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