Gel Permeation Chromatography (GPC) Technique

 
'
 
GEL
 PERME
A
TION CHROMATOGRAPHY
 
 
Introdu
c
t
i
on
Princ
i
ple
 
of
 
sep
a
ra
t
ion
Th
e
ory
 
of
 
s
ep
a
ra
t
ion
G
P
C
 
componen
t
s
Exper
i
m
en
t
al
 
setup
Sepa
r
a
t
ion
 
pro
c
edu
r
e
Advan
t
ag
e
s and disadv
a
nt
a
ges
Appl
i
c
a
t
i
ons
 
 
 
In
t
r
o
d
uc
t
ion
   
 
Gel permea
t
ion chro
m
a
t
ogr
a
phy
   
 
Gel chromatogr
a
phy
   
 
S
i
z
e exc
l
u
s
ion
 
chro
m
a
t
ography
   
 
Gel fi
l
tr
a
t
i
on
   
 
Mole
c
ul
a
r
-sieve
 
ch
r
o
m
a
t
ogr
a
phy
Definitio
n
:
T
e
chn
i
que
 
: 
sep
a
ra
t
ion
 
of
 
components bas
e
d
 
on
 
the di
f
fe
r
en
c
e in 
m
ol
e
cu
l
ar
weight 
o
r
 
size, and is
 
one
 
of
 
the e
f
fe
c
t
i
ve
 
m
e
t
hods
 
u
s
ed
 
to i
s
ol
a
te
 
and
 
an
a
l
y
ze
 
the bi
o
-
m
a
c
ro
m
ol
e
cu
l
ar
 
sub
s
t
a
nc
e
s.
 
B
r
i
e
f back
g
r
o
u
nd
 
L
i
ter
a
t
u
r
e
 
   
 
1951-1955
 
the SEC 
c
onc
e
pt
 
was f
i
rst
 
re
c
ognised but 
w
as
 
not c
l
e
a
rly
 
for
m
ul
a
t
e
d
  
 
1963-
 
Jim
 
W
a
t
ers
 
build the
 
first
 
co
m
m
er
c
i
a
l 
G
P
C 
e
quip
m
ent based
 
on John
 
Moore
s
 
design
 
1979-
 
Y
a
u
, Kirkl
a
nd
 
and Bly
 
publ
i
shed
 
the first def
i
ni
t
ive
 
t
e
xt on
 
SEC whi
c
h
 
con
t
ribu
t
ed
 
to
the suc
c
ess
 
of
 
SEC
 
and
 
a comple
t
e
 
re
v
ised ed
i
t
i
on
 
was pub
l
ish
 
in 2009.
 
Ji
m
 
W
a
t
ers
 
- 
Joh
n
 
C. M
oo
re
 
-
 
Pr
o
f.
 
Fred
 
V
.
 
-
 
L
arry
 
E
.
 
Ma
l
ey
 
Mo
d
el
 
GP
C
-
10
0
 
price
 
$12
,5
0
0
 
Princ
i
ple
 
of
It
s
 
a t
e
chn
i
que
 
th
a
t 
s
ep
a
ra
t
es
 
dissolved
m
ol
e
cu
l
es
 
on
 
the basis of the
i
r 
s
i
z
e by
pu
m
ping 
t
hese
 
m
ol
e
cu
l
es
 
through
spe
c
i
a
l
i
z
e
d
 
co
l
u
m
ns 
c
ont
a
in
i
ng
 
a
m
i
c
roporous p
a
ck
i
ng
 
m
a
t
er
i
a
l
(g
e
l).
Sta
t
ion
a
ry
 
phase is a porous po
l
y
m
er
 
m
a
t
rix
who
s
e
 
por
e
s
 
are comple
t
e
l
y
 
fi
l
l
e
d
 
with the
s
o
lv
e
nt to
 
be u
s
ed
 
as
 
the 
m
obile phase.
The pore size is
 
high
l
y
 
cr
i
t
i
c
a
l,
 
since the
basis
 
of
 
the sep
a
ra
t
ion
 
is 
t
hat
 m
ol
e
cu
l
es
above a c
e
rt
a
in
 
size are to
t
a
l
ly
 
ex
c
lud
e
d
from the por
e
s, and
 
the in
t
er
i
or
 
of
 
the por
e
s
is
 
a
c
c
e
s
s
ibl
e
,
 
part
l
y
 
or wholl
y
,
 
to 
sm
a
l
l
e
r
m
ol
e
cu
l
es.
The flow
 
of
 
m
obi
l
e phase will c
a
use
 
l
a
r
ger
m
ol
e
cu
l
es
 
to pass through the co
l
u
m
n
unhind
e
re
d
,
 
without pen
e
tr
a
t
i
ng
 
the gel
m
a
t
rix, w
h
er
e
as
 
s
m
a
l
l
e
r
 
m
ol
e
cu
l
es
 
will be
re
t
ar
d
ed
 
a
c
co
r
ding
 
to
 
th
e
ir
 
pen
e
tr
a
t
i
on
 
of
the ge
l
.
 
s
epara
t
ion
 
 
 
 
 
T
h
eory of
 
separa
t
ion
 
A
 
co
l
u
m
n
 
is made up
 
of
 
s
w
ol
l
en gel pa
r
t
i
c
l
es
 
and the solvent used
 
to 
s
well
 
the
 
gel
 
i
n a
 
suit
a
ble
 
tubu
l
ar
 
con
t
a
i
ne
r
.
An equ
a
t
i
on
 
is
 
giv
e
n
 
be
l
ow:
V
t
 
=
 
V
0
 
+
 
V
i
 
+
 
V
m
wher
e
,
V
t
 
= the to
t
al
 
volume of the co
l
u
m
n
 
(whi
c
h c
a
n
 
be
 
m
e
a
sured),
 
V
0
 
= the volume
 
of
 
l
i
quid
 
outside the gel 
m
a
t
rix (known 
a
lso
 
void or d
e
ad volu
m
e),
V
i
 
= the 
v
olu
m
e of l
i
quid
 
inside the
 
m
a
t
rix,
V
m 
= the volume of 
t
he gel
 
m
a
t
rix
 
GPC
 
components
 
S
t
a
t
i
onary
 
P
hase
The
 
Mobile Phase
The
 
Colu
m
ns
The Pu
m
p
Det
e
c
t
ors
 
1.
2.
3.
4.
5.
 
1. 
Stationary
 
p
h
ase:
Co
m
posed
 
of
 
se
m
i
-pe
r
m
e
a
bl
e
,
 
porous
 
pol
y
m
er
 
gel be
a
ds with
 
we
l
l
 
de
f
in
e
d ra
n
ge of
 
pore siz
e
s .
P
r
o
p
e
r
ti
e
s
 
of g
e
l 
b
ead
s
:
1-
 
Chemic
a
l
l
y
 
in
e
rt
2-
 
Me
c
han
i
c
a
l
l
y
 
stab
l
e
3-
 
Has 
i
de
a
l
 
and
 
ho
m
ogen
e
ous porous
 
struc
t
ure (wide pore size give low
 
resolu
t
ion).
4-
 
Uniform
 
pa
r
t
i
c
l
e
 
and pore
 
siz
e
.
5-
 
The pore size of the gel 
m
ust be c
a
re
f
ul
l
y
 
con
t
rol
l
e
d
.
 
Ex
a
m
pl
e
s of gel
   
 
Dext
r
an
(
Sephad
e
x)
 
ge
l
:
 
An α 1-6-pol
y
m
er
 
of
 
glu
c
ose
 
na
t
ur
a
l
 
gel
   
 
Agarose ge
l
:
 
A
 
1
,
3
 
l
i
nked 
β
-
D
-ga
l
a
c
tose
 
and 1,4 l
i
nked 3,
6
-anh
y
dr
o
-
α
,
 
L
-ga
l
a
c
tose
 
na
t
ur
a
l gel
   
 
Acr
y
l
a
m
ide
 
ge
l
:
 
A
 
pol
y
m
er
i
z
e
d
 
a
c
r
y
l
a
m
id
e
,
 
a 
s
y
nth
e
t
i
c
 
gel
 
2.
 
T
h
e
 
Mobi
l
e
 
Phase
Co
m
po
s
ed
 
of a l
i
quid
 
u
s
ed
 
to 
d
is
s
olve the bio
m
ol
e
cu
l
es
 
to 
m
ake the 
m
obile
 
phase
pe
r
m
i
t
t
i
ng
 
high
 
de
t
e
c
t
i
on
 
response
 
and wet the pa
c
king
 
surfa
c
e.
 
3. Colum
n
s
Co
mm
er
c
i
a
l
l
y
 
A
va
i
l
a
ble
 
Colu
m
ns 
i
nc
l
ude
  
 
A
n
a
l
y
t
i
cal
 
co
l
u
m
n
-
 
7.
5
–8
m
m
 
d
i
a
m
e
t
ers.
  
 
Prep
a
ra
t
ive
 
co
l
u
m
n
s
-22–25
m
m
  
 
U
s
ual co
l
u
m
n l
e
ngth
s
-25,
 
30, 50, and
 
60
 
cm.
  
 
Narrow
 
bore co
l
u
m
ns-
 
2–3
m
m
 
di
a
m
e
t
er
have been introduced
 
Ma
terial                                             
 
S
o
l
v
ent
 
S
y
n
t
h
etic
 
elast
o
m
ers                           
 
T
o
l
u
e
n
e
( 
po
l
y
bu
ta
d
ie
n
e
 
,
 
po
l
y
is
o
p
rene
 
)
 
PS,
 
PVC,
 
St
y
ren
e
-
B
u
ta
d
ie
n
e              
 
T
etrahydro
f
u
ran
 
(
T
HF)
R
u
bb
er
 
,
 
Ep
o
x
y
 
r
esi
n
s
 
Polyole
f
ins                                          
 
T
r
i
-
 
chl
o
ro
 
-
b
enzene
 
P
o
l
y
u
retha
n
e                                       
 
Di-
 
m
et
h
y
l
f
o
r
m
a
m
i
d
e
 
(DMF)
 
Protei
n
s,
 
po
l
y
sacc
h
arides                   
 
W
ater
 
/
 
B
u
f
f
ers
 
4.
 
T
h
e
 
p
u
mp
A
r
e ei
t
her 
s
y
ringe
 
pu
m
ps
 
or
 
re
c
iproc
a
t
i
ng
 
pu
m
ps
 
with
 
a highly
 
constant
 
flow ra
t
e.
 
5. Det
e
ct
o
rs
Co
n
c
e
ntration
 
sen
s
it
i
ve
 
det
e
ct
o
rs
• Bulk
 
P
roperty
 
Dete
c
tors-
 
Refra
c
t
i
ve
 
Index
 
(RI) Det
e
c
t
or
• Solute
 
Property
 
Det
e
c
t
or
s
-
 
Ultr
a
vio
l
et
 
(U
V
)
 
Ab
s
orpt
i
on Det
e
c
t
or
• Ev
a
por
a
t
i
ve
 
Det
e
c
t
or
s
-
 
Ev
a
por
a
t
i
ve
 
L
i
ght 
S
c
a
t
t
er
i
ng
 
De
t
e
c
tor (E
L
S
D
)
Mo
l
ar
 
m
a
ss s
e
nsit
i
ve
 
d
e
t
e
c
t
or
s
1. L
i
ght
 
Sca
t
t
e
ring
 
Det
e
c
t
ors
• Low
 
Angle L
i
ght
 
Sca
t
t
e
ring
 
(LA
L
S) De
t
e
c
tors
• Mult
i
ang
l
e
 
L
i
ght 
S
c
a
t
t
er
i
ng
 
(M
A
LS) de
t
e
c
tors
2.
 
V
iscosity Dete
c
tors-
 
Di
f
fer
e
nti
a
l
 
V
isco
m
e
t
ers
 
Ex
p
er
i
m
e
ntal
 
se
t
up
 
Separat
i
on
 
p
r
ocedu
r
e
 
1-
 
Prep
a
ra
t
ion of
 
co
l
u
m
n for gel fi
l
tr
a
t
i
on 
 
whi
c
h
 
invo
l
ves
 
Swe
llin
g
 
o
f
 
th
e
 
g
el
 
P
a
c
k
in
g
 
th
e
 
c
olu
m
n
 
 
W
a
s
hing
:
 
Af
t
er
 
p
ac
king
,
 
s
e
v
e
r
al
 
c
olu
m
n
 
volu
m
es
 
o
f
 bu
f
fer
 
solut
io
n
i
s
 
p
a
s
s
ed
 
th
r
ou
g
h
 
t
h
e
 
c
olu
m
n
 
to
 
re
m
ov
e
 
a
n
y
 
a
i
r
 
bubb
l
es
 
a
n
d
 
t
o
 
t
e
s
t
th
e
 
c
o
lu
m
n
 
ho
m
og
e
n
e
it
y
.
 
2-
 
Lo
a
ding the sa
m
ple onto
 
the
 
co
l
u
m
n using
 
a
 
s
y
ringe
 
3-
 
El
u
ti
ng
 
the
 
sa
m
ple
 
and de
t
e
c
t
i
on
 
of
 
componen
t
s
 
A
d
vantages
 
and
 
disadvantages
 
Advant
a
ges:
   
 
Short an
a
l
y
sis
 
t
i
m
e.
   
 
W
e
l
l de
f
in
e
d
 
sep
a
ra
t
ion.
  
 
Narrow bands
 
and
 
good
 
sen
s
i
t
ivi
t
y
.
   
 
Th
e
re
 
is no sa
m
ple
 
loss.
   
 
S
m
a
l
l amount of
 
m
obi
l
e phase requ
i
re
d
.
   
 
The
 
flow
 
ra
t
e c
a
n
 
be se
t
.
 
Disadv
a
nt
a
ges:
  
 
L
i
m
i
t
ed
 
nu
m
ber
 
of
 
pe
a
ks
 
th
a
t c
a
n
 
be
 
resolv
e
d
 
with
i
n
 
the short t
i
m
e 
s
c
a
le
 
of
 
the G
P
C
 
run.
  
 
F
i
l
tr
a
t
i
ons
 
m
u
s
t
 
be
 
perfor
m
ed before
 
u
s
ing
 
the instru
m
ent to
 
prevent
 
du
s
t
 
and 
o
th
e
r
pa
r
t
i
cu
l
a
t
es
 
from ruin
i
ng
 
the co
l
u
m
ns 
a
nd
 
in
t
er
f
er
i
ng
 
with the de
t
e
c
tors.
 
 
The
 
m
ol
e
cu
l
ar
 
m
asses
 
of 
m
ost
 
of the ch
a
ins
 
will be too close for
 
the G
P
C sep
a
ra
t
ion
 
to
 
show
an
y
th
i
ng
 
m
ore
 
th
a
n
 
bro
a
d
 
pe
a
ks.
 
A
p
pl
i
ca
t
ions
 
of 
G
PC
 
  
 
P
r
oteins
 
f
r
ac
t
io
n
a
t
i
o
n
  
 
P
u
rifica
t
i
o
n
  
 
Mol
e
cular
 
weig
h
t
 
deter
m
inat
i
o
n
.
 
  
 
Separat
i
on
 
of
 
su
g
a
r
,
 
p
r
ote
i
ns,
 
pe
p
t
i
des,
 
r
u
b
b
e
r
s
 
and others
 
on
 
the
 
basis
 
of
 
the
i
r
 
s
i
ze.
  
 
This
 
t
e
ch
n
iq
u
e
 
can be use
 
to deter
m
ine
 
the
 
q
u
a
t
er
n
a
r
y
 
structu
r
e
 
of p
u
rif
i
ed
 
p
r
oteins.
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Gel Permeation Chromatography is a technique used for separating components based on molecular weight or size. It utilizes columns packed with a porous material to separate molecules by size, allowing smaller molecules to penetrate the gel matrix while larger ones pass through unhindered. Learn about the principle, theory, components, and applications of GPC from its historical background to modern usage.


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  1. GEL PERMEATION CHROMATOGRAPHY '

  2. Introduction Principle of separation Theory of separation GPC components Experimental setup Separation procedure Advantages and disadvantages Applications

  3. Introduction Gel permeation chromatography Gel chromatography Size exclusion chromatography Gel filtration Molecular-sieve chromatography Definition: Technique : separation of components based on the difference in molecular weight or size, and is one of the effective methods used to isolate and analyze the bio- macromolecular substances.

  4. Brief background Literature 1951-1955 the SEC concept was first recognised but was not clearly formulated 1963- Jim Waters build the first commercial GPC equipment based on John Moore s design 1979-Y au, Kirkland and Bly published the first definitive text on SEC which contributed to the success of SEC and a complete revised edition was publish in 2009. Jim Waters - John C. Moore - Prof. FredV . - Larry E. Maley Model GPC-100 price $12,500

  5. Principle of separation It s a technique that separates dissolved molecules on the basis of their size by pumping these molecules through specialized columns containing a microporous packing material(gel). Stationary phase is a porous polymer matrix whose pores are completely filled with the solvent to be used as the mobile phase. The pore size is highly critical, since the basis of the separation is that molecules above a certain size are totally excluded from the pores, and the interior of the pores is accessible, partly or wholly, to smaller molecules. The flow of mobile phase will cause larger molecules to pass through the column unhindered, without penetrating the gel matrix, whereas smaller molecules will be retarded according to their penetration of the gel.

  6. Theory of separation Acolumn is made up of swollen gel particles and the solvent used to swell the gel in a suitable tubular container. An equation is given below: Vt = V0 + Vi+ Vm where, Vt = the total volume of the column (which can be measured), V0 = the volume of liquid outside the gel matrix (known also void or dead volume), Vi = the volume of liquid inside the matrix, Vm = the volume of the gel matrix

  7. GPC components Stationary Phase The Mobile Phase The Columns The Pump Detectors 1. 2. 3. 4. 5. 1. Stationary phase: Composed of semi-permeable, porous polymer gel beads with well defined range of pore sizes . Properties of gel beads: 1- Chemically inert 2- Mechanically stable 3- Has ideal and homogeneous porous structure (wide pore size give low resolution). 4- Uniform particle and pore size. 5- The pore size of the gel must be carefully controlled. Examples of gel Dextran(Sephadex) gel:An 1-6-polymer of glucose natural gel Agarose gel:A1,3 linked -D-galactose and 1,4 linked 3,6-anhydro- , L-galactose natural gel Acrylamide gel:Apolymerized acrylamide, a synthetic gel

  8. 2. The Mobile Phase Composed of a liquid used to dissolve the biomolecules to make the mobile phase permitting high detection response and wet the packing surface. Material Solvent Synthetic elastomers Toluene ( polybutadiene , polyisoprene ) PS, PVC, Styrene-Butadiene Tetrahydrofuran (THF) Rubber , Epoxy resins Polyolefins Tri- chloro -benzene Polyurethane Di- methylformamide (DMF) Proteins, polysaccharides Water / Buffers 3. Columns CommerciallyAvailable Columns include Analytical column- 7.5 8mm diameters. Preparative columns-22 25mm Usual column lengths-25, 30, 50, and 60 cm. Narrow bore columns- 2 3mm diameter have been introduced

  9. 4. The pump Are either syringe pumps or reciprocating pumps with a highly constant flow rate. 5. Detectors Concentration sensitive detectors Bulk Property Detectors- Refractive Index (RI) Detector Solute Property Detectors- Ultraviolet (UV)Absorption Detector Evaporative Detectors- Evaporative Light Scattering Detector (ELSD) Molar mass sensitive detectors 1. Light Scattering Detectors LowAngle Light Scattering (LALS) Detectors Multiangle Light Scattering (MALS) detectors 2. Viscosity Detectors- DifferentialViscometers

  10. Experimental setup

  11. Separation procedure 1- Preparation of column for gel filtration which involves Swelling of the gel Packing the column Washing:After packing, several column volumes of buffer solution is passed through the column to remove any air bubbles and to test the column homogeneity. 2- Loading the sample onto the column using a syringe 3- Eluting the sample and detection of components

  12. Advantages and disadvantages Advantages: Short analysis time. Well defined separation. Narrow bands and good sensitivity. There is no sample loss. Small amount of mobile phase required. The flow rate can be set. Disadvantages: Limited number of peaks that can be resolved within the short time scale of the GPC run. Filtrations must be performed before using the instrument to prevent dust and other particulates from ruining the columns and interfering with the detectors. The molecular masses of most of the chains will be too close for the GPC separation to show anything more than broad peaks.

  13. Applications of GPC Proteins fractionation Purification Molecular weight determination. Separation of sugar, proteins, peptides, rubbers and others on the basis of their size. This technique can be use to determine the quaternary structure of purified proteins.

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