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Laboratory Laboratory CentrifugesCentrifuges

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DefinitionDefinition A A laboratory centrifugelaboratory centrifuge

is laboratory equipment, is laboratory equipment, driven by a motor, driven by a motor, spins liquid samples at high speed. spins liquid samples at high speed. There are various types of centrifuges, depending on There are various types of centrifuges, depending on

the size and the sample capacity. the size and the sample capacity. They vary widely in speed and capacityThey vary widely in speed and capacity work by the sedimentation principle, where the work by the sedimentation principle, where the

centripetal acceleration is used to separate substances centripetal acceleration is used to separate substances of greater and lesser density.of greater and lesser density.

comprise a rotor containing two, four, six, or many comprise a rotor containing two, four, six, or many more numbered wells within which the samples more numbered wells within which the samples containing centrifuge tips may be placed.containing centrifuge tips may be placed.

Centrifuges are used inCentrifuges are used in chemistry, biology, and biochemistry for isolating and chemistry, biology, and biochemistry for isolating and

separating suspensions.separating suspensions.

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OperationOperation Increasing the effective gravitational force Increasing the effective gravitational force

will more rapidly and completely cause the will more rapidly and completely cause the precipitate to gather on the precipitate to gather on the bottombottom of the tube of the tube as a "as a "pelletpellet". ".

The The remainingremaining solution is called the solution is called the ""supernatesupernate" or "supernatant"." or "supernatant". The supernatant liquid is then either The supernatant liquid is then either

decanted from the tube without disturbing the pellet, or decanted from the tube without disturbing the pellet, or withdrawn with a Pasteur pipette. withdrawn with a Pasteur pipette.

The The rate of centrifugationrate of centrifugation is specified by the is specified by the acceleration applied to the sample, typically acceleration applied to the sample, typically measured in revolutions per minute (measured in revolutions per minute (RPMRPM) or ) or relative centrifugal force (relative centrifugal force (RCFRCF). ).

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OperationOperation

The The particles' settling velocityparticles' settling velocity in in centrifugation is a function of their centrifugation is a function of their size and shape, size and shape, centrifugal acceleration, centrifugal acceleration, the volume fraction of solids present, the volume fraction of solids present, the density difference between the the density difference between the

particle and the liquid, and the particle and the liquid, and the viscosity.viscosity.

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Rotor objectivesRotor objectivesGenerally spoken, there Generally spoken, there

are two main types of are two main types of rotors:rotors:

Fixed-angle rotorFixed-angle rotor The rotor (mainly made The rotor (mainly made

of aluminium) is very of aluminium) is very compact.compact.

There are boreholes There are boreholes with a specific angle with a specific angle (like 45°) within the (like 45°) within the rotor. rotor.

These boreholes are These boreholes are used for the sample used for the sample tubes.tubes.

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Swing-out rotorSwing-out rotor (= (= horizontal rotor)horizontal rotor) The rotor looks like a cross The rotor looks like a cross

with bucket. with bucket. Within these buckets, Within these buckets,

different tubes can be different tubes can be centrifuged. centrifuged.

For a safe centrifugation, a For a safe centrifugation, a specific adadpter for every specific adadpter for every tube shape is mandatory.tube shape is mandatory.

Rotor objectivesRotor objectives

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DesignDesignRotor objectivesRotor objectives

The rotor is closed by a rotor lid. The rotor is closed by a rotor lid. The rotor is located in a rotor The rotor is located in a rotor

chamber which is covered by a chamber which is covered by a metal centrifuge lid. metal centrifuge lid.

The open lid prevents the motor The open lid prevents the motor from turning the rotor when the from turning the rotor when the rotor chamber is open. rotor chamber is open.

During the run, the lid is locked. During the run, the lid is locked. The lid protects the user from The lid protects the user from

being injured by touching a rapidly being injured by touching a rapidly spinning rotor. spinning rotor.

The rotor chamber and the lid of The rotor chamber and the lid of high quality centrifuges are robust high quality centrifuges are robust enough to survive a rotor failure at enough to survive a rotor failure at full speed. full speed.

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DesignDesignRotor objectivesRotor objectives

After a rotor crash, a centrifuge should not be After a rotor crash, a centrifuge should not be reused as the enormous forces during a crash reused as the enormous forces during a crash may have damaged essential parts of the may have damaged essential parts of the device.device.

The rotor must be balanced by placing The rotor must be balanced by placing samples or blanks of equal mass opposite each samples or blanks of equal mass opposite each other. Since most of the mass is derived from other. Since most of the mass is derived from the solvent, it is usually sufficient to place the solvent, it is usually sufficient to place blanks or other samples of equal volume. blanks or other samples of equal volume.

As a safety feature, some centrifuges may stop As a safety feature, some centrifuges may stop turning when wobbling is detected (automatic turning when wobbling is detected (automatic imbalance detection, see Safety).imbalance detection, see Safety).

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TheoryTheory During circular motion During circular motion

the acceleration is the product of the the acceleration is the product of the

radiusradius and the and the square of the square of the angular velocityangular velocity and it is and it is traditionally named traditionally named

"relative centrifugal force" "relative centrifugal force" ((RCFRCF). ).

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TheoryTheory

The The accelerationacceleration is measured in multiples of is measured in multiples of ""gg“, the standard acceleration due to gravity at “, the standard acceleration due to gravity at the Earth's surface, or × "the Earth's surface, or × "gg", and it is given by", and it is given by

wherewhere

gg = acceleration = acceleration

rr = rotational radius (centimeter, cm) = rotational radius (centimeter, cm)

NN = rotating speed (revolutions per = rotating speed (revolutions per minute, r/min)minute, r/min)

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Calc. of RCF:Calc. of RCF: To calculate the RCF value at any point along the

tube or bottle: measure the radius, in mm, from the center of

the centrifuge spindle to the particular point. draw a line from the radius value on the right

hand column to the appropriate centrifuge speed on the left hand column.

the RCF value is the point where the line crosses the center column.

The formula:

R = Radius in mm from centrifuge spindle to point in tubeN = Speed of spindle in RPMRCF = (11.17 x 10-7) RN2

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Types of centrifugeTypes of centrifuge

There are at least five types of There are at least five types of centrifuge:centrifuge:

preparative centrifugepreparative centrifuge analytical centrifugeanalytical centrifuge angle fixed centrifugeangle fixed centrifuge swing head centrifugeswing head centrifuge haematocrit centrifugehaematocrit centrifuge

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Centrifuge tubesCentrifuge tubes Centrifuge tubesCentrifuge tubes or or centrifuge tipscentrifuge tips are are

tapered tubes of various sizes made of tapered tubes of various sizes made of glass or or plastic. .

They may vary in capacity from tens of mm, to They may vary in capacity from tens of mm, to much smaller capacities used in micro-much smaller capacities used in micro-centrifuges used extensively in molecular centrifuges used extensively in molecular biology laboratories. biology laboratories.

The most commonly encountered tubes are of The most commonly encountered tubes are of about the size and shape of a normal test tube about the size and shape of a normal test tube (~ 10 cm long). (~ 10 cm long).

Micro-centrifuges typically accommodate micro-Micro-centrifuges typically accommodate micro-centrifuge tubes with capacities from 250 μl to centrifuge tubes with capacities from 250 μl to 2.0 ml 2.0 ml These are exclusively made of plastic.These are exclusively made of plastic.

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Centrifuge tubesCentrifuge tubes Glass centrifuge tubesGlass centrifuge tubes can be used with can be used with

most solvents, but tend to be most solvents, but tend to be more more expensiveexpensive. They can be cleaned like other . They can be cleaned like other laboratory glassware, and can be laboratory glassware, and can be sterilizedsterilized by autoclaving. by autoclaving.

Plastic centrifuge tubesPlastic centrifuge tubes, especially micro-, especially micro-centrifuge tubes tend to be centrifuge tubes tend to be less expensiveless expensive. . Water is preferred when plastic centrifuge Water is preferred when plastic centrifuge tubes are used. They are more difficult to tubes are used. They are more difficult to clean thoroughly, and are usually inexpensive clean thoroughly, and are usually inexpensive enough to be considered enough to be considered disposabledisposable

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Centrifuge tubesCentrifuge tubes

Three microcentrifuge tubes: 2 mL, 1.5 mL and 200 μL (for PCR).

Four screw-top micro-centrifuge tubes.

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Centrifuge at NASA Ames Research Center.

Featured in movie “Space Cowboys,” 2000

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Mechanical FailureMechanical Failure

Is caused by age and by improper use Is caused by age and by improper use or inadequate care of centrifuge or or inadequate care of centrifuge or rotor. Especially the rotor.rotor. Especially the rotor.

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Care and AttentionCare and Attention

Safe high-speed spin Safe high-speed spin requires nearly requires nearly perfectly balanced perfectly balanced load.load.

Age, use, and misuse Age, use, and misuse contribute to rotor contribute to rotor flaws.flaws.

A rotor which comes A rotor which comes apart at high speed apart at high speed can be deadly.can be deadly.

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Tiny flaws areTiny flaws are

…NOT so tiny at 80,000 rpm

These are micro-fissures and cracks caused by stress and corrosion in the bottom of a tube cavity.

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22(Purdue 2003) This rotor came apart while coming up to speed (not yet spinning at full speed)

The 6 inch (15 cm) long chunk damaged (ruined) but did not completely penetrate the centrifuge lid and housing. The motor and spindle were also destroyed.

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To reduce wear and To reduce wear and chance of failurechance of failure

Follow instructions in manual and rotor care guideFollow instructions in manual and rotor care guide Use only compatible rotorsUse only compatible rotors Check routinely for rotor damageCheck routinely for rotor damage Don't overfill tubesDon't overfill tubes Cap tubesCap tubes Balance load carefullyBalance load carefully Check that rotor correctly seated on drive spindleCheck that rotor correctly seated on drive spindle Run at Run at << max safe speed max safe speed

continued……continued……

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To reduce wear and To reduce wear and chance of failure, continuedchance of failure, continued

Stay right there until full speed is reached.Stay right there until full speed is reached. Stop the centrifuge if anything seems unusualStop the centrifuge if anything seems unusual Never open until stopped (never ‘manual brake’)Never open until stopped (never ‘manual brake’) Clean rotor gentlyClean rotor gently Dry completelyDry completely Store rotor upside down (why?)Store rotor upside down (why?)

Maintain a careful rotor logMaintain a careful rotor log Derate and retire rotors for age/use Derate and retire rotors for age/use

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25(MIT 1999) This rotor split in half at 55,000 rpm after 3 h of what was supposed to be a much longer spin.

Grad student report “Rotor was manufactured in 1986 and was covered for 10,000 hours of operation or 5 years, whichever comes first.” No surprise that it flew apart 13 years later. Fortunately the centrifuge housing contained it.

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(Cornell, 1998)

The rotor failure was not contained.

Much other damage done, equipment ruined, chemicals spilled.

Nobody was near, or someone would have been badly hurt or killed.