1. Gel Permeation Chromatography Size Exclusion Chromatography
Wendy Gavin Biomolecular Characterization Laboratory Nuclear Magnetic Resonance Spectroscopy Laboratory

2. 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.

3. 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

4. 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

5. 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

6. Fingerprint GPC Basics
Though they are subtle, differences such as those shown in the molecular-weight distributions could cause marked variations in the performance of the polymer.

7. GPC Separates by Effective Size in Solution
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

8. 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

10. GPC System

11. Waters 2695 Module

12. Pumps Deliver Mobile Phase Handle Harsh Solvents Quaternary Pump
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

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

14. Sample Loop ( viable draw syringe)
250µL Sample loop
Introduce sample into chromatographic system
No disruption in flow
Reproducible
Known volume

15. Styrene Divinylbenzene Resin for organic solvents
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 K
HR4 MW range of 5K-600K

16. Biggest comes out first
GPC Column
Biggest comes out first

17. 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

18. 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

19. 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

20. 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
Amount of deflection is proportional to concentration
Universal detector but not very sensitive

21. Polystyrene Standards
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

22. 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 column set used

23. Sample must dissolve in Tetrahydrofuran
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

24. GPC Results

27. GPC Results

28. 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.

29. 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.