Biochemical Techniques II

Lecture Notes 2014/2015

SEDIMENTATION

 Sedimentation of suspended and other dissolved particles occurs due to

centrifugal force
 Sedimentation is based on two major principles
i. Separate solid matter as a pellet from dissolved solutes as a
supernatant
ii. Separates soluble macromolecules of different mass or density

Centrifugation
 A centrifuge is an equipment used for separation of macromolecules such as:
i. cells
ii. subcellular components
iii. proteins
iv. nucleic acids
 The separation is based on:
i. size
ii. shape
iii. density
iv. viscosity of the medium
v. speed of the rotor

NB: The method utilizes the density difference between particles and
the medium in which they are dispersed.
 Dispersed systems are subjected to artificially induced gravitational
fields (centrifugation)
o Substances whose densities are higher than that of solvent will sink
(sediment) and vice versa
o The greater the particles are in density, the faster they will move
o Whenever there is no difference in density (isopycnic condition), the
particle will stay steady
o a centrifuge provides a much greater force (centrifugal force) to
separate particles which exhibit tiny differences in density
o a centrifuge gives artificially induced gravitational fields to sediment
particles faster.
Composition of centrifuge
 a centrifuge comprises of:
i. an electric motor to pass electricity through the coil, i.e. converts
electricity into (angular) motion)
ii. a drive shaft to transmit mechanical power
iii. a rotor to hold tubes

11
  a centrifuge is also used to provide centrifugal force to drive other processes
such as ultrafiltration

Types of Centrifuge
 Classification of Centrifuges depend on:
i. maximum speed of sedimentation and an example is the Microfuge,
10,000g
ii. presence/Absence of vacuum and an example is the Ultracentrifuge,
600,000g
iii. temperature control refrigeration and an example is the High Speed
refrigerated centrifuge, 100,000g
iv. volume of samples and capacity of centrifugation tubes and an
example is the Large-capacity preparative centrifuge, 3,000-
7,000g
 Another classification of centrifuges is based on the type of rotor
used:
i. fixed angle rotor
ii. swinging bucket rotor

Density gradient sedimentation

Sedimentation through density gradient overcomes the following:

o peletting of small particles in the bottom of the tube due to:

 mechanical vibration

 thermal gradients

12
  convection currents

Types of density gradient centrifugation

• First sedimenting particles will be contaminated with slow sedimenting ones.

• Mechanical vibrations, thermal gradient, convection currents also affect
sedimentation properties of a molecule.

• To minimize the above problems, centrifugation through a dense gradient is

used

1. Rate Zonal Centrifugation = common technique for macromolecules

separation, purification, and isolation. Here the density of the separated
molecule is greater than the density of the solvent

2. Isopycnic (equilibrium) centrifugation where the solvent density

encompasses density of the particle.

• Samples are overlaid onto a pre-formed gradient (sucrose, CsCl, Ficoll,

hypaque, Percoll etc) and then centrifuged.

• Separation is based on the sedimentation coeffiecient (s value) of the

molecule.

• S value is determined by the molecular weight, density, size and shape

of the molecule.

• The selection of the correct centrifugation time is vital ( too short a

time is not enough to separate the molecule and too long a spin leads
to peletting of one or more species)

Ultracentrifugation

 Molecular Weight and Shape = fundamental physical properties of a

protein.
 Estimates of molecular weight can be obtained using SDS-PAGE or gel
filtration

 One very useful technique for measuring molecular weight and shape is
centrifugation.

 A PARTICLE THAT'S SUBJECTED TO A CENTRIFUGAL FIELD BY BEING SPUN IN A CENTRIFUGE IS SUBJECTED TO A

FORCE,

13
 where m is the mass of the particle, r is the distance of the particle from the
center of rotation, and ω is the angular velocity.
= buoyancy factor, which accounts for the fact that particle, is buoyed
up by the surrounding solvent.

is the specific volume of the particle (ml/g) (= 1/density of the particle).

 Movement of particle through the solvent is resisted by a frictional

coefficient, f that depends on the shape of the particle.
Frictional coefficient is an important factor in any transport process, such as
centrifugation or gel filtration.
A spherical particle has f = 1.0, whereas a cigar-shaped or cylindrically-
shaped particle will have f > 1.0.
 Movement of any particle under the influence of a centrifugal field is
characterized by its sedimentation coefficient, S, which is directly
proportional to its molecular mass, M, and inversely proportional to f.

, where N is Avogadro's number.

 Ultracentrifugation is used in two ways to characterize proteins:

In sedimentation equilibrium In sedimentation velocity

experiments, the centrifuge is operated experiments, the centrifuge is operated
at a relative low speed so that the at maximal speed, which causes the
forces of sedimentation and diffusion protein to sediment to the bottom of
balance and the protein distributes in the tube. The rate at which the
the centrifuge cell in a manner boundary moves gives S, which when
proportional to its molecular weight. combined with M gives f, a measure of
the shape of the protein.

• Spinning around an axis creates a centrifugal field

• A particle within a centrifugal field experiences a centrifugal force (Fc =

mω2r)

• Fc = the centrifugal force, m = mass of the particle, w = angular velocity and

r = distance from the axis

• Centrifugal force is opposed by: buoyant and frictional forces which can be
defined as:

14
Conclusions: sedimentation

Forces interacting in centrifugation

15
16
Sedimentation coefficient

• Movement of any particle under the influence of a centrifugal field is

characterized by its sedimentation coefficient, S, which is directly
proportional to its molecular mass, M, and inversely proportional to f.

• Movement of particle through the solvent is resisted by a frictional

coefficient, f, that depends on the shape of the particle.

17