ZOOL805E
Tools and Techniques in Biology
Centrifugation
Centrifugation technique
• Process that uses centrifugal force to separate and purify mixtures of biological particles in a
liquid medium.
• Key technique for isolating and analysing cells, subcellular fractions, supramolecular complexes
and isolated macromolecules such as proteins or nucleic acids
• Analytical ultracentrifuge (Svedberg, late 1920s) - mainly concerned with the study of purified
macromolecules or isolated supramolecular assemblies
• Preparative centrifugation (Claude et. al., 1940s) - the actual separation of tissues, cells,
subcellular structures, membrane vesicles and other particles of biochemical interest
Centrifugal vs Centripetal force
• Centrifugal force is an outward fictitious force
that is experienced by an object moving in a
circular path directed away from the center of
rotation.
• Equal in magnitude and dimensions with
another force (centripetal) that acts towards
the center of a circular path
• Depend on the mass of the object, the
distance of the object from the center, and
the speed of the rotation.
SEDIMENTATION
• increased rate of sedimentation in a centrifugal field (g>981 cm s–2)
• biological structures exhibit a drastic increase in sedimentation when they undergo acceleration in a
centrifugal filed
• The relative centrifugal field is usually expressed as a multiple of the acceleration due to gravity.
• Depends on a number of factors
• Essentially, the rate of sedimentation is dependent upon the applied centrifugal field (cm s -2), G, that is
determined by the radial distance, r, of the particle from the axis of rotation (in cm) and the square of the
angular velocity, ω, of the rotor (in radians per second):
G = ω 2r
• The average angular velocity of a rigid body that rotates about a fixed axis is defined as the ratio of the
angular displacement in a given time interval.
• One radian (1 rad) represents the angle subtended at the centre of a circle by an arc with a length equal to
the radius of the circle. Since 360˚ equals 2π radians, one revolution of the rotor can be expressed as 2π rad.
Accordingly, the angular velocity in rads per second of the rotor can be expressed in terms of rotor speed s
as:
ω = 2π s/60
and therefore the centrifugal field can be expressed as:
G = 4 π2(rev min-1)2 r/3600
= 4 π2s2 r/3600
• The centrifugal field is generally expressed in multiples of the gravitational field, g (981cms–2).
• The relative centrifugal field (g), RCF, which is the ratio of the centrifugal acceleration at a specified
radius and the speed to the standard acceleration of gravity, can be calculated from the following
equation:
RCF = G/g = 4 π2(rev min-1)2 r/ (3600 × 981)
• RCF units are therefore dimensionless (denoting multiples of g) and revolutions per minute are
usually abbreviated as r.p.m.:
RCF = 1.12 ×10-5 r.p.m.2 r
• Nomograph
• for the convenient conversion between relative
centrifugal force and speed of the centrifuge at
different radii of the centrifugation spindle to a
point along the centrifuge tube
• 3 columns
Types
• large-capacity low-speed preparative centrifuges
• refrigerated high-speed preparative centrifuges
• analytical ultracentrifuges
• preparative ultracentrifuges
• large-scale clinical centrifuges, and
• small-scale laboratory microfuges
Types of rotors
fixed-angle rotors vertical tube rotors swinging-bucket rotors
Operation of rotors: movement of
biological samples
1. Initial acceleration stage,
2. Main centrifugal separation phase,
3. De-acceleration and
4. Final harvesting of separated particles in the rotor
at rest.
• A great variety of gradients can be used with
swinging bucket rotors, making them the method of
choice when maximum resolution of banding zones
is required.
Cross-sectional diagram of a centrifuge tube positioned in a fixed-angle
rotor (a), a vertical tube rotor (b), and a swinging-bucket rotor (c).
Preparative Centrifugation
(a) During differential sedimentation of a
particulate suspension in a centrifugal field, the
movement of particles is dependent upon their
density, shape and size.
(b) For separation of biological particles using
a density gradient, samples are carefully
layered on top of a preformed density gradient
prior to centrifugation.
• For isopycnic separation, centrifugation is
continued until the desired particles have
reached their isopycnic position in the liquid
density gradient.
• In contrast, during rate separation, the
required fraction does not reach its isopycnic
position during the centrifugation run.
Diagram of particle behaviour during differential and
isopycnic separation
Diagrammatic presentation of the subcellular membrane system from skeletal muscle fibres
Scheme of the fractionation of skeletal muscle homogenate into various subcellular fractions. Flow chart of the
fractionation protocol of these membranes from tissue homogenates using differential centrifugation and density
gradient methodology (b).
ANALYTICAL CENTRIFUGATION
• Isolated biomolecules in solution only exhibit distinguishable sedimentation when they undergo
immense accelerations, e.g. in an ultracentrifugal field.
• A typical analytical ultracentrifuge can generate a centrifugal field of 250 000 g in its analytical
cell.
• Within these extremely high gravitational fields, the ultracentrifuge cell has to allow light passage
through the biological particles for proper measurement of the concentration distribution.
• Sedimentation of isolated proteins or nucleic acids can be useful in the determination of the
relative molecular mass, purity and shape of these biomolecules.
Schematic diagram of the optical system of an
analytical ultracentrifuge.
• The high-intensity xenon flash lamp of the Beckman
Optima XL-A analytical ultracentrifuge shown here
allows the use of wavelengths between 190 nm and
800 nm.
• The high sensitivity of the absorbance optics allows
the measurement of highly dilute protein samples
below 230 nm. (Courtesy of Beckman-Coulter.)
Analytical ultracentrifugation is most often employed in
• determination of the purity of macromolecules
• determination of the relative molecular mass of solutes in their native state
• examination of changes in the molecular mass of supramolecular complexes
• detection of conformational changes
• ligand-binding studies
