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DIFFERENTIAL AND ULTRA CENTRIFUGATION

By: Priya James M.Pharm (Q.A.) 1ST Sem

School of Pharmaceutical Sciences,RGPV, Bhopal.

CENTRIFUGATION

Centrifugation is a technique often employed during isolation or analysis of various cells, organelles and biopolymers, dissolved or dispersed in biologically relevant solvents.

Biologists use the technique of centrifugation to extract and isolate pure samples of individual cell organelles for study.

In this technique, the sample (comprising a liquid phase and a solute) is placed in a suitable vessel, and is spun in a centrifugal rotor.

THE FACTORS THAT DEFINE THE ENTIRE SEDIMENTATION SYSTEM:

The rate at which the rotor spins. The length of time for which the

centrifugal force is applied. The solvent density. The system temperature.

Ultracentrifugation

Preparative ultracentrifugation Analytical ultracentrifugation

Differential centrifugation

Density gradient centrifugation

Rate zonal centrifugation Isopycnic centrifugation

PREPARATIVE CENTRIFUGATION

It aims to isolate and purify specific particles such as sub. cellular organelles.

It is mainly of two types: Differential centrifugation Density gradient centrifugation

Molecules separate according to their size, density and centrifugal force.

DIFFERENTIAL CENTRIFUGATION

Differential centrifugation, also termed pelleting.

It fractionates particles according to their weight.

The suspension of isolated organelles is spun for different combination of speed and time.

The lowest speed and shortest time separates out the heaviest organelles and high speed and longer time separating the lightest organelles.

Method of differential centrifugation: Chopped liver tissue is homogenised in

an ice cold isotonic saline solution. The internal organelles of the cell are

released in the suspension called the homogenate.

The homogenate is transferred into centrifuge tubes and centrifuged.

The first sample is centrifuged at low speed for a short period of time.

The heaviest organelles (nuclei) collect at the bottom of the tube.

The Supernatant from the first sample istransferred to a second centrifuge tube.

Sample 2

The sediment or pellet from

the first sample

is removed and examined

This second sample is then centrifuged at moderate speed for a longer period of time.The moderately heavy organelles (mitochondria) collect at the bottom of the tube.

The sediment or pellet from

the first sample

is removed and

examined

The supernatant from the second sample is transferred to a third centrifuge tube.

Sample 3

This third sample is then centrifuged at high speed for an even longer period of time.

The light organelles (rough endoplasmic reticulum) collect at the bottom of the tube.

DENSITY GRADIENT CENTRIFUGATION

Isolated organelles are separated on the basis of their density.

Solutions of increasing density, such as sucrose solutions, are layered into a test tube with the most concentrated solution at the bottom of the tube.

The suspension of isolated organelles is pipetted onto the top of the most dilute solution.

As the tubes are spun, the organelles collect in the layer which corresponds to their own density.

GRADIENT MATERIALThe properties of an ‘ideal’ solute for gradients

to be used in rate zonal or isopycnic centrifugation:

The solute should be stable in solution. It should be totally inert towards biological

materials It should not be absorbed in the UV or visible

range. It should be easily and completely removable

from the purified particles. It should be inexpensive and readily available.

RATE ZONAL CENTRIFUGATION

Particles are separated in terms of their molecular mass.

The sample solution is layered on top of the density gradient, and under centrifugal force the particles will begin sedimenting through the gradient in distinct zones, each zone consisting of particles characterized by the same sedimentation rate.

Force must be terminated before any of the separated zones reaches the bottom of the tube.

Photograph of an actual tissue separation: sub fractionation of arat hepatic light mitochondrial fraction by rate zonal centrifugation using a vertical rotor.

ISOPYCNIC CENTRIFUGATION Isopycnic means "of the same density. Isopycnic centrifugation takes advantage

of differences in the effective buoyant densities of particles- it does not rely on their sedimentation rates and molecular mass.

Each particle sediments only to the position in the gradient at which its own effective buoyant density is equal to the actual gradient density.

Viruses (rotaviruses) purified by centrifugation through a caesium chloride density gradient. The original tube was 5cm long. 

ANALYTICAL ULTRACENTRIFUGATION

Analytical ultracentrifugation (AUC) is the study of the behaviour of macromolecules in solution under the influence of a strong gravitational force.

It is a centrifuge with one or several optical detection systems, which allow the observation of the fractionation process while the sample is centrifuged.

The commercially used analytical centrifuge is Beckman-Coulter AUC (Optima XL-A/I),

Rotors:The analytical rotors are built from one piece of titanium. Depending on the rotor type, four or eight holes are drilled into the titanium

Measuring Cells:The part of an analytical ultracentrifugation system that is in direct contact to the sample is the measuring cell.AUC cells must fulfill two criteria:They should not leak or distort even at high centrifugal fields.They should allow passage of light through the cell.

Detectors:

UV/Vis absorption optics is integrated in modern XL-A/I analytical ultracentrifuges.Fluorescence detector, is being recently used commercially. This class of detectors makes use of the sample’s specific light absorption or fluorescence.

Double-sector centerpiece. The sample solution is placed in one sector and a sample of the solvent is placed in the reference sector. The reference sector is usually filled slightly more than the sample sector, so that the reference meniscus does not obscure the sample profile.

Mechanism:

Light from the flash lamp passes to hit the slit assembly, where it is converted to monochromatic light.The lens-slit assembly moves stepwise as a unit driven by an electrical motor, and allows one to scan the sector of the measuring cell.While the rotor spins, the lamp is triggered to flash when the cell of interest passes the optical path.