centrifugation

Topics: Organelle, Laboratory techniques, Force Pages: 25 (2140 words) Published: February 26, 2014
Chapter 3 Centrifugation
Biochemistry and Molecular Biology (BMB)

3.1 Introduction
3.2 Basic Principle of sedimentation
3.3 Types, care and safety of centrifuges
3.4 Preparative centrifugation
3.5 Analytical centrifugation
Analytical Biochemistry (AB)

3.4.3 Ultracentrifugation
Koolman, Color Atlas of Biochemistry, 2nd edition
1

General Steps in Biochemical Separation

2

Separation of Macromolecules
Chromatography, precipitation
Electrophoresis, ultracentrifugation

3

Densities of biological material
Material

Density (g/cm3)

Microbial cells

1.05 - 1.15

Mammalian cells

1.04 - 1.10

Organelles

1.10 - 1.60

Proteins

1.30

DNA

1.70

RNA

2.00
4

Introduction (MBM 3.1)
Principles of centrifugation
A centrifuge is a device for separating particles from a
solution according to their size, shape, density,
viscosity of the medium and rotor speed
In a solution, particles whose density is higher
than that of the solvent sink (sediment), and
particles that are lighter than it float to the
top. The greater the difference in density, the
faster they move. If there is no difference in
density (isopyknic conditions), the particles
stay steady. To take advantage of even tiny
differences in density to separate various
particles in a solution, gravity can be replaced
with the much more powerful “centrifugal force”
provided by a centrifuge.
5

Centrifugation
A centrifuge is used to separate particles or macromolecules: -Cells
-Sub-cellular components
-Proteins
-Nucleic acids
Basis of separation:
-Size
-Shape
-Density
Methodology:
-Utilizes density difference between the
particles/macromolecules and the medium in which these are
dispersed
-Dispersed systems are subjected to artificially induced
gravitational fields
6

Type 1– Preparative Centrifugation
Collect (isolation) material:
cell, subcellular structure, membrane vesicles
1. Handle larger liquid volumes (i.e.
1 to several thousand litres)
2. Range of designs
3. Typical rotating speed: 500 - 2000
rpm

Immunofluorescent imaging of
human cells (U2OS) with pan
7
Cadherin antibody

Type 2– Analytical Ultracentrifugation (AUC)
Determine the mass, shape and stoichiometry ratio of noncovalent association of macromolecules (protein-protein, small molecule-protein, quaternary structure)
骨骼肌
1. Rotates at high speeds e.g.
30000 rpm
1.肌膜

肌膜

2. The high speeds used in
such devices generate
considerable amounts of
heat
3. Therefore cooling
arrangements are required
in ultracentrifuges

http://www.cgmh.org.tw/chldhos/intr/c4a90/new_page_50.htm

8

3.2 Basic Principle of Sedimentation (AB 3.4.3)
Relative centrifugal force

F = Mω 2 r

M: mass of particle
r: radius of rotation (cm) (ie
distance of particle from axis
of rotation)
ω :Average angular velocity
(radians/sec)
2π rev min -1
ω=
60
Rev: revolution per minute
(r.p.m.)
1 revolution = 2π radians
=360
9

Centrifugal Field
G=rω2

depends on the radical distance of the particle from the
rotation axis and the square of the angular velocity

G=



2

(rev min ) r
-1 2

3600

10

Angular Velocity
2π rev min -1
ω=
60

rev: revolution per minute (r.p.m.)

11

Because rotors are
Relative Centrifugal Force different from various
manufactures, we use
f c Mω 2 r (RCF) RCF value
RCF =
=
= ω 2 r × g -1
RCF to represent the
fg
Mg
"No. x g"
centrifugation force.
2
(multiples of earth's gravitational force).
2π rmp


RCF = 


60


-1
 r×g


Radius

Radius

Radius

Min

Ave

Max

RCF =
1.12 x 10-5 x (rpm)2 x r

RPM (x1000)

Radius

rpm:
revolution per minute
r: radius of rotor

RCF (x1000)
12

Relative centrifugal force
RCF =
1.12 x 10-5 x (rpm)2 x r

rmin
rmax

13

Interacting Forces in Centrifugation
Sedimenting force, mpω2r, is opposed by...

Fcentrifuge

mp = the mass of equal volume of solvent...
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