3.

0 Centrifugation
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 CLO 02
Illustrate the principles and applications of
basic centrifugation, chromatographic and
spectroscopic analytical techniques in the
isolation and characterization of biological
molecules.

(i) The LLO(s) (ii)

Understand the fundamental of Differentiate different type of
centrifugation centrifugation techniques and
their applications

(iii)
Knows different types of centrifuges,
rotors and it cares
Outlines

Differential &
Basic Principles
Density Types of
and Rotors and Care
Gradients centrifuges
Applications
centrifugation
Overview
• Biological centrifugation is a process that uses centrifugal force to
separate and purify mixtures of biological particles in a liquid medium.

• One of widely used laboratory technique for the separation of materials in

the field of, biochemistry, molecular biology, medicine, food sciences and
industry.

• Today, centrifugation techniques represent a critical tool for modern

biochemistry and are employed in almost all invasive subcellular studies.
General steps
Bioseparations
Centrifugation is everywhere….

It is a key technique for isolating and

analysing cells, subcellular fractions,
supramolecular complexes and isolated
macromolecules such as proteins or nucleic
acids.
Centrifuge vs Centrifugation
• A centrifuge is a device that separates particles from a solution
through use of a rotor.

• Centrifugation is a technique that uses centrifugal force to separate

and purify mixtures of biological particles in a liquid medium.
Why do we need to centrifuge?

• Its all about gravity and mass –particles

in a heterogenous solution will, given
enough time, separate based on their
size and density.
• Smaller , less-dense particles may also
migrate down, but not always; some
particles will never settle, but remain
suspended in solution

…….But do we have the patient to wait long?????

Centrifuges force this process along much more quickly and
efficiently
Principle of
Operation
• A centrifuge is a piece of equipment,
generally driven by an electric motor, that
puts an object in rotation around a fixed
axis, applying a force perpendicular to the
axis to separate substances of different
densities.
• Tubes in the centrifuge are tilted so
centrifugal force can pull denser substances
towards the bottom of the tube.
• Relative Centrifugal Force (RCF) measures
acceleration applied to the sample
FORCE IN A CENTRIFUGE IS PROPORTIONAL
TO TWO THINGS
• First, it depends on how fast the centrifuge spins

Speed use in centrifugation; the higher the speed the

higher the centrifugation force thus the smaller molecules
separated.

• Second, it depends on the radius of rotation

The greater the radius of
rotation, the more force that is
experienced by the molecule

Imagine this thing with longer string!!

RELATIVE CENTRIFUGAL FORCE (RCF)
• Also called x g
• RCF = 11.17 (r) * (n/1000)2

Where r = radius in cm from centerline

n = rotor speed in RPM, revolutions/
Try these….
1. Calculate the RCF for a centrifuge with a rotor with a spinning
radius of 6 cm that is spun at 4,000 RPM?

1. Calculate the RPM for a centrifuge with a rotor with a spinning

radius of 6 cm that needs a relative centrifugal force of 5,000 g?
Post centrifugation sample

Supernatant – liquid at the top

Pellet – particles at the bottom

How fast does a particle sediment?
• It depends on:
• The applied relative centrifugal force (RCF)
• Size, density and radius of particles
• Density and viscosity of the suspended medium
This is how separation is achieved

www.phys.sinica.edu.tw/TIGP.../AC_Chapter%203%20Centrifugation%200321.pdf
Applications

• Separation of two immiscible liquids – eg: to remove cellular debris from blood to prepare
cell free plasma or serum
• To concentrate cellular elements and other components for microscopic and chemical
analysis
• To separate protein bound or antibody bound ligand from free ligand in immunological assay
• To separate and isolate subcellular organelles, macromolecules - DNA, RNA, proteins or lipids
• For determination of purity and shape of biomolecules
• To determine the relative molecular mass analysis using density gradient
Basic modes of centrifugation
There are two (2) types:
1. Differential centrifugation
2. Density gradient centrifugation
1. Zonal
2. Isopycnic
Differential
Centrifugation

• Basic concept:
Particles of different
sizes will sediment at
different rates.
• Separate particles
into two phase; a
pellet and a
supernatant

Procedure used to isolate different particles by stepwise successive

centrifugations at increasing RCF
• Rough separation of
subcellular components

• Usually performed prior

to density gradient
centrifugation

• Carried out using Fixed

Angle rotor
 2. Density centrifugation
i) Zonal centrifugation a.k.a band or gradient centrifugation
• A density gradient is created in the tube with a suitable medium (eg. Sucrose and
glycerol) having high density at the bottom
• The sample is applied in a thin zone at the top of the centrifuge tube on a density
gradient.
• Upon centrifugal force, particles move at different rate depending on their size
and mass
2. Density centrifugation
ii) Isopycnic or sedimentation equilibrium
centrifugation
• Where as, in isopycnic, the sample and the
medium is uniformly mixed in the tube and
the rotate in the centrifuge

The particles travel through the gradient until

they reach a point at which their density
matches with the density of surrounding
sucrose medium forming separate bands
Confused? These are they key points….
• 1. Differential centrifugation
• Cell-free extract and supernatant are centrifuged at progressively higher speeds and longer
times

• 2. Density gradient centrifugation

• Cell free extract is centrifuged through a medium whose density gradually increases toward
the bottom of centrifuge tube

• a) Rate-zonal
• Sample applied at the top and centrifuged until most dense component approaches
bottom of centrifuge tube
• b) Isopycnic
• Sample is mixed and centrifuged until all components reach their equilibrium buoyant
density.
Types of centrifuges
• Centrifuge are generally divided into 3 types based on their maximum
attainable speed:
• 1. Low speed ; < 10, 000 rpm
• 2. High speed; 10,000– 30, 000 rpm with temperature control
• 3. Ultracentrifuges ; up to 100, 000 -150 000 rpm with temperature control
Types of Centrifuge
• Low –speed centrifuge
• Also called: microfuge, clinical, table top, bench top centrifuge
• Max speed < 10, 000 rpm (3000-7000 rpm)
• Do not have temperature regulatory system; operate at RT
• Fixed angle or swinging bucket can be used
• Commonly for rapid separation of coarse particles
• E.g. RBC from blood, DNA from protein, bacterial cells etc.
• The ample will be centrifuged until the particles are tightly packed into pellet at the
bottom of the tube.
• Liquid portion, supernatant is decanted
They are used to collect small amount of material that rapidly sediment
High-speed centrifuge- Preparative
• Max speed < 30, 000 rpm
• Often refrigerated, and requires vacuum to operate
• Fixed angle or swinging bucket can be used
• Generally used to separate macromolecules (proteins or nucleic acids)
during purification or preparative work.
• Can be used to estimate sedimentation coefficient and MW
Can be used to collect micro-organism cellular debris, larger cellular organelles and
proteins precipitated by ammonium sulphate
Ultracentrifuge
• The most advanced form : specialized and expensive
• Used to precisely determine sedimentation coefficient and MW of
molecules, molecular shape, protein-protein interaction
• Uses very high speed
• Uses small sample size (<1 ml)
• Uses relatively pure sample
• Built in optical system to analyze movements of molecules during
centrifugation
Can be used both for preparative & analytical works

▪In this centrifuge rotors are

mounted on a rigid shaft . Shaft is
made up of aluminum or
titanium alloy of high tensile
strength to withstand the great
force generated during
centrifugation.
▪It is used for both preparative work
and analytical work
Instrument Design
Type of Rotors
• The principle component of a centrifuge is the rotor, which is the
moving part that spins at high speeds.
• Common types of rotor used:
1. Fixed Angel Rotor
2. Swinging bucket
Fixed Angle Rotor
• It holds tubes in the exact same fixed angle all the time, usually 45
degree
• Mostly used in lab for simple pelleting applications
• Also useful for isopycnic separation
• Due to rigid design of the metal alloy material, fixed rotors can
withstand much higher gravitational forces.
• Sedimenting particles have only shorter distance to travel before pelleting
Swinging Bucket Rotors
• Has bucket that start off in a vertical position but during acceleration
of the rotor swing out to a horizontal position
• Hence, the solution in the tube, is aligned perpendicular to the axis of
rotation
• The tube returning to its original position during decelaration of the
rotor
Longer distance of travel may allow better
separation, such as in density gradient
centrifugation. Easier to withdraw
supernatant without disturbing pellet.
When and which rotors to use?
• Depending on the
• 1. Applications
• 2. Production
 Carefully read the manual before using
ROTORS ARE FRAGILE! centrifuge

• Must withstand huge forces

• Regular maintenance
• Do not exceed maximum speed!
• Derate (run slower) when necessary
• Balance, balance, balance
• Proper handling and care; protect rotor from
• Scratches, Moisture, Spills and detergents
• Purchase the correct rotors for your application
• Log book- accurate record-keeping of run times and speeds
 Follow manufacturer’s directions
Proper use of Rotors
• Wear safety PPE; gloves, glasses
1. Place correct size of tubes in centrifuge
2. Proper balancing and counterbalance: Ensure loads are evenly BALANCE!
• By mass NOT volume
• Put the tubes opposite each other in the centrifuge. If your have more than two
tubes, only the ones opposite each other have to be equal in mass.
3. If centrifuge has variable speeds, enter RPM (do not exceed max speed!)
4. Close lid
5. Turn timer and press start
6. Remove the tubes carefully after the centrifuge has completely stopped
spinning to prevent remixing
7. Make sure you know what you are doing
 NEVER exceed the speed limit and Balance,
balance and balance….
• Result of unbalanced centrifuge
How Did Scientists Find Cytochrome C? Preparation of Mitochondria from
mouse liver

The mouse livers were removed after sacrifice and dounce homogenized in ice-cold mitochondria
isolation buffer (MIB) containing 250 mM mannitol, 0.5 mM EGTA, 5 mM HEPES, and 0.1% (w/v)
BSA (pH 7.2) supplemented with the protease inhibitors of leupeptin (1 mg/ml), pepstatin A (1
mg/ml), antipain (50 mg/ml), and PMSF (0.1 mM). Unbroken cells and nuclei were pelleted by
centrifugation at 600g for 5 min at 4oC. The supernatants were further centrifuged at 10,000g for 10
min at 4oC to pellet the mitochondria. The mitochondria pellet was resuspended in 4 ml MIB and
loaded onto a continuous Percoll gradient consisted of 30% (v/v) Percoll (Sigma), 225 mM mannitol,
25 mM HEPES, 0.5 mM EGTA, and 0.1% (w/v) BSA (pH 7.2). The suspension/gradient was
centrifuged at 40,000g for 1 hr. The mitochondria were removed from the brownish band at 1.10
g/ml with a transfer pipette. The mitochondrial pellets were washed with MIB by centrifuging for 10
min at 6300g at 4oC. The mitochondria were then resuspended gently in mitochondria resuspension
buffer containing 400mM mannitol, 10 mM KH2PO4, and 50 mM Tris-HCl (pH 7.2) with 5 mg/ml BSA
and stored on ice for up to 4 hr.

http://www.swmed.edu/home_pages/wanglab/Wanglab-pic/protocols.htm
THANK YOU