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

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.

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

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

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

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.

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

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

GPC System

Waters 2695 Module

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

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

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

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 500-30K HR4 MW range of 5K-600K

Biggest comes out first GPC Column Biggest comes out first

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  

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

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

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

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

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

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

GPC Results

GPC Results

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.

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.