centrifugation

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
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General Steps in Biochemical Separation

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Separation of Macromolecules
Chromatography, precipitation
Electrophoresis, ultracentrifugation

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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
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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.
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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
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Type 1– Preparative Centrifugation
Collect (isolation) material: cell, subcellular structure, membrane vesicles
1. Handle larger liquid volumes (i.e.
1 to