GEL PERMEATION CHROMATOGRAPHY

1. GEL
PERMEATION
CHROMATOGRAPHY
Presented By:
MEHAK
14320022
M.Sc. Chemistry
Large size
molecule
Intermediate
size molecule
Small size
molecule

2. CONTENTS
• Chromatography
• definition
•Uses of Chromatography
• Gel Permeation Chromatography (GPC)
• GPC Separation Mechanism.
• Theory
• Main Components Of GPC
• Solvent Containers
• Pump
• Oven
• Samples
• Injector
• Column
• Detector
• Applications
• Refrences
Mehak 14320022 Nov 2'2015
2

3. Mehak 14320022 Nov 2'2015
3
Chromatography is a Greek
word chroma “colour” and
graphein “to write”.
chromatography is a family of
analytical chemistry techniques
for the separation of mixtures.
It was the Russian botanist
“Mikhail Tsvet” who invented the
first chromatography technique
in 1901.
CHROMATOGRAPHY

4. Mehak 14320022 Nov 2'2015
4
USES
OF
CHROMATOGRAPHY
It is used in
crime scene
investigations
In hospitals it
can be used to
detect alcohol
levels in a
patient's blood
stream
It is used for
environmental
agencies to
determine the level
of pollutants in
water supplies
It is used to purify
chemicals needed
to make a product
in a manufacturing
plant
It is used by
pharmacists to
determine the
amount of each
chemical found in
each product.

5. GEL PERMEATION CHROMATOGRAPHY (GPC)
Mehak 14320022 Nov 2'2015
5
• Gel permeation chromatography is a type of high performance liquid
chromatography (LC).
• GPC/SEC separates molecules on the basis of their size, hence ‘size
exclusion’.
• GPC/SEC is used to determine the molecular weight distributions of polymers
• GPC/SEC uses columns packed with very small, round, porous particles to
separate molecules contained in the solvent that is passed through them.
• The first GPC/SEC columns were packed with materials referred to as gels,
hence ‘gel permeation
• GPC/SEC is used to determine the molecular weight distributions of polymers

6. Figure : Principle of gel chromatography: A) mixture applied to the top of
the column; B) partial separation; C) complete separation; D)excluded
substance emerges from the column.
GPC SEPERATION MECHANISM
Mehak 14320022 Nov 2'2015
6

7. Mehak 14320022 Nov 2'2015
7
The polymer sample
is first dissolved in a
solvent.
Once they have been
dissolved, the
molecules coil up on
themselves to form a
coil conformation.
Although they are chains,
when we analyze them by
GPC they behave like tiny
spheres,
with the size of the sphere
dependent on the molecular
weight (higher molecular
weight polymers coil up to
form larger spheres)
These coiled up
polymer
molecules are
then introduced
into the mobile
phase and flow
into the GPC
column.
Much larger polymer coils
cannot enter the pores and
so are carried straight past
by the mobile phase.
If the polymer coils are a
little smaller than the biggest
pores they can enter the
larger, but not the smaller
pores as they pass by
If the polymer
coils are smaller
than the smallest
pores in the
beads, then they
can enter any of
the pores and so
can potentially
occupy all of the
stationary phase

8. Mehak 14320022 Nov 2'2015
8
www.separations.us.tosohbioscience.com/ServiceSupport/TechSuppo
rt/ResourceCenter/PrinciplesofChromatography/SizeExclusion/

9. Mehak 14320022 Nov 2'2015
9
Thus ,
Pass through the
column unhindered,
without penetrating the
gel matrix.
Thus excluded, they
travel mostly around
the exterior of the
packing.
These will be retarded
according to their
penetration of the gel.
Thus very small
molecules diffuse into
all or many of the
pores accessible to
them. Small molecule
exit the column last
Intermediate size
molecule exit at
intermediate times.
LARGE
MOLECULES
SMALL
MOLECULES
INTERMEDIATE
MOLECULES

10. • Total volume of column packed with a solid
matrix that has been swelled by water or other
solvent is given by
Vt = total bed volume
Vg = volume occupied by solid matrix.
VM = void volume of mobile phase i.e. unbound
solvent in interstices between the solvent loaded
porous particles.
VS = volume of solvent held in pores
Vt = Vg + VM + VS
Mehak 14320022 Nov 2'2015 10
THEORY

11. Ve = Vo + Kd.Vl
Mehak 14320022 Nov 2'2015 11
Time taken for solute
molecules to diffuse
into pore is less as
compared to time spent
by molecule near pore.
Separation process is
independent of
diffusion.
Under these conditions:
Assumed conditions:
Where,
Ve = vol. of effluent flowing through column between point of sample
injection & sample emergence from column.
Kd = distribution coefficient.
FOR LARGE MOLECULES : Kd = 0 , Vo = Ve
FOR MOLECULES THAT CAN PENETRATE ALL THE PORES :
Kd = 1 , Ve = Vo + Vl

12. Mehak 14320022 Nov 2'2015
12
MAIN COMPONENTS OF GPC

13. Solvent and Solvent containers
 The solvent must be able to dissolve the sample,
sometimes a polymer insoluble at room temperature will
dissolve at higher temperature.
 The solvent must not induce any other interactions between
the sample and the stationary phase, so that the separation is
solely on the basis of sample size.
Mehak 14320022 Nov 2'2015
13
The solvent container should be made of clear glass, or
amber glass for solvents affected by sunlight, with a
stopper to exclude dust and limit evaporation.
Solvent Container

14. PUMPS
Mehak 14320022 Nov 2'2015
14
The pump takes the solvent and delivers it to the rest of the
system at a constant, accurate and reproducible flow rate.
The pump has to be able to run the same flow rate
regardless of viscosity, so that results can be compared from
one analysis to another.
The pressure delivered by the pump also needs to be
smooth so that there are no pulses in the flow.

15. OVENS
Mehak 14320022 Nov 2'2015
15
GPC is usually carried out at room temperature, but some
instruments have heated and thermostatically controlled ovens in
which the columns and detectors are placed.
Higher temperatures, up to 220 °C, are necessary for some
solvents that have much higher viscosities, such as
trichlorobenzene or chloronaphthalene .
Operating the instrument at high temperatures reduces viscosity
and hence column back pressure, with a corresponding increase
in efficiency.

16. SAMPLES
Mehak 14320022 Nov 2'2015
16
To prepare a sample for analysis it is first dissolved in an
appropriate solvent, such as tetrahydrofuran (THF) for organic
GPC.
Since the separation obtained depends on the size of the sample
molecules, it is important that they are allowed to swell and then
fully dissolve in the solvent before being put through the
chromatograph .
Where possible, the eluent used to prepare the samples should
be the same as the solvent running through the system

17. INJECTION AND INJECTORS
Injectors introduce the polymer sample into the flowing
solvent stream.
It is important that the injector does not disturb the flow of the
mobile phase
Mehak 14320022 Nov 2'2015
17

18. Different Types Of Column
Packing
Column packing
Semi rigid
Cross linked
macromolecular
polymers.
Rigid
Controlled pore-
size glasses or
silica 18
Separation of the sample takes place inside the column, a
hollow tube tightly packed with extremely small porous
beads, typically polymer or silica.
The columns vary in length from 50 mm to 300 mm, and
internal diameters of 4.6 to 25 mm, depending on their
intended use.

19. Semi rigid polymers
These materials swell slightly
Limited to a max. pressure of 300 psi.
Bead diameters are usually 5 micrometer
Styrene divinylbenzene polymers are used for
compounds of molecular weight ranging from 100-500
million
Sulphonated polystyrene beads are compatible with
aqueous systems , non sulphonated with non aqueous
systems.
Mehak 14320022 Nov 2'2015 19

20. Rigid porous glasses or silica
Cover wide range of pore diameter
Chemically resistant
Used with aqueous and polar organic solvents.
Mehak 14320022 Nov 2'2015 20

21. DETECTORS
Mehak 14320022 Nov 2'2015
21
So, it has to be very
sensitive since the
changes they measure in
the mobile phase are very
small.
Detectors may
respond to a
change in the
mobile phase
due to the
presence of the
sample
So, it has much greater
sensitivity but often only
work with specific
samples
Detectors may
respond to a
property of the
sample alone

22. Mehak 14320022 Nov 2'2015
22
DETECTORS
Measure
concentration alone
> Differential refractive
index (DRI) detector *
> UV detector
> Evaporative light
scattering (ELS) detector.
whose response is
proportional to
concentration and
other properties of the
polymer molecules.
Static light scattering
detectors or viscometers.

23. Mehak 14320022 Nov 2'2015
23
• These detectors work by assessing
the difference in refractive index
between the mobile phase and the
pure solvent
• Since the refractive index of
polymers is usually constant above
molecular weights of about 1,000
g/mol, the detector response is directly
proportional to the sample
concentration.
WIDELY USED DETECTORS
• Use the fact that a beam of light will be
scattered when it strikes a polymer
molecule
•Low Angle Laser Light Scattering
(LALLS),
•Multi-angle Laser Light Scattering
(MALLS)
• Right Angle Laser Light Scattering
(RALLS).
•The advantage of these detectors is that
they give a response directly proportional
to the molecular weight of the polymer
molecules, and can provide size
information
Static Light Scattering Detectors:Differential Refractometer
(Universal detector) :

24. Mehak 14320022 Nov 2'2015 24
APPLICATIONS
Gum arabic, good and bad: Gum arabic is a polysaccharide
widely used in the food industry as a viscosity modifier or
gelling agent. The physical properties and processibility of
these water soluble polymers are related to their molecular
weight distributions, which can be determined by GPC.
Modifying PVC (Poly Vinyl Chloride) : Unplasticized PVC has a
high melt viscosity leading to some difficulties in processing. In
order to overcome these problems, additives are used as impact
modifiers to ensure more uniform flow and hence improve surface
finish.
The properties of the final material are dependent on the
molecular weight distribution of the PVC and the type and
level of the added plasticizers.

25. REFERENCES
• https://www.agilent.com/cs/library/primers/Public/5990-
6969EN%20GPC%20SEC%20Chrom%20Guide.pdf
• http://pubs.acs.org/doi/pdf/10.1021/ed083p1567.2
•http://www.separations.us.tosohbioscience.com/ServiceSupport/Te
chSupport/ResourceCenter/PrinciplesofChromatography/SizeExclus
ion/
• Instrumental Methods of Analysis, Willard, Merritt, Dean and Settle,
CBS Publisher and Distributors.,1986.
• Principles of Instrumental Analysis, Skoog, Holder, Nieman, Fifth
edition Thomson Books ,1998.
Mehak 14320022 Nov 2'2015 25

26. Mehak 14320022 Nov 2'2015 26