Ultra-centrifugation

Dr. Farzana Shahin

Assistant Professor
Biological Techniques
 Ultracentrifugation
It is an important tool in biochemical research.
Rapid spinning imposes high centrifugal forces on
suspended particles, or even molecules in solution.
Causes separations of such matter on the basis of
differences in weight.
 Example;
Red cells separated from plasma of blood, nuclei
from mitochondria in cell homogenates, one
protein from another in complex mixtures.
Isolation of macromolecules such as DNA, RNA,
Lipids etc.
Its rotational speed up to 150,000 rpm.
It is creating a centrifugal force up to 900,000 x g.
 TYPES
There are two types of ultracentrifugation:
 Analytical ultracentrifugation:- The aim of
Analytical ultracentrifugation is use to study
molecular interactions between
macromolecules or to analyze the properties of
sedimenting particles such as their apparent
molecular weight.
 Preparative ultracentrifugation:- The aim of
Preparative ultracentrifugation to isolate and
purify specific particles such as subcellular
organells.
 Analytical ultracentrifugation
 Two kinds of experiments are commonly performed on
these instruments:
 Sedimentation velocity experiments:- Aim of SVEs to
interpret the entire time-course of sedimentation, and
report on the shape and molar mass of the dissolved
macromolecules, as well as their size distribution.
 Sedimentation equilibrium experiments:- SEEs are
concerned only with the final steady-state of the
experiment, where sedimentation is balanced by
diffusion opposing the concentration gradients,
resulting in a time-independent concentration profile.
 Preparative ultracentrifugation
• It is to isolate specific particles which can be reused.
 Differential ultracentrifugation:
 Differential centrifugation is a common procedure in
microbiology and cytology used to separate certain
organelles from whole cells for further analysis of specific
parts of cells.
 Density gradient ultracentrifugation:
 Based on denstiy difference.
 There are two types of density gradient ultracentrifugations
under preparative ultracentrifugation such as.
1. Zonal or rate
2. Isopycnic
 Preparative ultracentrifugation
ZONAL or RATE Centrifugation:
• Mixture to be separated is layered on top of a
gradient (increasing concentration down the tube).
• Provides gravitational stability as different species.
• Move down tube at different rates.
 ISOPYCNIC Centrifugation:
• Isopycnic means “of the same density”.
• Molecules separated on equilibrium position.
• Each molecule floats or sinks to position where
density.
 Function of analytical centrifuge
It uses small sample size (less than 1mm).
Inbuilt optical system for read out.
 It uses relatively pure samples.
 It is used to determine sedimentation coefficient
and molecular weight of the molecules.
 Functions of preparative centrifuge
Large sample size can be used.
 No inbuilt optical system for read out.
 Less pour sample can be used.
 It can be used to determine sedimentation
coefficient and molecular weight of
molecules.
 It is generally used to separate organelles and
cell component.
 Rotor
 Four types of rotors are available for ultracentrifugation,
1. Fixed-angle rotor,
2. Swinging-bucket rotor,
3. Vertical rotor
4. Near-vertical rotor.
 Rotors are made from either aluminum or titanium, or from
fiber-reinforced composites.
 A titanium rotor is designated by T or Ti, as in the Type 100 Ti,
the SW 55 Ti, or the NVT 90 rotor.
 A composite rotor (fiber) is designated by C, as in VC 53.
 A aluminum rotor is designated by AC, as in VAC 50.
 Rotors without the T, Ti, C, or AC designation (such as the Type
25) are fabricated from an aluminum alloy.