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Agilent Technologies — Gulf Coast Conference 2011
38 slides from Agilent Technologies's conference presentation, October 2011
Slide 1: GPC/SEC Practical Tips and Tricks
GPC/SEC Practical Tips and Tricks Thomas Dent Applications Scientist Agilent Technologies October, 2011 Gulf Coast Conference 1
Slide 2: Section 1: Introduction – Goals
Section 1: Introduction Goals • Brief introduction to GPC/SEC • Highlight considerations for column and solvent selection • Examples of problematic chromatography • Propose possible solutions • Components of a GPC
Slide 3: Nomenclature
Nomenclature Gel Permeation Chromatography GPC Size Exclusion Chromatography SEC Gel Filtration Chromatography GFC All refer to the separation based on size or hydrodynamic volume in solution of a polymer or biopolymer in solution
Slide 4: Why do GPC?
Why do GPC? MWD determined by GPC • GPC is the best technique for characterizing polymer molecular weight distribution • GPC provides key information to predict the process-ability and material properties of a polymer…. • Often MW is not enough information log M Both Samples = Mp 100,000
Slide 5: Understanding the Molecular Weight Distributions of Polymers
Understanding the Molecular Weight Distributions of Polymers • The MW distribution controls many physical properties • As distribution decreases the strength and toughness of the polymer increases • However distribution decreases the polymer becomes more difficult to process • GPC provides key infor
Slide 6: Why do GPC?
Why do GPC? Isolation… …Quantitation
Slide 7: GPC Separation Mechanism
GPC Separation Mechanism • Polymer is introduced onto the column • The 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 poro
Slide 8: Size Exclusion Mechanism
Exclusion Partial Permeation Total Permeation
Slide 9: Section 2: Column Selection
Section 2: Column Selection • The columns perform the separation • The choice and care of columns is critical to good chromatography • Column selection criteria include • mw range • pore size (related to mw range) • solvent compatibility • particle size • column volume • Let’s Look at examples 9
Slide 10: Effect of Particle Size on Resolution
Effect of Particle Size on Resolution • Smaller particle size leads to greater efficiency and resolution • Smaller particle size also leads to shear degradation • Therefore only use small particle sizes for relatively low molecular weight separations • High molecular weight separations require large
Slide 11: Effect of Pore Size
Effect of Pore Size Much of polymer is too large to fit inside the pores. Therefore the polymer is excluded…
Slide 12: Effect of Column Length on Resolution
Effect of Column Length on Resolution Mp values Eluent: THF (stabilized) Injection 1 Injection 2 FlowRate: 1.0 ml/min 1. 7500000 6. 2560000 Detector: UV 2. 841700 7. 320000 Calibrants: PL EasiCal PS-1 3. 148000 8. 59500 4. 28500 9. 10850 5. 2930 10. 580 1 x PLgel Column 3 x PLgel Column 12
Slide 13: Section 3: Solvent Selection
Section 3: Solvent Selection • Sample solubility • Solvent/column compatibility • Avoid non-size exclusion effects (match solvent, column and polymer polarity) • Compatible detection (RI iso-refractive solvent/sample, UV cut off) • Safety (eg toxicity, elevated temperature, etc) • Let’s look at exam
Slide 14: Typical Range of Solvents Used (with PLgel)
Typical Range of Solvents Used (with PLgel) (ascending order of polarity)
Slide 15: Polarity Considerations
Polarity Considerations Δ = [(cal/cm3)1/2] Interactions are typically avoided when the polarity of the solvent is with in +/- 2 units of the packing media polarity PS/DVB based GPC columns
Slide 16: Improper Column Chemistry: Column Interaction
ImproperChemistry: column chemistry Column Interaction • Phenol Formaldehyde resin analyzed on a PS/DVB column (red) and PolarGel (blue) • PS/DVB column interactions due to low surface polarity had strongly affected the chromatography Samples: P-F resins Columns: 2 x PolarGel, 2 x PS/DVB Injection:
Slide 17: Modification: Non-SEC Interaction
Modification Ss Non-SEC Interaction controlled with modifiers Hostavin N30 Polymeric UV stabilizer containing secondary amine groups Column: 2xPLgel 3µm MIXED-E Flow Rate: 1.0ml/min Detector: PL-ELS 1000
Slide 18: Modification examples: Starch
Modification examples: Starch Aggregation controlled with modifiers analysis Columns : 4 x 20µm MIXED-A 300x7.5mm Eluent : DMSO + 5mM NaNO3 Addition of salt is often required Flow rate : 1.0 ml/min for polar organic solvents to Temperature : 80°C suppress ionic interaction Detector : DRI effects 12
Slide 19: Solvent Selection with DRI Detectors
Solvent Selection with DRI Detectors Polydimethylsiloxane (PDMS) is soluble in several common GPC solvents. PDMS has a refractive index of 1.407 and therefore it is iso-refractive with THF Toluene (n = 1.496) and chloroform (n = 1.444) give good DRI signals and are therefore preferred solvents for G
Slide 20: Poor Column Lifetime
Poor Column Lifetime Degraded by materials in the mobile phase • Use high purity HPLC grade solvents • Filter eluent with 0.02 – 0.45 um filter prior to use • Use THF & TCB stabilized with antioxidant (BHT) Deterioration can occur due to contaminant build-up on the column • This can be avoided by us
Slide 21: Section 4: Sample Preparation
Section 4: Sample Preparation • Use an aliquot of the mobile phase to prepare the solution • Sample concentration depends on molecular weight (high MW lower concentration) • For polymers, avoid high shear stirring • Dissolution time and temperature will depend on molecular weight and crystallinity o
Slide 22: Sample Concentration
Sample Concentration • The viscosity of the polymer solution is dependant on both the molecular weight and the concentration • A high viscosity in the separation zone leads to reduced mass transfer and band broadening • This results in decreased resolution and in extreme cases peak splitting
Slide 23: Effect of Concentration on Peak Shape and Resolution
Effect of Concentration on Peak Shape and Resolution 0.05% 0.10% Column: PLgel 10µm MIXED-B 300x7.5mm Eluent: THF Flow Rate: 1.0ml/min Detector: UV Polystyrene standards 1. 8,500,000 4. 34,500 0.15% 0.20% 2. 1,130,000 5. 5,100 3. 170,000 6. 580
Slide 24: Injection Volume
Injection Volume • GPC columns have a relatively large volume (typically 300 x 7.5 mm) • Injection volumes for GPC can therefore be higher than for HPLC • As a rule of thumb, 20-50 μl per 300 x 7.5 mm column length will have little effect on band broadening • Minimize injection volume for high effic
Slide 25: Effect of Injector Loop Size on Resolution
Effect of Injector Loop Size on Resolution 20 µl loop Column : PLgel 3 µm MIXED-E 300 x 7.5 mm Eluent : THF Flow rate : 1.0 ml/min Sample : Epikote 1001 epoxy resin 200 µl loop Injection loop is a major contribution to system dead volume, use reduced injection volume and increase concentration to ma
Slide 26: Section 5: Components of a GPC System
Section 5: Components of a GPC System: Pump: delivers flow down the column Injection valve: Allows us to introduce our samples GPC column set: Performs the separation Detector: detects the material leaving the columns Optional extras: autosamplers, degassers, etc. 26
Slide 27: Pump
Pump • Isocratic pump (single channel) • GPC/SEC is always isocratic • Pulseless or low pulse flow required to ensure good detector baselines • Service typically includes replacement of worn check valves and piston seals: same service as any LC 27
Slide 28: Flow Rate and Efficiency
Flow Rate and Efficiency Eluent : THF Columns : PLgel 100Å Test probe : ODCB Optimum flow rate may differ with column particle size 28
Slide 29: Effect of Flow Rate on Resolution
Effect of Flow Rate on Resolution • Flow rate strongly affects resolution • Every column has an optimum flow rate, as in all LC systems • However in GPC the mass transfer effect is much more prominent 29
Slide 30: Column Ovens
Column Ovens • Ovens are used to heat and maintain the temperature in a GPC separation • They come in a range of specifications, from low temperature all the way up to very high temperatures (25 – 220 o C) • Temperature can be critical in GPC for stability, solubility and to reduce back pressure • S
Slide 31: Why use Elevated Temperature?
Why use Elevated Temperature? GPC applications employing elevated temperature generally fall into three categories : • To reduce solvent viscosity for improved chromatography • To achieve and maintain sample solubility • To provide a stable thermal environment for detectors 31
Slide 32: Effect of Temperature on Separations on Viscous Solvents
Effect of Temperature on Separations on Viscous Solvents Column : PLgel 5 µm MIXED-C 300 x 7.5 mm Eluent : DMF Flow rate : 1.0 ml/min Increased temperature : Reduced operating pressure Improved resolution, particularly at high MW PEO/PEG standards 990,000 252,000 86,000 18,000 4,800 200 32
Slide 33: Effect of Temperature on Column Pressure
Effect of Temperature on Column Pressure Column PLgel 5 µm MIXED-D Column pressure falls as 300 x 7.5 mm temperature increases due to Eluent Toluene reduced viscosity Flow rate 1.0 ml/min 33
Slide 34: Concentration Detectors for GPC
Concentration Detectors for GPC There are several concentration detectors that are used in conventional GPC • Differential refractive index (DRI) • Ultraviolet (UV) • Infrared (IR) • Evaporative light scattering (ELSD) 34
Slide 35: Information Rich Detectors for GPC
Information Rich Detectors for GPC • Static Light Scattering Detection • Viscometry • FTIR 35
Slide 36: Band Broadening
Band Broadening Large dead volumes • Always use LDV end fittings and connectors • Minimize lengths and diameters of tubing wherever possible Mobile phase is too viscous • May need to increase operational temperature Detector cell(s) volume is too large • If possible, use a smaller cell volume Column
Slide 37: Effects of Band Broadening
Effects of Band Broadening Modern high performance GPC columns have minimized the effect of band broadening in the separation. However poor system design with large amounts of dead volume can still cause loss of resolution. System dead volume should be minimized, especially when using very high effi
Slide 38: Questions
Questions aS gilent Technologies