The document discusses centrifugation and its applications in bi separations. Centrifugation uses centrifugal force generated by high-speed rotation to separate particles, cells, organelles, and other biological materials based on density. It describes how mitochondria are isolated from mouse liver tissue using differential centrifugation at increasing speeds and centrifugation in a Percoll density gradient to purify the mitochondria. Safety considerations for operating centrifuges to avoid rotor failure and aerosol release are also covered.

1. CENTRIFUGATION

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OUTLINE
 Basic theory
 Applications
 Instrumentation

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SEPARATIONS
 This begins new topic: separations
 Prior to this, talked about measurements and
solution making

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BIOSEPARATIONS
 Separating and purifying biological materials
 Filtration and centrifugation
 Chromatography and electrophoresis also
common methods

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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.

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MANY APPLICATIONS
 Separate two immiscible liquids
 Isolate cellular organelles
 Isolate DNA, RNA, and proteins
 Isolate small particles including
 Bacteria
 Viruses
 Cells

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SUPERNATANT AND A PELLET
 Supernatant is the liquid at the top
 Pellet is particles at the bottom

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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 – think about “crack the whip”

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RELATIVE CENTRIFUGAL
FORCE, RCF
 Also = Xg
 RCF = 11.17(r)(n/1000)2
 Where r = radius in cm from centerline
 n = rotor speed in RPM, revolutions/minute

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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?

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ANSWERS
 38,600 X g
 65,043 X g
 91,482 X g
 Don’t report RPM, report RCF because
everyone’s centrifuge is different

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HOW FAST DOES A PARTICLE
SEDIMENT?
 It depends on:
 RCFs in the centrifuge
 Size of particle
 Particle density
 Liquid density
 Liquid viscosity

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 It turns out that if:
 A particle is the same density as the liquid around
it, the particle doesn’t move
 A particle is more dense than the liquid, it moves
down the tube
 A particle is less dense than the liquid, it moves
up!

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TWO BASIC MODES OF
CENTRIFUGATION
 Most familiar is differential centrifugation
 Also density gradient centrifugation

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 On the next slide there is an excerpt from a
research article. Explain how centrifugation
is used in this research project.

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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

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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 or whenever
aerosols must be avoided

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TERMINOLOGY
 Desktop, or clinical centrifuges <10,000 RPM
 Superspeeds, 10,000 – 30,000 RPMs
(around 50,000 Xg)
 Ultracentrifuges up to 80,000 RPM and
500,000 Xg

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INSTRUMENT DESIGN

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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!!!!!

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TWO MAIN SAFETY
CONCERNS
 First is rotor coming off shaft - disaster

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ROTORS ARE FRAGILE
 Must withstand huge forces
 In an ultracentrifuge, a 1 gram particle
“weighs” 0.65 tons
 Any imperfection will weaken rotor
 Therefore:

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 Expert design
 Proper use
 Retire at correct time
 Derate (run slower) when necessary

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PROPER USE OF ROTORS
 Every rotor has a maximum speed
 As high speed rotors age, derate
 With ultrarotors retire them after certain age
or number of revolutions
 Log books record every use and revolution
 Overspeed discs on bottom

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 Purchase the correct rotors for your
application
 Derate them as necessary – follow
manufacturer’s directions
 Balance, balance , balance
 Check your textbook, p. 561 for guidelines

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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

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 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

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GENERAL RULES
 Protect the rotors from:
 Scratches
 Moisture
 Spills
 Alkaline detergents (like Countoff, for
radioisotopes)

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 Follow manufacturer’s directions and NEVER
NEVER NEVER NEVER! spin faster than is
supposed to go
 Select tubes that can handle the samples and
speeds you are using
 Keep your hands and hair out of centrifuges!
 Keep your hands out of rotors
 Wear glasses
 Make sure you know what you are doing

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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