Paper 9: Techniques Used in Molecular Biophysics I (Based on Hydrodynamics)

Module 7: Introductions, Principles & types of centrifugation

Introduction:

Centrifugation is one of the widely used techniques in the most of the biological research areas
such as molecular biology, Immunology, cell biology, etc. Currently it is used for isolation of
cells, subcellular organelles, DNA, RNA, proteins, or lipids and downstream processing. A
centrifuge exerts excess centrifugal force (g-force) to separate suspended particles from the
solution. The particles of different size separated on the basis of the difference in their
centrifugal acceleration, size and shape, the volume fraction of present solids, a difference
density between the particle and solution, and the viscosity of medium. Simply it separates solid
particles from the concentrated suspensions of solutions for further treatment during isolation
and purification.

Objective: In this module we discuss following things in detail,

1. Centrifugation and its principle

2. Theory of Operation
3. Centrifugal force
4. Types of Rotors
5. Types of Centrifuges

1. Centrifugation:

Centrifugation exerts a centrifugal force to separate the particle of heavier density from the liquid
medium. In laboratory centrifuge is generally used to:

 separation of cellular debris of blood to get the cell free plasma or serum
 separate different cellular fractions and other components for further analytical or
chemical analysis.
 To separate labeled protein/antibody of ligand bounded protein from the
unlabelled/unbound fractions in biochemical assays.
 To separate aqueous and organic phase dissolved solute/solvent.
 To separate lipid parts from other components of plasma.

Principle

The particles to be separated are suspended in a specific liquid media and kept in a centrifuge
tubes or bottles fitted to the rotor of a centrifuge machine and positioned in such a way that
perceive a maximum of centrifugal force. Due to this centrifugal force particles that are differ in
size, shape and density got settled at the bottom of tube. In practice, particles or cells in any
liquid solution settles to the bottom of a container due to its own gravity (1 x g) in a given time.
Thus to settle such particles a very long time required. Sometimes, many particles which are of
extremely small in size may not settle at all in solution, unless subjected to high centrifugal
force. When these suspensions are rotated at a certain speed a centrifugal force causes the
particles to move away from the axis of rotation radically. The force exerted on the particles
(relative to gravity) is called Relative Centrifugal Force (RCF). For example, an RCF of 1000 x g
indicates that the centrifugal force applied is thousand times greater than gravitational force of
earth.

2. Theory of centrifuge ope ration

The force FC acts on the particles in the liquid sample, approaching them towards the outside of
centrifuge tube or bottles and then separating it from the liquid component in the form of pellet
to the bottom of tube. The magnitude of the centrifugal force, F C can be show to be given by
equation (1).

FC = m w 2 R (1)

FC= Magnitude of the centrifugal force (units Newton)

R= Radius of the circular motion of the centrifuge (unit metres)
w= Angular velocity of the centrifuge (units radian s −1 )
m= Mass of the particle being spun.

Centrifugal force acting on particle is given by,

(2)

ρp = density of a particle

Force due to buoyancy FB,

(3)

ρs = density of displaced fluid

Force due to drag FD

FD = 6πηrv (4)

r =radius of the moving sphere (Units m)

η= Viscosity of the solution (Units Pa.s)
v = Velocity of the moving sphere (Units m.s−1)

Since, FD = FG - FB

Combining Equation 2,3 and 4,

πη (5)

From equation 1, g= w2 R

Hence,

πη (6)

The linear velocity v of the tube as it moves in a circle is given by equation,

v= wR (7)

The time for all particles to assemble in the form of pellet in the centrifuge tube T is thus given
by dividing equation l, the depth of fluid in the centrifuge tube by vT , the terminal velocity.

η
(8)

These symbols represent

T= Time taken to precipitate all suspended particles (unit seconds)

r = Radius of suspended particles (m)
v= Viscosity of the solution (units Pa.s)
w=Angular velocity of the centrifuge (unit radian s−1 )
ρs=Density of solution (kg m−3 )
ρp =Density of suspended particles (kg m−3 )
R= Radius of the circular motion of the centrifuge (m)

During biological experiments samples are always suspended in the form of solution. Hence, the
rate of sedimentation also depends on the,

 Density of particle
 Mass of particle
 The extent to which its shape deviates from spherical
 Density and viscosity of the medium used

3. Centrifugal Force

The force exerted onto a particle in solution when centrifuged is proportional to the radius of the
centrifuge rotor R and speed in terms of revolution per minute. Since the earlier varies with
different centrifuge models it makes difficult to compare centrifuge times and speeds across
different devices.

Hence, centrifugation can be better described in terms of relative centrifugal force (RCF) as
compared to its rotational speed in terms of revolution per minute. Thus, RCF is described as the
ratio of the force acting on a particle to the force acting on the particle by gravity during
centrifugation process. Simply, RCF indicates that how many g forces acting on a particle when
processed in a centrifuge.

In centrifugation it is important to understand a difference between the speed of centrifugation

(RPM) and the relative centrifugal force (RCF or g) because these terms are often confused. The
centrifugal force generated during centrifugation can be calculated from the following equation:

RCF = 1.118R (RPM/1000)2 (9)

where, R is the distance from the centre of rotation in centimetres, indicating that the centrifugal
force increases as the particles move down the centrifuge tube because of an increase in the value
of R. The RCF is directly proportional to the speed of rotation (rpm) and the distance of the
particles from the center. Hence, RCF is actually the ratio of the weight of the particle in the
applied centrifugal field to the weight of the same particle when acted by gravity (g) alone.
Therefore it is necessary to quote the rotor speed, radial dimensions and time for which
centrifugation was done. This is a general rule that greater the centrifugal force the shorter will
be separation time. However, centrifugation also caused a hydrostatic force within the solution
that may causes disruption of some biological particles such as ribosomes. Hence, to separate a
particular molecule a particular speed is applied.
4. Types of Rotor

Rotors can be broadly classified into three categories including swinging-bucket rotors, fixed-
angle rotors, and vertical rotors. All these rotors have different advantages and limitations
depend on the kind of separation method and sample. The amount of g- force or RCF to a rotor
tube increases directly with the radius of tube hence the geometry of tube with respect to the axis
of rotation is essentially important for selecting the suitability of a rotor for a particular types of
separation.

Figure illustrates different types of rotors used in centrifugation. 1. swinging-bucket rotors, 2.

fixed-angle rotors, and 3. vertical rotors.

4.1. Swinging-bucket rotor: In this type of rotor centrifuge tubes swing out towards the
horizontal as centrifugation process started. An increase in radius provide the longest path of
particle movement causes the fast separation of solid in the form of pellet at the bottom of tube.
Tubes are placed in the rotor to assume a horizontal plane when the rotor is moving and a
vertical position when it is at rest. During such centrifugation solid particles travel
homogeneously and constantly along the tube thus the sediment is distributed uniformly against
the bottom of the tube and remains there when rotor stops. This liquid part can easily taken off.

4.2. Fixed-angle rotor: The degree of inclination is permanent in the fixed-angle rotor to the
vertical. Generally tubes are held at a fixed position (in a angle from 25° to 40°) to the vertical
axis of rotation. Mostly one rotor can hold eight centrifuge tubes. Such rotor has an advantage of
not moving parts on the rotor. This arrangement causes solute to be forced settle at the side of
tube with a faster separation of the solute from fluid. In this type of rotor meniscus is free to
reorient hence it is recommended to fill the tube at particular level to avoid spillage of sample.
As rotor stops, gravity causes the deposit to slide down the tube, usually a poorly packed pellet is
formed.

4.3. Vertical rotors: This type of rotor is totally different in which sample tubes are fixed
vertical position. This type of rotor is not used for pelleting applications. However, such rotors
are most efficient for isopycnic (density) separations due to the short pathlength. Vertical rotors
are widely used for the separation of plasmid DNA, RNA, and lipoprotein isolations.

k’-factor of rotors

The k’- factor is defined as the time taken for a particle to deposit through a sucrose gradient. In
general a efficient rotors that operated at a high RCF and have a low sedimentation path length,
it has a lowest k’- factors. The centrifugation times (t) and k’- factors for two different rotors (1
and 2) are related by:

k1t 2
t1 
k2

k’ factors are a useful way of comparing the performance of ultracentrifuge rotors and the
equation permits the calculation of the time required for particle banding in one rotor compared
to another.

5. Types of Centrifuges

5.1. Low Speed Centrifuge

This is a simple centrifuge, can achieve a maximum speed of 4000-6000 rpm. Depend on its
application this is further divided into two types,

a) Small bench centrifuges (Microcentrifuge): Generally used for separation of small volume
that is rapidly sediment (1-2 min) maximum angular speeds of 10,000–15,000 rpm. This system
does not require special cooling system. Used to rapid sedimentation of samples before it is
applied to other technique.

b) Large capacity refrigerated centrifuges: This type of centrifuges contains a refrigerated

rotor chambers and a large volumes (10 to 100 ml) can be handled depending on the types of
rotors are mounted. It is most commonly used to separate bulky precipitates, erythrocytes from
blood sample, cell organelle, etc.

5.2. High speed refrigerated centrifuge

High-speed centrifuges used to handle wide range of sample volumes (10 ml to 1 Liter) with a
higher angular velocities of 25000 rpm (60000g). High-speed centrifuges may accommodate
verities of interchangeable rotors (fixed angle or swinging buckets rotors) with different
adapters to hold various sizes of tubes. This type of centrifuge is widely used for the separation
of cell debris, cell organelles and proteins precipitates.

5.3. Continuous flow centrifuge

Continuous flow centrifuge is relatively simple and high speed centrifuge with a special design
non interchangeable rotor system (long and tubular) with a capacity of 1-1.25 dm3 /min. The
continuous flowing particles sediment at wall and excess clarified medium (supernatant)
overflows through an outlet port. Application of continuous flow centrifuge are generally in the
bacterial and yeast cultures in which cells culture volume is very high (about 100-500 dm3 ).

5.4. Ultracentrifuge

Ultracentrifugation exerts very high centrifugal force with maximum angular velocity of ~
70,000 rpm with a fixed head rotors. It is mainly used for the separation of lipoproteins,
ribosomes, proteins, and viruses. Ultracentrifuges can separate molecules both in batch or
continuous flow system. It is a widely accepted classical method of biochemistry, cell and
molecular biology. Because of its high speed and long time run an internal cooling system is
necessary to run ultracentrifugation.

Ultracentrifugation provides two opposite views of solution behavior although the same
instrument is used with a different experimental protocols are employed. In general
sedimentation velocity provides first-principle, hydrodynamic information about the size and
shape of a molecule. However, sedimentation equilibrium provides first-principle,
thermodynamic information about the solution such as molar mass, stoichiometries, association
constants, and solution non- ideality.

Ultracentrifuge are again of two types,

a) Preparative ultracentrifuge

Preparative ultracentrifuge is a highly sophisticated refrigerated sealed system evacuated to

minimize excess heat generated during centrifugation with a maximum rotor speed of 30000-
80000 rpm (60,000g). This ultracentrifuge employed with an infrared temperature sensor. It is
used to separate lipoprotein from plasma and deproteinisation of physiological fluids for amino
acid analysis and macromolecule/ligand binding, steroid hormone receptor assays, etc.

b) Analytical ultracentrifuge

Analytical ultracentrifuge is a highly protective refrigerated and evacuated system contains

optical system to observe the sedimenting material throughout the centrifugation process. This
can have a maximum rotor speed of 70000 rpm. Analytical ultracentrifuges are comprised of
three different types of optical system, alternative Schlieren system, a light absorption system,
and Rayleigh interferometric system. It is firmly based on equilibrium and non-equilibrium
thermodynamics and represent a gold standard for characterization of hydrodynamic properties
of macromolecules and its complexes. Analytical ultracentrifuge is most accepted technique for
molar- mass and binding-constant determinations.

Table 1 summarizes the applications that can be classified by the relative centrifugal force.

Summary:
In this module you have learned about centrifugation techniques and its basic concept. The centrifuge
is a simple device used for separation of solid particles from its solution based on their shape, size,
density, rotor speed and viscosity of the medium. Particle with a high density is settled first than
lighter particle during the centrifugation process. Hence, greater the difference in density, the faster
they move toward to end of tube. On the other hand, if there is no or limited difference in density
(isopycnic conditions), the particles keep on steady and not settled. To take the advantage of
centrifugation even a minute differences in density various particles are separated from a solution by a
powerful “centrifugal force” provided by the centrifuge. Centrifuges are of different types and it is
used for different purposes such as amount of volume applied and degree of force is necessary. A basic
concept of centrifugal force and its application is described well.

End of Module 7

Thank you

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