GeNei*

Gel Flltralion Chromalography QeNei*

CONTENTS

PageNo.

t objective 3

i Pfinciple 3

* KitDescription 6

+ Matedals
Provided 7

'l Procedure B

* Obse.vation
& Intepretation I

* Ordedng
Information 10

@B.ngrior€C.n.l,2006
Gel FjltrationChromalography GeNei'"' Gel Flltfation Chromatography GeNei'"

Obiective:
To learn lhe separaiionol biomoleculeson the baSisot
their size by Gel filtration chromatogr€phy.

PrinciDle:
) Chromatographyis a sepafationmelhod thal reles on
I dillorencesin partitioningbehaviorbetweena flowingmobate
phaseand a slaiionaryphaselo separatethe components
oi a mixture.A column holds the slallonaryohase and lhe
mobileohase carriesthe samole.

Gel-fillralionchromatography,also calledsize-exctr.rsion
or gel-permeation chromatography, separatesmolecules
basedon lhe differencesin lheir size.Th9 sampleis applied
on top of the column containing porous bead6. As lhe
moleculespass through the coltrmn of porous beads ot
crosslinkedagarose,they get sepa€ted as fottows:
. Large molecules cannol enter lhe pores and elule as
the lirsi peak in the chromalogram.
They etulelast and
lhis is callediotal exclusion-
. Interm€diatemoleculesmay enter the pores and may
have an average residence time in the panicles
dependingon lheir size and shap€.Ditferentmolecules
thereforehave d fferenttotal transittimes thfouqhihe
column.This podion ol a chromatogramrs calt6drhe
selectivepermeationregion.
. Smallmoleculssenterlhe pores and have the long€st
residencetim€ on ihe column and elute loqelher as
lhe lasl peak in lhe chromatogram.This tasi peak in
the chromalogramis the tolal permeation timit
(Ret6rfigure 2),

O BanglloE G.n6i,20Oo 6 Brngaror.G.n.'. 2006

 Gel FittrationChromalography GeNei"' Gel FiltrationChromatography GeNei"'

Columncontainsstationaryphase

Lad
Porousgel bead sryrle <- ,qddBrfBr ---------+

Smailermoleculesretardedby
U L'H H H U tJ
enteringpores in beads
tl tln n | l n n
l Ilili]l i l lll
Molecules too large to enter
!Y)r)f
t J)r)rY)/
!EHl t u
U U U WH b
pores pass rapidly through

Fig 1: SchematicDiagramofa celFittrationChromatography

Column Componentscollectedin differenttubes

MoleculeSize
l ffiLlE
H{t
l
t*n
iii:::j

Fig 2i Schemalicrepres€ntationof.sample separationby

gel tiltrationchromatography

O B,nsaror.G.nsr,2006 4 o Bansaroio
G.n.i, 2006
GslFiliretionChromatography GeNei'"

OrderingInfgrmation

Product Size

CeNeld Gel Filtration 1 Pack

ChromatographyTeachingKit
(Consumables
for 5 experiments)

O Aang.lo'ec.n.r,2006
 Gel FiltrationChromatography GeNei'" Gd FiltrationChromatography GeNei"
Note:
Observation:
. Read
,the entire procedure before arting the Note down the orde. in which various rholeculesare
. collectedand inteeret the rcsults.
Reconshtute.onesampte vat per expertme wih
u.z mt ot Outtcr to get a hamogenous solutpn Store
lhe sample at 4aC and use wihin 3 motns. Interprelation:
. Do not let the cotumn go dry at any tjme. . Blue coloutedco-1pone1tis bruedext,ar navirg a
. Atv/ays open the top cap first and lhen the battan cap molecularweightof2,000000 Da. Theseare verylaige
to stai the flow of bufler in column. molecules thatexilfaslfrornthecolumnandarecollected
gmilarly Io stopthe ftot ofbuflerorto as firsl fraction.
storcthecolumn, .
nx tne bonom cap fist and lhen the top cap. Brownishred coloufedcomponentcollecieoas second
Column can Lre usealfive tines ontu f'acdon_rsh€e.noglobi.lhavinga mo,ecutarweighl of
Store the column at 4"C after eaci use 64500 Da. These motecutesare ol jnter/.red;ate stze
and they enterthe poresbut theirretentionlime is low.
These moleculestake an averagetime lo exil lhe
Procedqrei
1. Fixihecolumnverticay to a siand. . Pink colouredcomponenlis vitamin 812 having
2. Equilibrate thecotumnwilh4 mtGetfi ratronbufter.Drarn molecular weight of 376 Da, These are very small
out the buffercomptetety. moleculesthatenteror permeatelhe poresof th; beacls
3. Load 0 2,ml ot lhe sampleonto rhe cotumn a,ongme and are retainedin the bead for a longertjme. They
srdesof lhe colLmn lake a long time to exit the columnand are coilected
4. Allowthe sampleto sinkcompletely andthenaddo.z ml as lhifd fraction.
of buffer
5. Allowihe bufferto flowout completety.
6. Keep iiliing the columnwith bufier,ti all the coloured
bromo,ecules have otutedo.rl. (Approximalety20 mt of
ounerrs required).
7_ Collect the colored fractions in difierent lubes.
(Beferfig 2).
8. E,x lhe,boltoncap and then lhe top cap to stop ^e
llow of buffef Sloreat 4.C for next use_

o B'nsaro'eG.nei.2oo6
@Bangdrorc
c€n.r,2005
 Gel Fillration Ch.omatography GeNei" Gel Fillration Chromatography GeNei"
Kit Description:
Materials Provided:
ln this kit, a mixlureot ihree drfferentbiomotecules
rangjngin molecutarsize from 376 Da to 2000 kDa is The list belowprovidesinformationaboutthe materials
supplied.Theseafe separaiedon the basisof theirstze suppliedin thekit.Theproductsshouldbe stoed as suggested.
thfough use the kil wilhin6 monthsof afiival.
a gelfiltration
cotumn. Themovemenl ofthesamples
throughlhe columnis easijymonitoredas the bioffolecules
are coloured,which also aids in.collectionof the resolved Quantity
KT39
(s expts.)
K739 : The kit is designe.J to carry aul
5 experinents, i el filllalion column (2 mr) 1 No. 4'C
)el fillralionbutler 125m J 4'C
Duration of experiment: Approxtmatetyt hour
5x0-2ml 4'C

MaterialsRequired:
Glassware:Tesltubes.
Reagent : Distilledwater
Other Requirements : Columnstand; Micropipelte.Tips.

o Bansaro16
Genei,2006
o Brns.roBGonei,2006