Ultracentrifugation is a technique that uses high centrifugal forces generated by rotational speeds of up to 150,000 rpm to separate particles in solution based on differences in size, shape, density, and viscosity. It is an important tool in biochemical research used to isolate molecules like DNA, RNA, lipids, and separate organelles from cells. There are two main types - analytical ultracentrifugation which studies molecular interactions and properties, and preparative ultracentrifugation which isolates and purifies particles using techniques like density gradient centrifugation. Proper rotor selection and maintenance of the centrifuge are important for safe and effective use of this technique.

1. ULTRACENTRIFUGATION
BCH 503
5th semester

2. Centrifuge
• A centrifuge is a device for separating particles
from a solution according to their size, shape,
density, viscosity of the medium and rotor
speed.

3. ULTRACENTRIFUGATION
• It is an important tool in biochemical research. Which
through rapid spinning imposes high centrifugal forces
on suspended particles, or even molecules in solution,
and causes separations 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. And
also 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.

5. TYPES:
1. 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.
2. Preparative ultracentrifugation:- The aim of
Preparative ultracentrifugation to isolate and purify
specific particles such as subcellular organelles.

6. Analytical ultracentrifugation
Two kinds of experiments are commonly performed on
these instruments:
1. Sedimentation velocity experiments:- Aim of SVEs to
interpret the entire time-course of sedimentation,
and report on the shape and molar mass & size
distribution of the dissolved macromolecules.
2. 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

7. Preparative ultracentrifugation
• 1. 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.
• 2. Density gradient ultracentrifugation:- Based
on density difference. There are two types of
density gradient ultracentrifugation's under
preparative ultracentrifugation such as.
• 1.RATE ZONAL & 2.ISOPYCNIC

8. Density Gradient Centrifugation:
1} RATE ZONAL 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.
2} ISOPYCNIC Centrifugation:
• Isopycnic means “same density”.
• Molecules separated on equilibrium position.
• Each molecule floats or sinks to position where its
density equals.

9. Functions of analytical and preparative
ultracentrifugation:
Analytical
• Uses small sample size (less than 1 ml).
• Built in optical system to analyze progress of molecules during
centrifugation.
• Uses relatively pure sample.
• Used to precisely determine sedimentation coefficient and MW of
molecules.
Preparative
• Larger sample size can be used.
• No optical read-out collect fractions and analyze them after the run
• Less pure sample can be used.
• Can be used to estimate sedimentation coefficient and MW.
• Generally used to separate organelles and molecules. Most
centrifugation work done using preparative ultracentrifuge.

10. Rotor
• Four types of rotors are available for ultracentrifugation,
• 1. Fixed-angle rotor,
• 2. Swinging-bucket rotor,
• 3. Vertical rotor and
• 4. Near-vertical rotor.
• Rotors are made from either aluminum or titanium, or
from fiber-reinforced composites.
• A aluminum rotor is designated by AC, as in VAC 50
• 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.
• Rotors without the T, Ti, C, or AC designation (such as the
Type 25) are fabricated from an aluminum alloy.

11. Care of Centrifuges and Rotors
• Carefully read the operating manual or receive proper instructions
before use any centrifuge.
• Select the proper operating conditions on the instrument.
• Check the rotor chamber for cleanliness and for damage.
• Select the proper rotor. Many sizes and types are available.
• Be sure the rotor is clean and undamaged.
• Filled centrifuge tubes or bottles should be weighed carefully and
balanced before centrifugation.
• Rotor manufactures provide a max. allowable speed limit for each
rotor. Do nor exceed that limit.
• Keep an accurate record of centrifuge and rotor use.
• If an unusual noise or vibration develops during centrifugation,
immediately turn the centrifuge off.
• Carefully clean the rotor chamber and rotor after centrifugation.