TETRAS Educare Study Notes Vol 30 Centrifugation
Transcript of TETRAS Educare Study Notes Vol 30 Centrifugation
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TETRAS EDUCAREVolume 30
2011- 2012
Handout for Lifescience Aspirants
Compiled By:
Shanmugam V. M.
Faculty, Dept. o Biotech., !yoti "i#a$ College, %o$u&'oad,
Bangalo&e ( )*0 0+).p&oheli .$m$ gmail.com
Handout for Lifescience Aspirants
Compiled By:
Shanmugam V. M.
Faculty, Dept. o Biotech., !yoti "i#a$ College, %o$u&'oad,
Bangalo&e ( )*0 0+).p&oheli .$m$ gmail.com
BIOCHEMICAL ANDANALYTICALTECHNIQUESCENTRI U!ATION
BIOCHEMICAL ANDANALYTICALTECHNIQUESCENTRI U!ATION
Not for pri"ate circu#ation$ on#% for &NC students$An% 'ueries and su((estion fee# free to )rite and *ai# to *e at pro+e#i,-s*s.(*ai#-co*
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C E N T R I F U G AT I O N
INTRODUCTION:
One of the most common separation technique used for the preparation of sub-fractions (such as biomolecules such as proteins, carbohydrates, nucleic acids, enzymes
etc.,) and sub cellular organelles (such as lysosomes, ER, olgi comple!, pero!isomes,
mitochondria, plastids, ribosome, etc.) in many biological science lab is the centrifugation
"ith the help of centrifuge. # centrifuge is an instrument that spins liquid samples at high
speeds and thus creates a strong centripetal force of normal gra$ity. %arious factors
affect the effecti$e separation by the centrifugation such as size and shape of the
molecules, density and $iscosity of the medium and rotor speed.
&entrifugation techniques are of t"o types: preparati$e centrifugation and
analytical centrifugation
'. Preparative centrifugation technique is concerned actually "ith the separation,
isolation and purification of "hole cells, sub-cellular organelles, plasma
membranes, polysomes, ribosomes, chromatin nucleic acids, $iruses, etc. for
subsequent biochemical in$estigations. hey require large amount of the samples
for getting desired amount of the fractions.
. Analytical centrifugation normally concerned "ith the study of the sedimentation
characteristics of biological macromolecules and molecular structures, rather than
the collection of the samples or particular fractions. hese require small amount
of samples.
PRINCIPLE
# difference bet"een the density of a molecule or particle and the density of the
surrounding fluid can be e!ploited in a separation process. *olutions in "hich large
density differences e!ist "ill often settle under the influence of gra$itational acceleration
alone.
THEORY INVOLVED IN CENTRIFUGATION:
he molecules are getting sediment in the medium based on their se$eral factors.
he sedimentation "ith some centrifugal force application is the basic principle in the
centrifugation.
he rate of sedimentation is dependent upon the applied centrifugal field ( or +)
being directed radially out"ards, this can be calculated as the product of the square of 2 Shanes quick N fact les centrifugation
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the angular $elocity of the rotor ( , in radian s -' ) and the radial distance (r, in
centimeters) of the particle form the a!is of rotation.
&entrifugal force (angular $elocity) ! radial distance
F = 2r
*ince, one re$olution of the rotor is equal to radians, the angular $elocity can
be e!pressed as
= (2 rev min -1 ! "#
/ence, the equation of centrifugal force "ill become
F = ($ 2 (rev min-1 2 r ! %"##
0ut, the + or centrifugal force is e!pressed as a multiple of the earth1sgra$itational field ( g 23' cm s - ).
RELATIVE CENTRIFUGAL FORCE (RCF &
he ratio of "eight of the particle in the centrifugal field to the "eight of the
same particle "hen acted by the gra$ity alone is 4no"n as relati$e centrifugal force (R&+)
(commonly called as number time1s g).
RCF = ($ 2 (rev min-1 2 r ! (%"## ' )1
RCF = (1*11) ' 1# -+ (rev min-1 2 r
(From above equation, RCF is depends upon rpm (revolution per minute) and the
radius of rotation, r).
5here r is the radius of rotation (the radius of the rotor) in cm and rpm is the
centrifuge speed in re$olutions per minute. he number 6'.''2 ! '7 -86 is a con$ersion
factor that allo"s us to use 6rpm6 and 6g6 units.
he R&+ is dependent upon the radius of the rotation of the rotor, the speed at
"hich it rotates, and the design of the rotor itself (fi!ed angle or s"inging buc4et). Rotor
speed and design can be held constant, but the radius "ill $ary from the top of a
centrifuge tube to the bottom. 9f a measurement for the radius is ta4en as the mid-point,
or as an a$erage radius, and all forces are mathematically related to gra$ity, then one
obtains relati$e centrifugal force, labeled as ! g .
hree r-$alues gi$en (by the manufacturer) for a rotor: the ma!imum, from the
center of rotation to the bottom, top and middle of minimum and a$erage r. hese
correspond to the distances for the sample tube.
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RELATION ,HIP ET.EEN RCF (/ 0 r m
R&+ is gi$en by equation
r* *m = (RCF ' %"## / !$ 2r
9t can be shortened by as
r* *m = * +$ (RCF ' / ! r
his is the equation for the con$ersion of g to rpm and $ice $ersa.
0y plotting the $alues for and g
r* *m = 2 )* % RCF ! r
5here r is the radial distance bet"een from the a!is of rotor.
Other "ise Relati$e centrifugal force is the measurement of the force applied to a
sample "ithin a centrifuge. his can be calculated from the speed (R;
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,EDI3ENTATION COEFFICIENT OR ,VED ERG UNIT (,
*edimentation coefficient is defined as sedimentation rate or $elocity (>) of a
particle per unit of centrifugal field. he basic unit of sedimentation coefficient is ' ! '7 -'? sec. his is also called as one *$edberg unit (*), in recognition of . *$edberg1s
pioneering "or4.
he larger molecule or particle, the larger is its *$edberg unit or sedimentation rate.
*ome e!amples, for $iruses, *$edberg units are @7 to '777*, for lysosomes @7777* etc.
TYPE, OF ROTOR,&
Rotors for a centrifuge are fi!ed angles, s"inging buc4ets, continuous flo", or
zonal. +i!ed angles generally "or4 faster substances precipitate faster in a gi$en
rotational en$ironment. he most common is a rotor holding 3 centrifuge tubes at an angle
of ?@ A& from the $ertical.
1. Fixed angle rotors : 9n a fi!ed angle rotor, the materials are forced against the side
of the centrifuge tube, and then slide do"n the "all of the tube. his action is the
primary reason for their apparent faster separation, but also leads to abrasion of
the particles along the "all of the centrifuge tube.
2. Swinging bucket (hori ontal rotors! : +or s"inging buc4et rotors, the materials
must tra$el do"n the entire length of the centrifuge tube and al"ays through the
media "ithin the tube. *ince the media is usually a $iscous substance, the s"inging
buc4et appears to ha$e a lo"er relati$e centrifugal force, "hich is it ta4es longer
to precipitate anything contained "ithin. 9f, ho"e$er, the point of centrifugation is
to separate a molecules or organelles on the basis of their mo$ements through
$iscous field, then the s"inging buc4et is the rotor of choice.
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refrigerated rotor chambers and $ary only in their ma!imum carrying capacity, all
being capable of interchangeable s"inging buc4et type rotors and fi!ed angle rotors.
he temperature can be maintained in the range 7 o& to @7o&. hese instruments
normally used for the collection of microbial cells, larger cellular organelles and
proteins precipitate by ammonium sulfate.
8. U;?r: en?ri B/e - he ultracentrifuge is capable of reaching e$en greater
$elocities and requires a $acuum to reduce friction and heating of the rotor. 9t can run
at speeds up to C8,777 rpm, sufficient to allo" fractionation of biomolecules, for
e!ample: plasmid F#, chromosomal F#, and RF#. Gltracentrifuges are usually
refrigerated and are $ery e!pensi$e and delicate pieces of machinery. here are t"o
types of ultracentrifuge: analytical and preparati$e ultracentrifuge.
a. Preparative centrifuge : he preparati$e ultracentrifuges are capable of
spinning the rotor up to 37,777rpm (by producing a relati$e centrifugal
force up to D7,777g). he centrifuge chamber is refrigerated, sealed and
e$acuated to minimize any e!cessi$e rotor temperature. he temperature
monitoring is done by the sensor de$ice "hich control temperature and
refrigeration system. #n electronic circuit "ill determine the motor
imbalance because of unequal loading of samples in centrifuge tubes. +or
safety purpose, the ultracentrifuges are al"ays enclosed in hea$y armor
plating.
E!: airfuge ("hich is laboratory, air dri$en table top preparati$e
ultracentrifuge called airfuge, this is capable of acceleration of a
magnetically suspended ?.Ccm diameter rotor, accommodating D centrifuge
tubes on a friction free cushion of air in a non e$acuated chamber. he
rotor speed is up to '77,777rpm or 'D7,777g. 9t found application in
biochemical and clinical research "here the small amount of samples can
be prepared "ith ultra pure in nature by using airfuge)
b. Analytical centrifuge : this type of centrifuge is normally used for the
molecular mass determination in centrifugation step itself (either by
sedimentation equilibrium method or by sedimentation coefficient method).
hese centrifuges are capable of operating at forces as great as D77,777g
H=- '77g "ith temperature control "ithin appro!imately 7.' o&.
#nstru$entation%
#nalytical ultracentrifuge basically consists of follo"ing parts,
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a) ;ieren :mer: ;in ri :;
vie in/ >@?@/r: >i
Len ;en ;en mirr@r
;:?e
3@?@r @n en in/ ;en
e e ie e
Di:/r:mm:?i re re en?:?i@n @ An:; ?i :; en?ri B/e
he thermistor ma4es electrical contact "ith the control circuit by means of pool
of mercury, on "hich the rotor tip touches.
he rotor chamber consist upper condensing lens and a lo"er collimating lens. he
lo"er lens "ill allo"s the passage of the light so as the sample is illuminating by parallel
light. he upper lens and camera lens focus the light on the film. Rotor contains t"o
cells, namely the analytical cell and counterpoise cell.
Optical system: there are three of optical systems they are,
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'. Ultraviolet light absorption s stem (by measuring incident and transmitting light
after absorption by the sample)
. !"hlieren opti"al s stem (based on the refracti$e indices of the particles under
centrifugal force at different zones, the *chlieren system plots the refracti$e inde!gradient against the distance along the analytical cell, "hich is useful for the study
of sedimentation $elocity measurements).
?. Ra leigh interferen"e s stem (it employs the double sector cell. One sector
contains the sol$ent and other the solute. he optical measurement is based on
the difference in refracti$e inde! bet"een the reference sol$ent and the solution
by displacement of interference fringes)
Application of analytical centrifuge :
'. o determine the relati$e molecular mass of the macromolecules such as proteins
and F#
. o in$estigate the purity of molecules, %iruses and proteins.
?. o detect the conformation changes in macromolecules.
TYPE, OF CENTRIFUGATION&
&entrifugation is classified based on different criteria1s, such as based on purpose
of the centrifugation, speed at "hich the centrifuge is operating, method of application of
samples. 0ut based on the purpose of centrifugation is classified as #nalytical
centrifugation and preparati$e centrifugation.
Analytical centrifugation : analytical centrifugation is normally employed for the
molecular mass determination and chec4ing the purity of the molecules. Optical methods
are employed in this centrifugation for obser$ing molecules during centrifugation. he
samples present in the centrifuge cells ha$ing "indo"s that lie parallel to the plane of
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rotation of the rotor head. #s rotor rotates, the images of protein or molecules are
proIected by the optical system to the photographic plate. he concentration of $arious
molecules are calculated based on the absorption of light at $arious "a$elength ("hich
follo"s the 0eer1s Ja"), "hich can be detected and recorded further by the recorder"hich send bac4 the signals to the microprocessor.
he molecular mass of the molecules can be determined by either sedimentation
coefficient or sedimentation equilibrium method.
'. he formulae for the calculation of molecular mass by sedi$entation coefficient
$ethod are
3 = RT,! D (1 - 4 6
5here: R molar gas constant, absolute temperature, *
sedimentation coefficient, diffusion &oefficient of the molecule, >K partial
specific $olume of the molecule, L ensity of the sol$ent at 7 o&.
. he formulae for the calculation of molecular mass by sedi$entation coefficient
$ethod are
3 = 2RT;n (C 2! C1 ! 2 (1 - 4 6 (' 22 7 ' 12
5here: R molar gas constant, absolute temperature, >K partial
specific $olume of the molecule, L ensity of the sol$ent at 7 o&. &' and & are
the concentrations of solute at distance ! ' and ! respecti$ely from the centre of
rotation.
Preparative centrifugation%
here are many types of preparati$e centrifugation, "here ifferential and density
gradient centrifugation is $ery important.
1* &ifferential 'entrifugation ('ell fractionation! : the process of the separation ofcell organelles from the tissues or cells is referred as cell fractionation. o isolate
such organelles different organs (such as 4idney, li$er, brain etc) are homogenized in
the suitable homogenization buffer at @o&. he resulting suspension containing many
intact organelles are called homogenate. he homogenate "as later ta4en for the
fractionation by the biochemical technique called as differential centrifugation.
he principle for the separation is based on the differences in the sedimentation
rate of particles of different sizes and density.
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he differential centrifugation is carried out based on the series of four different
centrifugation at greater speeds. Each step yields a pellet and supernatant. he
supernatant is further subIected for further centrifugation to yield different cell
fractionation. #t the end of each step, the pellet is "ashed "ith homogenization
buffer and recentrifuation under same conditions, this procedure minimize the
contamination problem of other sub cellular organelles. . /ence this procedure "ill
results in four pellets of nuclear, mitochondrial, lysosomal and microsomal fractions.
he purity and identification of compound can be done by some mar4ers, such for
nucleic acids F# is the mar4er, acid phosphatase for lysosomal fractions, etc.
xa$ple for the differential centrifugation%
'7M "=$ li$er homogenate in 7. 8< sucrose (87ml)
&entrifuged '777g,ra$ 3cm, '7min
;ellet (nuclear) supernatant
&entrifuged '?77g,
r a$ 3cm, '7min
;ellet (mitochondrial) supernatant
&entrifuged 'D?77g,
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ra$ 3cm, 7min
;ellet (lysosomal) supernatant
&entrifuged '77,777g,r a$ 3cm, ?7min
;ellet (microsomal) supernatant(cytosolicfraction)
2* &ensity gradient centrifugation : in this method separation of molecules are done
by medium "hich has gradients. *ome of gradient medium used for the
centrifugation are &aesium chloride for F#, nucleoproteins, $iruses, etc. &aesium
sulphate for 0anding of F# and RF#, peptidoglycans, *ucrose for separation of sub
cellular particles, ficoll for separation of "hole cells, sub cellular particles and
$iruses. e!tran for banding of microsomes etc. /ere the separation is based on
the buoyant densities of the particle. his method is used as a better separation
than the differential. here are t"o types of density gradient centrifugation: the
rate zonal and isopycnic techniques.
a. )ate onal density gradient centrifugation : here the separation is basedon the size, shape, centrifugal force applied and $iscosity density of the
medium. he gradient used in rate zonal centrifugation technique has the
ma!imum density at the bottom but its density is lesser that the most
defense sedimenting particle to be separated. &entrifugation is initially
carried out at lo" speed later, the greater speed is achie$ed for the
effecti$e separation. his method "as used for the separation of proteins
"hich are identical densities, differing slightly in their size and used for the
separation of F# N RF# hybrids, ribosomal subunits.
b. #sopycnic centrifugation : this depends only on the buoyant density of
particle. 9t does not depend on the shape or size of the particle and is
independent of time. 9n this method the ma!imum density of the gradient
"as al"ays e!ceeds the density of the densest particle. # continuous
gradient "as used here. he sedimentation of the particle occurs until the
buoyant density of the particle and the density of gradient are equal. #t
this stage or point of isodensity, no further sedimentation occurs.
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9rrespecti$e of ho" long the centrifugation continues. 9t is "idely used for
the separation of molecules "hich are ha$ing same molecular shape or
similar size but differing in density. *ub cellular organelles such as olgi,
ER, mitochondria and pero!isomes can be effecti$ely separated from this
method.
FOOT NOTE& PRACTICAL POINTER,
'. &entrifuge sample containers come in a $ariety of shapes, sizes, and materials. 0e
careful to use the correct tubes. lass tubes al"ays require an adapter to cushion
them because direct contact bet"een the glass and a metal rotor "ill result in a
crac4ed or shattered tube. #dapters may slide o$er the tube li4e a slee$e or may be
cushions that sit in the bottom of the rotor slot. ( he 6cushion6 adapters may be found
in clinical centrifuges but are not used in high speed centrifuges.)
. ubes that are o$er-filled can spill their contents inside the rotor. +or fi!ed-angle
rotors it is especially important not to fill the tubes more than =? to ?=@ full. *pills
need to be "iped up immediately to pre$ent corrosion of the rotor. 9t is al"ays a good
practice to "ash out a rotor "hen you are finished "ith it e$en "hen there s no
apparent spill liquid from the tubes often spatters. Rinse the rotor "ith distilled
"ater, dry "ith paper s to let any moisture drain. 5ipe up any liquid in the chamber
to"els, and store upside do"n on top of paper to"el of the centrifuge, then use a
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paper to"el (dampened "ith distilled "ater) to "ipe do"n the chamber. ry the
chamber "ell if the refrigeration has been used, don t lea$e the door open any longer
than necessary because condensation "ill build up on the chamber "alls.
?.