Gel Permeation Chromatography

Size Exclusion Chromatography

Wendy Gavin
Biomolecular Characterization Laboratory
Nuclear Magnetic Resonance Spectroscopy
Laboratory

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 GPC Basics
Gel permeation chromatography (GPC) is one of the
most powerful and versatile analytical techniques
available for understanding and predicting polymer
performance. It is the most convenient technique for
characterizing the complete molecular weight
distribution of a polymer.

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 Principle of GPC

A solute, based on its size in solution,

penetrates to a greater or lesser extent,
the chromatographic stationary phase.
Study the physical properties of a wide
range of compound classes including:
• Polymers
• Petrochemicals
• Naturally occurring products
• Biomolecules

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 GPC is commonly used for:

Sample Preparation
• Isolate compound of interest from other
contaminants or interferences.
Determination of Molecular Weight
• Correlate molecular mass or size information
using calibration standards
Molecular Interactions and Property
Determination
• Materials characterization of polymers

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 GPC Basics
These values are important, since they affect many of the characteristic
physical properties of a polymer. Subtle batch-to-batch differences in
these measurable values can cause significant differences in the end-use
properties of a polymer. Some of these properties include:

  Tensile strength Adhesive strength

  Elastomer relaxation time Cure time
  Brittleness Elastic modules
  Flex life Melt viscosity
  Impact strength Hardness
  Toughness Softening temperature
  Drawability Tear Strength
  Adhesive tack Stress-crack resistance
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 GPC Basics

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Though they are subtle, differences such as

those shown in the molecular-weight
distributions could cause marked variations in
the performance of the polymer.
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 GPC Mechanism
GPC Separates by Effective Size in Solution
Big Ones Come Out First
Assume all molecules are simple spheres
Smaller spheres will spend more time in
stationary phase
Large spheres will be excluded from the pore of
stationary phase
No interaction between sample and stationary
phase

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 GPC Mechanism
Macromolecules in solution are not simple
spheres
Tend to form random coils
Pliable molecules may enter part or all of the
stationary phase
May have interaction between sample and
stationary phase

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 GPC System

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 Waters 2695 Module

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 Pumps
Deliver Mobile Phase
• Flow rate accuracy and repeatability
Handle Harsh Solvents
• Polymers dissolve in aggressive solvents
Quaternary Pump
• Deliver up to four different mobile phases
Isocratic
• Constant mobile phase
Gradient
• Changing mobile phase composition

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 Autosampler

Holds 120
sample vials
Each sample
vial holds
1.5mls
Need at least
500µL sample
volume
Use correct
sample vials

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 Sample Loop ( viable draw syringe)

250µL Sample
loop
Introduce sample
into
chromatographic
system
No disruption in
flow
Reproducible
Known volume

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 3 Styragel HR Columns

• Styrene Divinylbenzene Resin for organic

solvents
• Susceptible to Shrinking and Swelling
• Always change flow rate slowly
• 3 Columns:
• HR 1 MW range of 100-5K
• HR3 MW range of 500-30K
• HR4 MW range of 5K-600K
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 GPC Column

Biggest comes out first

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 GPC Separation
 Polymer is prepared as a dilute solution in the eluent and
injected into the system

 The GPC column is packed with porous beads of controlled

porosity and particle size

 Large molecules are not able to permeate all of the pores and
have a shorter residence time in the column

 Small molecules permeate deep into the porous matrix and

have a long residence time in the column

 Polymer molecules are separated according to molecular size,

eluting largest first, smallest last

 As a result of the GPC separation mechanism, polymer

molecules elute from the column in order of size in solution

 Largest elute first, smallest elute last

 The separation is purely a physical partitioning, there is no

interaction or binding

 The separation is isocratic

 

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 Refractive Index Detector
Waters 2414 RI Detector

Universal Detector
• Don’t need UV chromophore or
Fluorophore
Less sensitive than other
detection methods
Exploits the improbability of
solvent and solute having same
RI
Not suitable for gradients
May have positive and negative
peaks
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 Refractive Index Detector

Monochromatic light at fixed

wavelength
Measures refractive index of the
analyte compared to the solvent
Flow cell has two parts:
• One for Reference mobile phase
• One for Sample
• Difference appears as peak in
chromatogram

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 o Measure of molecule’s ability to deflect light in flowing
mobile phase in a flow cell relative to static mobile phase
contained in reference cell
o Amount of deflection is proportional to concentration
o Universal detector but not very sensitive

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 Calibration
Polystyrene Standards

Inject series of polystyrene standards

from molecular weight of 850 to 400K.
Create calibration curve from standards
to use with your sample.
Check old curve
by injecting standards
Occasionally need
to create new curve

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 Calibration Curve
 Chromatograph a series of
well characterized, narrow
polydispersity polymer
standards

 Plot peak retention time (RT)

versus peak log molecular
weight (logM)
 
 Fit the data using a
mathematical function
 
 The calibration curve will be
characteristic of the GPC
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 Sample Preparation
 Sample must dissolve in Tetrahydrofuran
• At room temperature
• No sonication
• Prepare at least few hours ahead of time
 Filter through 0.45micron filter
 Sample about 2-20mg/mL
 Need 2mL THF
 Be consistent
• Treat samples the same
• Same amount, same volume, same
volume injected
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 GPC Results

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 GPC Results

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 • Most important numbers are Mw and Mn
• Mn provides information of flexibility
• Mw on strength of the material
• Molecular weight averages describe the
polymer at different points in the peak.

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 GPC Results
There are other techniques to obtain these
molecular weight averages:
 Number average, Mn, may be obtained by
membrane osmometry, or end group
analysis, (titration, NMR, etc.)
 Weight Average, Mw, may be obtained by
light scattering
 Z Average, Mz, and Z + 1 Average, Mz + 1,
may be obtained by ultracentrifugation
Once we have calibrated our GPC system, we
can obtain all of these averages with a single
injection.
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