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Gel Permeation Chromatography

Balaji Prapancham

Outline

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

Outline
Definitions

Chromatography Gel Permeation Chromatography

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

Definitions
Chromatography
Selective retardation of one or more components of a fluid solution The fluid uniformly percolates through a column of a finely divided substance or capillary passage ways. E.g. Paper chromatography

Definitions
Gel Permeation Chromatography

A chromatographic method in which particles are separated 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

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

Outline

Definitions Objective

Principle

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

Principle

Principle

Different sizes will elute (filter) through at different rates. Column

Consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. Pores may be depressions on the surface or channels through the bead. Smaller particles enter into the pores, larger particles don't.

The larger the particles, the less overall volume to traverse over the length of the column, and the faster the elution.

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

Working Features

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 solvent should be filtered before it ever hits the pump Common solvents for GPC are tetrahydrofuran (THF) and toluene.

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 solvent should be filtered before it ever hits the pump Common solvents for GPC are tetrahydrofuran (THF) and toluene.

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 solvent should be filtered before it ever hits the pump Common solvents for GPC are tetrahydrofuran (THF) and toluene.

Features

Pump

Designed to deliver very constant, accurate flow rates At microprocessor-controlled rates Designed not to produce any pressure pulses Prevent major junk from getting into the columns

Filters

Features

Pump

Designed to deliver very constant, accurate flow rates At microprocessor-controlled rates Designed not to produce any pressure pulses Prevent major junk from getting into the columns

Filters

Features

Pump

Designed to deliver very constant, accurate flow rates At microprocessor-controlled rates Designed not to produce any pressure pulses Prevent major junk from getting into the columns

Filters

Features

Pump

Designed to deliver very constant, accurate flow rates At microprocessor-controlled rates Designed not to produce any pressure pulses Prevent major junk from getting into the columns

Filters

Features

Pump

Designed to deliver very constant, accurate flow rates At microprocessor-controlled rates Designed not to produce any pressure pulses Prevent major junk from getting into the columns

Filters

Features

Pump

Designed to deliver very constant, accurate flow rates At microprocessor-controlled rates Designed not to produce any pressure pulses Prevent major junk from getting into the columns

Filters

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 been loaded

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 been loaded

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 been loaded

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

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

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

Features

Detectors

Viscosity Light Scattering Ultraviolet detectors Differential Refractive Index detector placed at the end to reduce pressure on it

Outline

Definitions Objective Principle Features

Analysis - What techniques are used Conventional GPC - How it works Molecular Mass Sensitive Detectors

Characterization Applications Advantages & Drawbacks

Analysis

Spectroscopic Techniques

Refractive Index Light Scattering Ultraviolet Spectroscopy Viscosity Flow rate

Viscometry Techniques

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 molecular weight or polymer size. Not very reliable

Molecular Mass Sensitive Detectors

Detectors sensitive to molecular weight used to overcome limitations 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 calibration curve

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

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 proper analysis 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 same concentration and molecular weight

Molecular weight can be directly determined without a calibration curve

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

Applications

Polymer characterization

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

Proteomics

Outline

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

Advantages & Drawbacks

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

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

Thank you !!!!

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