those of sucrose. For the separation of nuc1eic acids, inorganic salts
(mainly cesium chloride or rubidium chloride) are used.

▪ Cell disruption is the process of obtaining intracellular fluid by opening the cell
wall
▪ It is a unit operation closely linked to both upstream and downstream processes
▪ Types:
Mechanical
Non-Mechanical

Mechanical:
❑Forces acting in suspension (High-pressure homogenization, hydrodynamic
cavitation, mechanical agitation)
❑Forces acting through solid-solid interaction (Bead mill, mortal & pestle, bead
beating)
Non-mechanical approaches:
❑Physical (Temp., osmotic shock, dessication, sonication)
❑Chemical (pH extremes, detergents, solvents, antibiotics, chelating agents)
❑Biological (cell wall lytic enzymes, wall inhibitors)

High-pressure homogenization
▪ Suspension is pressurized using a positive-displacement pump.
▪ The pressure is rapidly released by passage through a fine orifice or annular gap.
▪ Thereafter, the cell suspension typically impacts a solid surface to enhance cell
breakage further

▪ A cavitation event similar to that generated by ultrasound can be induced through
fluid-flow patterns.
▪ According to Bernoulli’s equation, on flow through an orifice, the increasing velocity
required to satisfy the continuity equation is accompanied by a decreasing pressure
in the fluid
▪ Where the pressure decreases to the vapor pressure of the suspending medium or
below,the formation of vapor cavities results in the phenomenon of cavitation with
its associated cell damage or disruption on cavity oscillation and collapse

▪ Mortar & Pestle: plant samples frozen in liquid nitrogen
When material has been disrupted, metabolites can be extracted by adding solvents
▪ Blenders: high speed to disrupt cell walls;
Similar to centrifugation material suspended in a liquid medium

❑ Suspension is agitated vigorously in the presence of a particulate solid phase,
typically glass beads
❑ Disruption is achieved through interparticle collision and solid shear
❑ The bead mill consists of a horizontal or vertical cylinder fitted with a central drive
shaft and several impellers
❑ The cylinder is partly filled (typically 80%) with small glass or ceramic beads
❑ Agitation of the microbial slurry in this system in the presence of the beads at
impeller tip speeds of approximately 15 m s −1 results in cell disruption
❑ As with the homogenizer, the energy added to the system is partly used in cell
breakage, but largely dissipated as heat

a) Temperature:
High heat inactivates cell by disrupting cell wall and release intracellular products.
The effect of heat depends upon various factors such as pH, temperature, chelating
agent, ionic strength, presence of enzymes (proteolytic and hydrolytic), time etc.
Disadvantage:Cannot be used for heat labile substances
b) Osmotic shock:
• In this method, either hypotonic or hypertonic solution is used.
• The cell suspension is placed on either of the solution which create osmotic shock.
• Hypotonic solution:
• Plasmolysis occur, the water enters the cytoplasm of cell and well burst.
• Hypertonic solution
• Plasmolysis occur, the cell shrinks due to loss of water from cell.

c) Sonicator:
• About 50Khz frequency is applied on cell suspension which causes the formation
of tiny bubbles within liquid.
• It is very fast method.
• It breaks cells in 30-60 second and yeast cell (2-3mins).
• Disadvantages:
• Heat generation
• Noise pollution
• Expensive process
• Generate free radicals that might interfere desired product.

a) Extreme acidity or alkalinity cause cell lysis
b) Solvents used for the release of intracellular compounds include alcohols such as
ethanol, isopropanol, and butanol (at concentrations of 10–80%), dimethyl
sulfoxide, toluene (2%), and methyl ethyl ketone.
c) Detergent: to lyse or permeabilize cells for release of soluble components through
perturbing the protein–lipid interactions through interaction with the nonpolar
hydrophobic tail and polar hydrophilic head of the detergent molecule
❖Anionic detergents (e.g., sodium dodecyl sulfate, SDS) disorganize the cell
membrane
❖Cationic detergents are suggested to act on the lipopolysaccharide component of
the cell envelope as well as interacting with the phospholipids.
❖Nonionic detergents such as Triton X-100 and Pluronic F-68 cause a partial
solubilization of proteins in the inner membrane structure, resulting in
permeabilization

d) Antibiotics:
Polymyxin, azoles, Nystatin are cell membrane inhibitor and destroy cell membrane
inhibitor and destroy cell membrane formation causing release of cellular content.
e) Chelating agents:
chelates cations (bivalent) and make unavailable for cell causing disruption of cell
membrane. (Mg2+, Ca2+) such as EDTA

▪ glycosidases that hydrolyze polysaccharide chains;
▪ acetylmuramoylL-alanine amidases that cleave polysaccharide polypeptide
linkages
▪ endopeptidases that lyse polypeptide chains.
Each of these attacks the peptidoglycan wall, requiring the prior removal of the outer
membrane of Gram-negative bacteria
▪ Lysozyme hydrolyzes β-1-4 glucosidic linkages of polysaccharide chains of
peptidoglycan

❖Ensure all sample tubes are evenly filled. If additional tubes are required for
balancing, fill them with water or a liquid of similar density to the sample,
and ensure the mass is balanced to the nearest 0.1 grams.
❖For each tube inserted in the rotor, add a tube of equal weight directly
opposite it. This will ensure the center of gravity remains in the center of the
rotor.
❖Rotate the rotor 90° and add two additional tubes directly opposite one
another.
❖Repeat.

HOW TO BALANCE 3 TUBES, 5 TUBES, OR 7
TUBES IN A CENTRIFUGE WITH 12 POSITIONS?
▪ There are two ways to balance three tubes. The first option is to insert three
sample tubes next to each other, and create three balance tubes to be situated
directly across from the sample tubes.
▪ Alternatively, three sample tubes may be spaced evenly around the rotor.

❖To balance five tubes, create one balance tube and place
two sets of three tubes across from each other

❖To balance seven tubes, create one balance tube and
place two sets of four tubes across from each other

❖Pay close attention to noise, vibration, shaking, or grinding and
stop the unit immediately if this occurs
❖Inspect critical components, and look for signs of wear
including scratches, or effects of chemical exposure on the rotor
❖Regularly clean the centrifuge with neutral cleaning solutions
(alcohol or alcohol-based disinfectant) applied with a soft cloth
to rotors and accessories. Daily cleaning should include the
interior portion of the centrifuge, the rotor chamber, and
surfaces with electronic components, such as touchscreens and
keypads
❖It is important to be aware of the different types of samples
used with the centrifuge and any specific products or protocols
necessary for cleaning spills

Centrifugation/Cell-disruption techniques

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