GELPERMEATION
CHROMATOGRAPHYPresented By:
MEHAK14320022M.Sc. Chemistry
Large size
molecule
Intermediate size molecule
Small size
molecule
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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
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Mehak 14320022 Nov 2'2015
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
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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.
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GEL PERMEATION CHROMATOGRAPHY (GPC)
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• 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
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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
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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
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www.separations.us.tosohbioscience.com/ServiceSupport/TechSupport/ResourceCenter/PrinciplesofChromatography/SizeExclusion/
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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
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• 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
THEORY
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Ve = Vo + Kd.Vl
Time taken for solute molecules to diffuse into pore is less as compared to time spent by molecule near pore.
Separation process isindependent 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
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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.
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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
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PUMPS
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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.
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OVENS
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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.
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SAMPLES
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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
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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
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Different Types Of Column Packing
Column packing
Semi rigidCross linked
macromolecular polymers.
RigidControlled pore-size glasses or
silica
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.
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Semi rigid polymersThese 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.
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Rigid porous glasses or silica
Cover wide range of pore diameter
Chemically resistant
Used with aqueous and polar organic solvents.
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DETECTORS
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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
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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.
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• 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) :
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APPLICATIONSGum 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.
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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/TechSupport/ResourceCenter/PrinciplesofChromatography/SizeExclusion/
• 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.
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