Centrifugation

CENTRIFUGATION
 OUTLINE
• Basic theory
• Applications
• Instrumentation
 SEPARATIONS
• This begins new topic: separations
• Prior to this, talked about measurements and
solution making
 BIOSEPARATIONS
• Separating and purifying biological materials
• Filtration and centrifugation
• Chromatography and electrophoresis also
common methods
 BIOSEPARATIONS

• Ultrafiltration

• Dialysis

• Precipitation

• Sedimentation
 PRINCIPLE
Rate of settling of a particle, or the rate of
separation of two immiscible liquids, is
increased many times by the application of a
centrifugal field (force) many times that of
gravity.
 MANY APPLICATIONS
• Separate two immiscible liquids
• Isolate cellular organelles
• Isolate DNA, RNA, and proteins
• Isolate small particles including
• Bacteria
• Viruses
• Cells
SUPERNATANT AND A PELLET
Supernatant is the liquid at the top
Pellet is particles at the bottom
 FORCE IN A CENTRIFUGE IS
PROPORTIONAL TO TWO THINGS

• First, it depends on how fast the centrifuge

spins
• Second, it depends on the radius of
rotation

• F = mω2r
 Radius of Rotation

The farther a particle is from the center of rotation,

the more force it experiences
 RELATIVE CENTRIFUGAL FORCE, RCF
RCF = (RPM)2 × 1.118 × 10-5 × r.

Where r = radius in cm from centerline

RPM = revolutions/minute
 CALCULATING RCF
Suppose rmin = 3.84 cm
raverage = 6.47 cm
rmax = 9.10 cm
N = 30,000RPM
Then, what is the RCF
on a particle at
rmin, rave, and rmax?
 ANSWERS
1.118 x 3.84 (30,000)2 x 10-5= 38,638 X g

1.118 x 6.47 (30,000)2 x 10-5 = 65,101 X g

1.118 x 9.10 (30,000)2 x 10-5 = 91,564 X g

Don’t report RPM, report RCF because

everyone’s centrifuge is different
Nomogram for conversion of RPM to RCF.

Q. You are following a

protocol that says:
Spin cells at 400 rpm for
10 minutes. What else do
you need to know?

RCF=11.2 x r (RPM/1000)2
r in cm
HOW FAST DOES A PARTICLE
SEDIMENT?

It depends on:
RCFs in the centrifuge
Size of particle
Particle density
Liquid density
Liquid viscosity
Sedimentation by centrifugation:

If a particle has the same density as the liquid

around it, the particle doesn’t move
If a particle is more dense than the liquid, it moves
down the tube
If a particle is less dense than the liquid, it moves
up!
 THE BASIC MODES OF CENTRIFUGATION
DENSITY
DENSITY is the mass per unit volume of a substance, often expressed in g/ml.

DIFFERENTIAL CENTRIFUGATION
DIFFERENTIAL CENTRIFUGATION separates particles on the basis of their size.
By a series of centrifugations at various speeds and times, different-sized particles
are sedimented and collected from an initially homogenous suspension.

DENSITY GRADIENT CENTRIFUGATION

DENSITY GRADIENT CENTRIFUGATION is separation performed in a supporting
column of solution in which the density and solution concentration increase toward
the bottom of the centrifuge tube.

DISCONTINUOUS, OR STEP, GRADIENT

DISCONTINUOUS, OR STEP, GRADIENT is composed of layers, with abrupt
changes in density and/or concentration from one layer to the next.
Differential centrifugation for fractionation

Fractionation of a cell homogenate to isolate nuclei or smaller parts of cell.

Density Gradient Centrifugation
Separation by centrifugation through a sucrose
gradient.

Two types of
hepatitis B capsids

Separation and crystallization of T = 3 and T = 4

icosahedral complexes of the hepatitis B virus core
protein. Acta Cryst. (1999). D55, 717-720
Step Gradient-discontinuous gradient centrifugation

4000 rpm

Histopaque
 INSTRUMENTATION
Lots of types
Some go faster and some slower
Some can take a lot of volume, others little
Some allow temperature control
Some allow you to add sample as centrifuge is running
Some are specific for pathogens, the aerosols must be
avoided
 TERMINOLOGY
1. Desktop or clinical centrifuges <10,000 RPM

2. Highspeeds, 10,000 – 30,000 RPMs (around

50,000 x g)

3. Ultracentrifuges up to 80,000 RPM and

500,000 x g
INSTRUMENT DESIGN
 SAFETY!!
Centrifuges look sturdy, sort of like washing
machines
But, they are probably the most dangerous
instrument any of you will use
Also surprisingly easy to damage
BE CAREFUL!!!!!
 ROTORS ARE FRAGILE
Must withstand huge forces
In an ultracentrifuge, a 1 gram particle “weighs”
0.65 tons
Any imperfection will weaken rotor
Therefore:
BE CAREFUL!!!!!
Centrifugation basics
Always balance opposing tubes
Place test tube in centrifuge holder.

Balance with another test tube

filled to the same level
in the opposite holder.
Centrifuge Basics-contd.
Make sure you are using the correct rotor for the centrifuge.

Never exceed the maximum g force for the rotor.

Make sure you are using the correct centrifuge tube for the job.

Tubes may break if centrifuged at a too high speed.

Even plastic tubes can break if not in appropriate adaptors.

Pre-cool centrifuge and rotors.

NEVER walk away from a centrifuge until it reaches top speed.

 Rotor Collapse

The higher the speed

the more accurately
balanced the centrifuge
must be.
 PROPER USE OF ROTORS
Every rotor has a maximum speed
As high speed rotors age, derate
With ultra-rotors retire them after certain age or
number of revolutions
Log books record every use and revolution
Overspeed discs on bottom
Purchase the correct rotors for your application
Derate them as necessary –
Follow manufacturer’s directions
Balance, balance , balance
 BIGGEST CHALLENGES IS TO READ
THE CATALOGS
Rotor and tubes must match application
Rotor and tubes must match centrifuge
Rotor and tubes each have maximum RPMs at
which can use them – and may be different
Use the slower value
May need adaptors to fit certain tubes into
certain rotors

This is because rotors are engineered to take varying

size and styles of tubes
Makes them versatile, but also requires complex
combinations of adaptors and tubes
 GENERAL RULES
Protect the rotors from:

Scratches
Moisture
Spills
Alkaline detergents
 MUCH MORE SUBTLE, BUT ALSO
DANGEROUS,
Aerosols
Inevitable with normal centrifuge and very high
levels are released if accident occurs
Special centrifuges use special rotors, caps, and
seals that prevent leakage
Containment is always desired