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Gel Permeation Chromatography

Balaji Prapancham

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Outline

Definitions Objective Principle Features Analysis Conventional GPC Molecular Mass Sensitive Detectors

Characterization Applications Advantages & Drawbacks

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Outline

Definitions Chromatography Gel Permeation Chromatography

Objective Principle Features Analysis Conventional GPC Molecular Mass Sensitive Detectors Characterization Applications Advantages & Drawbacks

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Definitions

Chromatography

Selective retardation of one or more components ofa fluid solution

The fluid uniformly percolates through a column of afinely divided substance or capillary passage ways. E.g. Paper chromatography

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Definitions

Gel Permeation Chromatography

A chromatographic method in which particles areseparated based on their size, or in more technical

terms, their hydrodynamic volume. Organic solvent as the mobile phase. The stationary phase consists of beads of porous

polymeric material

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Objective

Analysis of synthetic and biologic polymers Purification of polymers Polymer characterization Study properties like

Molecular weight Polydispersity Index Viscosity Conformation Folding Aggregation Branching Copolymer composition Molecular size

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Outline

Definitions Objective

Principle How the GPC works

Features Analysis Conventional GPC Molecular Mass Sensitive Detectors Characterization Applications Advantages & Drawbacks

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Principle

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Principle

Different sizes will elute (filter) through at differentrates.

Column Consists of a hollow tube tightly packed

with extremely small porous polymer

beads designed to have pores of differentsizes.

Pores may be depressions on the surfaceor channels through the bead.

Smaller particles enter into the pores,larger particles don't.

The larger the particles, the less overall volume totraverse over the length of the column, and the

faster the elution.

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Outline

Definitions Objective Principle Features

The setup inside the GPC Significance of each part of the GPC

Analysis Conventional GPC Molecular Mass Sensitive Detectors Characterization Applications Advantages & Drawbacks

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Working Features

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Features

Solvent Should be kept dry Should be degassed in some

applications. The samples should be made from

the same solvent

For GPC/light scattering, the solventshould be filtered before it ever hits

the pump Common solvents for GPC are

tetrahydrofuran (THF) and toluene.

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Features

Solvent Should be kept dry Should be degassed in some

applications. The samples should be made from

the same solvent

For GPC/light scattering, the solventshould be filtered before it ever hits

the pump Common solvents for GPC are

tetrahydrofuran (THF) and toluene.

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Features

Solvent Should be kept dry Should be degassed in some

applications. The samples should be made from

the same solvent

For GPC/light scattering, the solventshould be filtered before it ever hits

the pump Common solvents for GPC are

tetrahydrofuran (THF) and toluene.

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Features

Pump Designed to deliver very constant,

accurate flow rates

At microprocessor-controlled rates Designed not to produce any

pressure pulses

Filters Prevent major junk from getting into

the columns

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Features

Pump Designed to deliver very constant,

accurate flow rates

At microprocessor-controlled rates Designed not to produce any

pressure pulses

Filters Prevent major junk from getting into

the columns

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Features

Pump Designed to deliver very constant,

accurate flow rates

At microprocessor-controlled rates Designed not to produce any

pressure pulses

Filters Prevent major junk from getting into

the columns

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Features

Pump Designed to deliver very constant,

accurate flow rates

At microprocessor-controlled rates Designed not to produce any

pressure pulses

Filters Prevent major junk from getting into

the columns

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Features

Injector Loop Allows you to load the sample loop

which is a piece of tubing precut for a

precise volume the output of the pump flushes through

the loop

Carries the sample to the columns. sends a signal to the detector to

indicate that the sample has beenloaded

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Features

Injector Loop Allows you to load the sample loop

which is a piece of tubing precut for aprecise volume

the output of the pump flushes throughthe loop

Carries the sample to the columns. sends a signal to the detector to

indicate that the sample has beenloaded

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Features

Injector Loop Allows you to load the sample loop

which is a piece of tubing precut for aprecise volume

the output of the pump flushes throughthe loop

Carries the sample to the columns. sends a signal to the detector to

indicate that the sample has beenloaded

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Features

Columns Contain the beads through which the

sample is allowed to pass

Reference column is also present Very expensive Never change the pumping rate by a

large amount

They are very delicate

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Features

Columns Contain the beads through which the

sample is allowed to pass

Reference column is also present Very expensive Never change the pumping rate by a

large amount

They are very delicate

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Features

Detectors Viscosity Light Scattering Ultraviolet detectors Differential Refractive Index detector

placed at the end to reduce pressureon it

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Outline

Definitions Objective Principle Features Analysis - What techniques are used Conventional GPC - How it works Molecular Mass Sensitive Detectors Characterization Applications Advantages & Drawbacks

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Analysis

Spectroscopic Techniques Refractive Index Light Scattering Ultraviolet Spectroscopy

Viscometry Techniques Viscosity Flow rate

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Conventional GPC

Analysis Molecules separated according to their hydrodynamic volume. Molecular weights (MW) and molecular weight distribution can be

determined from the Measured retention volume (RV)

A calibration curve (log MW against RV), using known standards

RI signal = KRI . dn /dc . C

KRI = apparatus-specific sensitivity constant dn /dc = the refractive index increment C = concentration. Limitation

Their signals depend solely on concentration, not on molecularweight or polymer size.

Not very reliable

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Molecular Mass Sensitive Detectors

Detectors sensitive to molecular weight used to overcomelimitations of Conventional GPC

E.g., light scattering and viscosity detectors Advantages over Conventional GPC

True molecular weight distributions can be obtained Structural information Size distribution

molecular weight can be directly determined without a calibrationcurve

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Outline

Definitions Objective Principle Features Analysis Conventional GPC Molecular Mass Sensitive Detectors Characterization

The equation used in each technique Light Scattering and Viscometric techniques

Applications Advantages & Drawbacks

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Characterization

Light Scattering

LS signal = KLS . (dn/dc)2 . MW . C

KLS = sensitivity constant dn/dc = refractive index increment MW = molecular weight C = concentration

dn/dc depends on the Polymer Solvent combination and if it is low, then properanalysis cannot be done.

PS in THF (dn/dc = 0.185), PLA in THF (dn/dc

0.049), the signal is only about 7% of the PS-in-THF signal at the sameconcentration and molecular weight

Molecular weight can be directly determined without a calibration curve

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Characterization

Viscosity Detectors

Visc. signal = KVisc . IV . C

Provides the intrinsic viscosity (IV) of the polymer directly. Better sensitivity compared to Light Scattering Can measure to lower molecular weights than Light Scattering Radius of Gyration can be found from Intrinsic Viscosity and

Molecular weight

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Applications

Polymer characterization Molecular weight Polydispersity Index Viscosity

Folding Aggregation Branching Copolymer composition Molecular size

Proteomics Purification Conformation Hydrodynamic volume

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Outline

Definitions Objective Principle Features Analysis Conventional GPC Molecular Mass Sensitive Detectors Characterization

Applications Advantages & Drawbacks

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Advantages

Can be used to find shape also Rapid, routine analysis Identify high mass components even in low

concentration Can analyze polydisperse samples Branching studies can be done Absolute molecular weights can be obtained

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Drawbacks

There is a size window Bad response for very small molecular

weights

Standards are needed. Sensitive for flow rate variation. Internal

standard should be used whenever possible.

High Investment cost

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Thank you !!!!