tetras educare study notes vol 30 centrifugation

8/11/2019 TETRAS Educare Study Notes Vol 30 Centrifugation

1/14

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*


2/14

Study handout o& li e $cience a$pi&ant$ "o#u*e /0

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


3/14

3Study handout o& li e $cience a$pi&ant$

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.

Shanes quick N fact les Centrifugation


4/14


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;


5/14

)Study handout o& li e $cience a$pi&ant$

,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.



6/14



7/14

/Study handout o& li e $cience a$pi&ant$

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,



8/14


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,

Shanes quick N fact les centrifugation


9/14

+Study handout o& li e $cience a$pi&ant$

'. 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



10/14


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.

10 Shanes quick N fact les centrifugation


11/14


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,



12/14


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.

12 Shanes quick N fact les centrifugation


13/14


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



14/14


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.

?.

tetras educare study notes vol 30 centrifugation

Documents