Centrifuge

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Centrifuge
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What is a centrifuge?

• A centrifuge is a piece of equipment, generally
  driven by a motor, that puts an object in
  rotation around a fixed axis, applying force
  perpendicular to the axis. The centrifuge works
  using the sedimentation principle, where the
  force of acceleration is used to separate
  substances of greater and less density. There are
  many different kinds of centrifuges, including
  those for very specialized purposes.
• Used in the clinical laboratory to separate
  substances of different masses.
• The substances to be separated can be a solid and
  liquids.

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A laboratory table-top centrifuge
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Functions of centrifugation

• Remove cellular elements from blood to provide
  cell-free plasma or serum for analysis.
• Concentrate cellular elements and other
  components of biological fluids for examination
  or chemical analysis.
• Remove chemically precipitated protein from
  analytical specimens
• Separate protein-bound or antibody-bound ligand
  from free ligand in other assays.
• Extract solutes in biological fluids from
  solvents.
• Separate lipid and proteins from one another.

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Types of centrifuges

• Horizontal-head or swinging bucket
• Fixed-angle
• Ultracentrifuge

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Horizontal-head or swinging bucket centrifuges

• This type of centrifuge allows the tubes placed
  in the cups of the rotor to assume a horizontal
  position when the rotor is in motion and a
  vertical position when its at rest.
• During centrifugation, particles travel
  constantly along the tube while the tube is
  positioned at a right angle to the shaft of the
  centrifuge.
• Thus the sediment is distributed at the bottom of
  the tube.
• Supernatant liquid is removed with a pipet.
• The spinning rotor will generate heat as a result
  of air friction.

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Fixed-angle centrifuge

• In the rotor of fixed angle centrifuge, tubes are
  held in fixed positions at angles from 25 to 40
  degrees to the vertical axis of rotation.
• On the start of centrifugation, particles are
  driven outward horizontally but strike the side
  of the tube .
• This is because the sediment packs against the
  side of the tube with the surface of the sediment
  parallel to the centrifuge.
• As the rotor slows down and stops, gravity causes
  the sediment to slide down the tube.

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Ultracentrifuge

• This is a high speed centrifuge that uses a
  fixed-head rotor.
• The most common application is the separation of
  lipoproteins.
• Due to the separation may require hours or days
  and generate heat as a result of high-speed
  friction, ultracentrifuge requires a refrigerated
  chamber.
• A table-top model operating at the force of
  165,000 times gravity.
• This model is used in the laboratory to clear the
  serum so that accurate analyses could be obtained.

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Centrifuge components

• All centrifuges have a motor, drive shaft and
  head or rotor, in the form of a chamber with a
  cover.
• A power switch, time, speed control, tachometer
  and brakes are the components that control the
  centrifuge.
• Also available in refrigerated units.
• Some centrifuges are equipped with an alarm that
  sounds when error occurs such as tube imbalances.
• Automatically shut down under extreme conditions,
  preventing tube breakage and the potential
  exposure to biohazardous agents.

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• All modern centrifuge have required safety latch
  that prevents the operator from opening the
  instrument before rotor has stopped.
• Swinging bucket rotors use pairs of buckets that
  swing freely.
• The buckets are designed to accept a variety of
  cushioned inserts, allowing centrifugation of
  small tubes or large bottles.
• Different fixed-angle rotor is required for
  different sized containers.

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• The motor in a large centrifuge is usually a
  direct-current, heavy duty, high torque, electric
  motor.
• In smaller centrifuges the current is usually
  alternating.
• Power is transmitted to the rotor by the brushes.
• The rotor shaft is sealed to minimize vibration
  and the need for lubrication.
• Centrifuge speed is controlled by a potentiometer
  that modulates the voltage supplied to the motor.
• Speed is determined by the mass of the load in
  the rotor.

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• The tachometer measures rotor speed in rpm.
• The brake decelerates the rotor by reversing the
  polarity to reach a pre-programmed speed. This is
  achieved when the rotor decelerates without
  braking after a set time has elapsed.
• Refrigerated centrifuge is used when the heat
  generated during centrifugation could cause
  evaporation or denaturation of protein or leakage
  of cellular components in the sample.
• The temperature can be controlled between -15
  degree C and 25 degree C, allowing centrifugation
  at higher speeds and for prolonged periods.

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The selection of centrifuge tubes

• Its important to have knowledge about the
  selection of tubes.
• Plastic tubes (polystyrene and polypropylene)
  have a higher speed tolerance and can withstand
  RCFs as high as 5000 x g.
• Tubes with tapered bottoms, which form more
  compact pellets require certain conditions such
  as preparing urine sediment for analysis and some
  radioimmunoassay procedures.
• The tubes must fit in the buckets, small tubes in
  too large carrier results in improperly packed
  pellets.
• The top of the tube must not protrude so far
  above the bucket that the rotor is impeded.

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• Balancing of tubes within the buckets is crucial.
• Modern centrifuges automatically decelerate and
  shut down when buckets are improperly balanced.
• Improper balancing can cause the centrifuge to
  vibrate, disrupting the formed pellet.
• Biohazardous materials should be centrifuged with
  the caps or stoppers to reduce contamination and
  also aerosols.

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Maintenance and quality assurance

• The cleanliness of a centrifuge is important to
  minimise the possible spread of infectious
  agents, such as hepatitis viruses.
• When the tubes broke, both the racks and the
  chamber of the centrifuge must be cleaned
  carefully.
• Any spillage should be treated as blood-borne
  pathogen hazard.

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• The load in a laboratory centrifuge must be
  carefully balanced. Small differences in mass of
  the load can result in a large force imbalance
  when the rotor is at high speed. This force
  imbalance strains the spindle and may result in
  damage to centrifuge or personal injury.
• Centrifuge rotors should never be touched while
  moving, because a spinning rotor can cause
  serious injury. Modern centrifuges generally have
  features that prevent accidental contact with a
  moving rotor.
• Because of the kinetic energy stored in the rotor
  head during high speed rotation, those who have
  experienced the loss of a rotor inside of an
  ultracentrifuge compare the experience to having
  a bomb explode nearby.

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• The speed of the centrifuge should be checked at
  least once every 3 months via an external
  tachometer.
• The measured speed should not differ by more than
  5 from the rated speed under specified
  conditions.
• The centrifuge timer should be checked weekly
  against a reference timer (stopwatch) and should
  not be more than 10 in error.
• The temperature of a refrigerated centrifuge
  should be measured monthly.
• The brushes in the centrifuge motor should be
  checked at least every 3 months and replace them
  when they considerate wear.
• However in modern motor, brushes have been
  eliminated, removing a source of dust that would
  causes motor failure.

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references

• Anonymous (2007) Centrifuge at
  http//en.wikipedia.org/wiki/centrifuge date
  assessed 10th February 2007.
• Ashwood, ED. R. and Burtis, C.A. (2001) Tietz
  Fundamentals of Clinical Chemistry Saunders.

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• Thank you!