LABORATORY CENTRIFUGE

By
Dr. Anwar Ullah

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 CENTRIFUGE

 Basic principle of centrifuge- sedimentation

 A centrifuge is a piece of equipment that accelerates the rate of sedimentation by

rapidly spinning the samples, thus creating centrifugal force many times that of
gravity.

 It is key technique for isolating and analyzing the cells, subcellular fractions and
isolated macromolecules such as proteins and nucleic acids.

 History

 The first analytical ultracentrifuge was developed by Svedberg in 1920 2

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 FORCE OF CENTRIFUGE
 Centrifugal force is generated whenever an object is forced to move in a circular path.

 Two factors that determine this force are:

1. The speed of rotation in the centrifuge expressed in revolutions per minute (RPM),
and

2. The radius of rotation, r – the distance from the center of rotation to the material of
interest

 Relative centrifugal field (RCF) is termed as the force acting on samples inside the
centrifuge, with units that are multiples of earth's gravitational field, g. 3
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 The centrifuge equation to determine the speed (rpm) is:
 where,
 RCF = relative centrifugal force x gravitational field, g
 (Note: "__ x g" meaning the force inside the centrifuge is x times greater than
the normal force of gravity)
 RPM = speed of rotation in revolutions per minute(rev/min), and
 r = the radius of rotation in centimeters(cm)
 This equation may also be expressed as:

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 RATE AND TIME OF SEDIMENTATION
 Rate of sedimentation depends on factors other than CF
 Mass of particle -Density & Volume
 Density of medium
 Shape of particle
 Friction
Time of sedimentation depends on:
 Size of particle
 Density difference b/w particle and medium
 Radial distance from the axis of rotation to liquid meniscus (rt)
 Radial distance from the axis of rotation to the bottom of the tube(rb)
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 TYPES OF CENTRIFUGES

 Desktop centrifuges

 High speed centrifuges

 Ultra centrifuges

 Analytical

 Preparative

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 SMALL MICROFUGES
Work with speed- 8000-13000 rpm & RCF 10000g
 Used for rapid sedimentation of small volumes (1-2 ml)

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 LOW SPEED CENTRIFUGES

Simple and small.

Typically
attains a speed of less than 10,000
RPM and force less than 8000xg
Do not have any temperature
regulatory system.
 .
 Normally used to collect rapidly
sedimenting substances such as blood
cells, yeast cells or bulky precipitates
of chemical reactions.
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 HIGH SPEED CENTRIFUGES

 Maximum speed up to 30000 RPM

providing 100000g centrifugal forces.
 Equipped with refrigeration to
remove heat generated.
 Temperature maintained at 0-40C by
means of thermocouple.
.
 Used to collect microorganism, cell
debris, cells, large cellular
organelles, precipitates of chemical
reactions, mitochondria, lysosomes)
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 ULTRACENTRIFUGE

 Operate at a speed up to 120,000 RPM

providing centrifugal force of 700,000g

 Rotor chamber is sealed and evacuated

by pump to attain vacuum.  .

 Refrigeration system (temp 0-40C).

 Rotor chamber is always enclosed in a

heavy armor plate.
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 CENTRIFUGE ROTOR TYPES
 The rotor sits on top of the drive shaft and holds tubes, bottles, or other sample
containers.

 Inside the centrifuge, the drive shaft rotates, causing the sample containers to spin
rapidly, which thereby causing the particles inside those sample containers to
sediment.

 Commonly used styles of centrifuge rotors: (1) Horizontal or swinging bucket,

(2) Fixed angle, (3) Vertical. 

 Each rotor is configured to perform different functions.

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 TUBE ADAPTERS

 Manufactures have designed adapters

configured to accommodate tubes and
bottles of different shapes and sizes to be
place in the same rotor.

 The use of adapters is also necessary in

cases where tubes do not fit securely in
the rotor compartment which can a. Adapter that holds
50 mL tubes
b. Adapter that holds c. Adapter that holds a d. Capped adapter
13 cm tubes 250 mL bottle that holds 15 mL tubes

ultimately shift during centrifugation.

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 BALANCING THE ROTOR
 To avoid any possible damage, when operating a centrifuge, the operator has to
balance the rotor properly.

 Properly balancing the rotor ensures that the rotor spins evenly on the drive shaft
so to avoid damage to the rotor, sample tubes, and centrifuge.

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 APPLICATIONS OF CENTRIFUGATION
 Centrifugation can be either preparative or analytical. 

 Preparative centrifugation provides separated materials for later use.

 Analytical centrifugation is used in specialized research settings to determine

the molecular weight, purity, shape, and physical characteristics of proteins and
other macromolecules.

 Preparative centrifugation can be operated in several different modes, but is most

commonly done in the laboratory as differential centrifugation.

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 I. DIFFERENTIAL CENTRIFUGATION
 Differential centrifugation separates a
sample in two or more phases, using an
initial lower force to sediment larger,
denser particles as a pellet while smaller,
less dense particles remain suspended.

 The supernatant is removed from the

pellet, placed in a clean centrifuge tube, and
centrifuged at a higher force so that the
remaining suspended particles are pelleted. 16
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 II. DENSITY CENTRIFUGATION

 Density centrifugation involves sedimenting a sample through a density gradient.

 A density gradient is a column of fluid that increases in density from the top to
the bottom.
 Gradients may be either step gradients or continuous gradients.
 A step gradient is formed in layers with the densest layer on the bottom.
 Each layer has a different density and is clearly demarcated from the layers around
it.
 With a continuous gradient, there is a smooth decrease in density from bottom to
top, with no sharp boundaries between layers.

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 GRADIENT MATERIALS
 Sucrose (66%, 50C)
 Silica sols
 Glycerol
 CsCl
 Cs Acetate
 Ficol (high molecular wt. sucrose
polymer & epichlorohydrin)
 Sorbitol
 Polyvinylpyrrolidone

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 TYPES OF DENSITY CENTRIFUGATION
 Rate zonal centrifugation ( size). A time dependent method in which particles
are separated as they sediment through a density gradient

 Buoyant Density Centrifugation (density). Also called Isopycnic

centrifugation-a mode in which particles are separated based on their density
alone (makes use of a density interface or barrier)

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 Rate-Zonal Isopycnic

Synonym S-zonal, sedimentation velocity

Density equilibrium, sedimentation
equilibrium
Gradient •Shallow, •Steep,
•Maximum gradient density less than the least •Maximum gradient density greater than that of
dense sedimenting specie, the most dense sedimenting specie,
•Gradient continuous. •Continuous or discontinuous gradients.

Centrifuga- tion •Incomplete sedimentation,

•Complete sedimentation till equilibrium
•Low speed,
is achieved,
•Short time
•High speed,
• Long time.

Separation RNA- DNA hybrids, ribosomal subunits, DNA, plasma lipoproteins, lysosomes,
etc., mitochondria, peroxisomes, etc., 20
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 III. CONTINUOUS CENTRIFUGATION

 Another mode of centrifuge operation is continuous centrifugation.

 Continuous centrifugation is used for separation of cells from large volume

fermentation broth.

 Here, the sample is continuously fed into a rotor that is rotating at operating
speed.

 Particles sediment in the rotor while supernatant is conveyed out of the rotor
continuously
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THANK YOU