CENTRIFUGE

DR.R.MEERA
SECOND YEAR POSTGRADUATE
DEPARTMENT OF BIOCHEMISTRY
OBJECTIVES
• What is centrifuge?
• What are the types of centrifuge?
• What are the components of centrifuge?
• What is the principle behind centrifuge?
• What are the applications of centrifuge?
• How to operate centrifuge –do’s and don’ts?
CENTRIFUGE
• A centrifuge is a device that accelerates
gravitational separation of substances
that differ significantly in their masses.
• It is used to prepare samples, blood and
body fluids, in clinical chemistry for
analysis and also to concentrate urine
sediment in urinalysis for microscopic
viewing.
TYPES OF CENTRIFUGE –
Depending on rotor inside

• Fixed/vertical angle centrifuge

• Swinging bucket centrifuge
• Ultracentrifuge
TYPES OF CENTRIFUGE-
Based on usage
COMPONENTS OF CENTRIFUGE
COMPONENTS

Major components

Rotor/centrifuge head
A drive shaft
A motor
OTHER COMPONENTS

1.Tube holders with cushion pads

2.Power switch
3.Timer
4.Speed control -tachometer
5.Brake
6.Protective shield
7.Refrigerator in some centrifuges
8.programmable microprocessor
9.Recall memory options
10.audible and visible alarms
• Rotor shaft driven by gyro system ,bearings sealed to
minimize vibration
• Potentiometer-controls speed of the centrifuge-which
changes voltage supplied to the motor
• Tachometer-indicates speed of the rotor[rpm]
• Brake –reversing polarity of the current and cause rapid
deceleration
• Timer-shows preprogrammed speed and decelerate without
braking .
HOW IMPORTANCE IS TEMPERATURE
MAINTAINENCE?
• TEMPERATURE-simple run to separate serum / plasma from cells rise 5
degree rise in temperature
Change in temperature depends on
• Initial ambient temperature
• Rotor speed
• Duration
• Rotor design
More temperature changes –heat-evapourates and denaturation occurs -
change in analyte due to loss of water---so that refrigerater unit is
mounted beside the centrifuge
 MOTORS
• Small centrifuge –ALTERNATING CURRENT /AC motors
• Large centrifuge-high torque ,heavy duty, DIRECT CURRENT/DC –
electrical motor

REFRIGERATION
• Refrigeration-to reduce heat generation
• Temperature controlled between -15 to 25 degree C
TUBES

• Recommended tubes used for proper operation of centrifuge

• POLYPROPELENE TUBES –withstand 5000 xg
• Tapered bottom
• Top of tube should not protrude out of the bucket
POLYPROPELENE TUBES WITH
CONICAL BASE
Depending on the
motor that spins
type of rotor
maximum speed of sedimentation
presence and absence of vacuum
potential for refrigeration
maximal volume of sample capacity
table top /floor models
--the type of centrifuge is decided
Depending on -Types of rotor

1.Fixed angle rotor

2.vertical angle rotor
3.swinging bucket rotor
FIXED ANGLE ROTOR CENTRIFUGE
FIXED ANGLE ROTOR - tubes are held in vertical position at fixed
angle 14 to 40 degree

VERTICAL ANGLE ROTOR- tubes are at vertical angle 7 to 10

degree

Particals are driven outward- initially parallel and later at bottom FIXED ANGLE ROTOR
as tight pellets

Advantage:Allows more rapid sedimentation of small particles.

Can run at a higher speed –micro hematocrit centrifuges to

operate at 11,000 to 15,000rpm with RCF 14000x g

Disadvantage :creative resistance and air friction-generate heat

VERTICAL ANGLE ROTOR
SWINGING BUCKET /HORIZONTAL HEAD ROTOR

Rotor contains four buckets,each enable to swing

between Xshaped metal limbs.
ULTRACENTRIFUGE
• Contain fixed head rotors/swinging bucket rotors
• separation of lipoproteins and chylomicrons
• Seperation generates high heat due to long run so combined with
refrigerators.
• It is an miniature air turbine with small rotor capable of achieving a
centrifugal force of 165000 times the gravity [x g]- separate
chylomicrons from serum.
PRINCIPLES OF
CENTRIFUGATION
• Speed of centrifugation –revolutions per minute[rpm] {does not describe
the force required to separate two phases}
• So correct term is- relative centrifugal force /relative centrifugal field[RCF]
• unit expressed as no of times greater than gravity
• Eg.500 x g
RCF =1.118*10-5* r*n2
1.118*10-5 is empirical factor
r is horizontal distance[the radius in centimeters]
n is the speed of rotation[rpm]
RCF applied to fixed angle rotor is much less than horizontal head rotor –
because the tube in a fixed angle head is unable to swing outward
OPERTATION OF CENTRIFUGE
• NCCLS –NATIONAL COMMITTEE OF CLINICAL LABOURATORY
STANDARDS –PROPOSES RCF OF 1000 TO 1200 X g for 10 min+ 5min
 BALANCING
•
Centrifuge balancing is the process of ensuring that a centrifuge
is properly balanced and aligned be
fore use
• Tubes should be positioned opposite each other in the centrifug
e rotor to keep the gravity in the center
• Each bucket is balanced with respect to pivotal axis
• The weight of the rack+tubes+their contents should be equal to
the opposite side ,should not differ more than 1%
• Smaller the difference-smooth centrifugation
• Before operating-check for balancing
• Tubes filled with water can be used for balancing weights
• Weight of racks should not exceed the limits given by
 IMBALANCE
.
• When centrifuge goes through acceleration and
deceleration slow speed –cause slight vibration
• Imbalance leads to vibration
• Increased wear on centrifuge and frequent
breakage of the tubes
TIME REQUIRED TO SEPARATE
PARTICLES
Depends on
• Rotor speed
• Radius of the rotor
• Effective length travelled by the sediment particles[depth of the liquid
in tube]
FORMULA FOR CALCULATING SPEED REQUIRED OF A ROTOR
WHOSE RADIUS DIFFERS FROM THE RADIUS WITH A PRESCRIBED
RCF WAS ORIGINALLY DEFINED

Length of the time for centrifugation can also be calculated so that running with an
alternate rotor of different size is equivalent to running with the original rotor.

Deceleration time even with breaking takes as long as 3 min

Nomogram for relative centrifugal force[RCF] to revolutions per
minute[RPM]

• NOMOGRAM is a
graphical calculating
device, a two-
dimensional diagram
designed to allow
the approximate
graphical
computation of a
mathematical
function.
APPLICATIONS OF CENTRIFUGATION
Separation of thick precipitates from solution:
1. Precipitating proteins from serum during colorimetric estimation
of serum urea, creatinine
2. Separation of serum from clotted blood.
3. Seperating protein bound or antibody bound ligand from from
ligand in immunochemical assay.
4. Seperating lipid components such as chylomicrons from other
components of plasma or serum and lipoproteins from each other.
5. Separation of erythrocytes from oxalated (or heparinized) blood.
6. To determine packed cell volume (PCV) or hematocrit values ..
Applications ctd...
• Isolation of subcellular organelles: The subcellular particles can be
separated by differential centrifugation.
The purity (or contamination) of the subcellular fraction can be checked
by the use of marker enzymes.

Example: DNA polymerase is the marker for nucleus, glutamate

dehydrogenase for mitochondrion, glucose-6-phosphatase for
ribosome, and hexokinase for cytosolic fraction.
Application ctd…
• Determination of molecular weight (in the fields of protein and nucleic
acid chemistry) by studying their sedimentation characteristics.

• Estimation of purity of macromolecules, i.e. purity of DNA preparations,

viruses and proteins.

• Detection of conformational changes in macromolecules like DNA and

proteins.
Do’s
• Clean the centrifuge before every use –
• To minimize the spread of infectious material,inside and outside surfaces cleaned
with tenfold dilution of household bleach/disinfectant
TUBE BREAK
• Eventhough we practice correct procedure –some tubes break
• It is a potential hazard-blood borne pathogen-clean with germicidal agents
• ROTOR HEADS AND BUCKETS- AUTOCLAVED
• Any breakage with gray dust indicates tube breakage
• It should be decontaminated
• Broken glass embedded in cushions if not cleaned and replaced ,leads to frequent
breakage of tubes
Do’s
LUBRICATION ,MAINTENANCE ,REPLACEMENT OF BRUSHES ARE TO BE
FOLLOWED
• Speed of the centrifuge-checked once in 3 months .
• Measuring speed should not differ more than 5% than the rated
speed under specified conditions
• Weekly timer should be checked comparing with stop watch error not
more than 10%
• In refrigerated one –monthly temperature check
• Commutators and brushes-once 3 monthly and replaced if needed
DON’TS
• Don’t remove the tube stopper before centrifuge –to prevent aerosol
contamination
• Wooden sticks to remove fibrin clots on the top of the tube avoided-
potential cause for hemolysis
In our lab we have REMI 8C