Practical Steps in
GPC Method
Development
Jean Lane
Applications Engineer
LC Columns & Consumables
Technical Support
March 23,2016
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Overview
Solvent
Criteria for Solvent Selection
Polymer Standards - choosing correctly
Modifiers – why to use them?
Sample & Instrument Considerations
Criteria for Dissolution and Concentration
System Optimization – dead volume, fittings etc
Columns
Organic or Aqueous
What exactly do I want from my analysis?
Reproducibility
Resolution
Speed
Detectors
Which is appropriate for my sample or my column?
Is OTHER information for my sample needed?
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GPC Separation Mechanism
The GPC column is packed with porous
beads of controlled porosity and particle
size
Polymer is prepared as a dilute solution in
the eluent and injected into the system
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
GPC compare to HPLC
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A Typical GPC System: Optimizing your GPC Method
• When establishing your GPC method and to ensure optimum accuracy,
reliability and throughput, we need to consider ALL elements of our
GPC/SEC system
• Solvent
• Sample
• Flow Rate
• Temperature
• Column
• Calibration
• Detection
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Criteria for Solvent Selection
Polymers are often employed due to their strength and
toughness. Aggressive solvents and long dissolution times
often required while ensuring:
• True sample solubility to avoid non-size exclusion
effects
• Compatibility with columns
• Permit adequate detection (e.g. refractive index, UV
cut off)
• Safety (e.g. toxicity, elevated temperature, etc.)
Additives can be employed
• Minimize non-size exclusion interactions between the
sample and the column
• Stabilize the solution of the polymer (ionic aggregation)
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2 Key Questions
• What solvent is your polymer • What is the expected molecular
soluble in? weight range of your polymer?
Type Typical Solvents MW MW Range (g/mol or Da)
• THF High Up to several millions
• Chloroform
Organic Intermediate Up to hundreds of thousands
• Toluene
• TCB
Low Up to tens of thousands
• THF/water
Mixed or Polar
• DMF Very Low A few thousand
Organic
• NMP
• Water
• Buffer in water
Aqueous
• Water/methanol
(up to 50%)
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March 23, 2016
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Polymer Standards – What is the Eluent/Mobile Phase?
Solvent Type GPC/SEC Standards Type
• Polystyrene (PS)
Organic
• Polymethylmethacrylate (PM)
• Polymethylmethacrylate (PM)
Mixed or Polar Organic
• Polyethylene glycol/oxide (PEG/PEO)
• Polyethylene glycol/oxide (PEG/PEO)
Aqueous • Polysaccharide (SAC)
• Polyacrylic acid (PAA)
• EasiVial – pre-prepared for fast and easy,
accurate concentration, 12-point column What TYPE of kits
calibration for organic and aqueous best suits my needs?
solvents
• EasiCal – easy 3-step process for
accurate 10-point calibration, for organic
solvents
• Calibration kits and individual standards –
Polystyrene, PMMA, PEG/PEO, PAA,
Polysaccharide
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Calibrations Standards and Importance
of Solvent Selection
Ex: PS/DVB columns are
excellent in many solvents,
but remember that although
the column may be used in
certain solvents this does
not mean SEC will occur -
the example here is
polystyrene standards
running in NMP, DMF, etc.
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9 March 23, 2016
Eluent Modification in Organic GPC
Hostavin N30
Polymeric UV stabiliser
containing secondary amine
groups
Column: 2xPLgel 3µm MIXED-E
Flow Rate: 1.0ml/min
Detector: PL-ELS 1000
Solvent Modifiers
2
• Addition of salt is often
required for polar organic
solvents to suppress ionic
interaction effects
Chromatogram 2
(chromatogram 2)
Chromatogram 1
Columns: 4 x PLgel 20 µm
MIXED-A
6
5
5
44
44 7.5 x 300 mm
minutes
Eluent: DMSO + 5 mM NaNO3
Flow rate: 1.0 mL/min
Temperature: 80
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Sample
Criteria for Dissolution
• Sample concentration depends on MW Range Concentration Time
molecular weight 100 – 10 k 3 – 5 mg/mL 30 mins
• Avoid high shear – stirring/sonication 10 k – 500 k 2 mg/mL 1 – 4 hrs
• Dissolution time and temperature, if 500 k – 800 k 1 mg/mL 3 – 8 hrs
required, will depend on molecular 800 k – low millions ≤0.5 mg/mL o/n - day
weight and crystallinity of polymer
Millions ≤0.25 mg/mL 1 – 3 days
• Use an aliquot of the eluent to
prepare the solution • All values offered as guide only
• Use same solvent for reproducibility • Narrowly distributed samples require lower
concentration
• Reduction of RI imbalance peak
• Filtering of samples to remove
insoluble material (0.2 - 1.0 µm • In GPC sample preparation is an
filters) important consideration
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Effect of Concentration on Peak Shape &Resolution
Column: 2 x PLgel 5 µm MIXED-C
7.5 x 300 mm
Eluent: THF
Flow Rate: 1.0 mL/min
Inj vol: 200ul
Detector: RI
Sample: broad Polystyrene
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Temperature
Use of Elevated Temperature
• GPC applications employing Eluent Temp (°C)
elevated temperature generally THF, Water, Chloroform 30 - 40
fall into these categories: DMF, DMSO, DMAc 60 - 80
• To reduce solvent viscosity for TCB 140 - 160
improved mass transfer and
improved chromatographic • All values offered as guide only
separation
• To reduce system pressure and
prevent column damage • Elevated temperature is a useful
• To provide a stable thermal approach in GPC
environment for GPC columns and
detectors (especially RID)
• To achieve and maintain sample
solubility
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Temperature
Effect on Separations using Viscous Solvents
• Increased temperature:
• Reduced operating pressure
• IMPROVED resolution, particularly
at high MW
Column: PLgel 5 µm MIXED-C
300 x 7.5mm
Eluent: DMF
Flow rate: 1.0 mL/min
PEO/PEG standards
990,000 252,000
86,000 18,000
4,800 200
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Sample
Effect of Injector Loop Size on Resolution
Column : PLgel 3 µm MIXED-E
20uL 300 x 7.5mm
Eluent : THF
Flow rate : 1.0 mL/min
Sample : Epikote 1001 epoxy resin
200uL • Injection loop is a major
contribution to system dead
volume, use reduced injection
volume and increased
concentration to maintain
sensitivity
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Reducing Dead Volume
Wide bore connecting tubing
• ID for tubing narrow as
possible
• Tubing Connections Short
Narrow bore connecting tubing
• Use proper fittings for
connections
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Proper Connections
• Problems with improper connections
• Source of leaks
• Mistaken for chromatography issues
• Making connections can vary with skill/technique
• Different manufacturers supply different types of fittings
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Potential Fittings Issues
• Leak
• Peak shape problem
• No dead volume
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Conventional GPC/SEC Workflow
• Calibrate the GPC column with a set of narrow polymer standards
• Plot retention time (RT) versus peak log molecular weight (logM)
• Calibration is used to generate molecular weight (averages and
distribution) of unknowns run on the same system/column-set
• Molecular weights are relative to the standards used
GPC sample chromatogram Molecular weight distribution
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March 23, 2016
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Pump Flow Rate & Reproducibility
Effect on MW Results
• A small change in flow rate can have a large effect on GPC
molecular weight results
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What are GPC Columns Made Of?
• Silica Packings
• Mechanically stronger
• Exhibit enthalpic properties due to presence of silanols
• Typically have lower pore volumes
•Polymeric Packings
• High pore volume and vendor specific differences in mechanical stability
• Due to polarity of stationary phase observed interactions are reduced
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What would you like to improve about your GPC/SEC?
Resolution is too low Analysis time is too long
0
Peak shapes are poor Results are not reproducible
0
4.5 8.5
Minutes
HrgpH
.0
r0
g8
pH
1.0
rg0
p8
2.007
Agilent Technologies
March 23, 2016
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GPC Columns
Making a Choice
What is the Molecular Weight range for my sample?
Organic or Aqueous eluents being used
What ARE your requirements for your GPC analysis?
i. Resolution is important
ii. Reproducibility of sample chromatography and results
iii. Speed of analysis and/or sample throughput is
something to improve on
Choose a column(s) based on your KEY requirements
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Effect of Column Selection: Pore size
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Combination of Individual Pore Size Columns
Traditional approach to increasing MW
operating range of column set
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PLgel MIXED Bed Calibrations
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Difference in Linearity
PlusPore PS/DVB Columns
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Effect of Increased Pore Volume
Columns 2xPLgel 3µm 100Å 300x7.5mm
2xOligoPore 300x7.5mm
Eluent THF
Flow rate 1.0ml/min Both columns have a similar exclusion
limit but OligoPore has greater pore
volume than PLgel 100Å. Hence the
slope of the curve is shallower leading to
greater resolution for columns of the
same efficiency
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Examples of Resolution Using Pluspore
Columns
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Comparison for Conventional Columns vs Cols for Fast GPC
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If Speed of analysis is important……..
Rapide columns reduce analysis
times while maintaining the
excellent solvent compatibility and
mechanical stability
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Polystyrene Mw 580 – Oligopore 250x4.6mm
1.2ml/min 0.6ml/min 0.3ml/min
Different flow rates overlaid to
show that faster doesn’t
sacrifice resolution. The
chromatograms have been
normalised to better illustrate
the differences
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High Speed MesoPore Columns
Easy Method Transfer from Standard to
rapid GPC on MesoPore 250x4.6mm
GPC columns
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Detection
Concentration Detectors
• Most common detectors for GPC/SEC are concentration detectors:
RID UV/DAD ELSD
• These provide information on the amount of polymer eluting from the
column at any given time
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Detector Selection: ex RI vs ELSD
RI:
Low response for sample
Unable to detect additives
System interference peaks present
ELSD:
Improved response
Additives detected
No system interference peaks
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Sensitivity of DRI Versus ELSD
Columns 2 x PLgel 5µm MIXED-C 300x7.5mm
Eluent THF
Flow rate 1.0ml/min
ELSD is essentially
Loading 0.1%, 20µl
independent of dn/dc,
improvement in sensitivity will
Mp values depend on a number of solute
parameters
1. 7,500,000
2. 841,700
3. 148,000
4. 28,500
5. 2,930
Case Study – Customer Sample “Kraton”
Standard GPC columns & Conventional RI
Customer wanted to reduce
runtime and maintain resolution
50min
Narrow-bore GPC columns & Conventional RI
Loss of resolution with shorter
run time & conventional RID
Narrow-bore GPC columns &
Resolution achieved using narrow bore
Micro RID
GPC columns & micro RID
15min
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Ultra low dispersion for improved resolution
EasiVial Polystyrene Green
EasiVial Polystyrene Red conventional RI detector
micro-RI detector
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Increasing the information from GPC/SEC
GPC/SEC provides critical information for the polymer chemist:
Distribution of chain lengths
(Relative molecular weights)
Further parameters can be determined by employing advanced
detectors
The molecular weight (accurate or absolute)
The polymer’s size
The polymer’s shape
Can be used to investigate polymer branching
Expanding Conventional GPC/SEC
Viscometer and Light Scattering Detectors
Advanced detectors give a greater understanding of the analyte
as well as overcoming the limitations of Conventional GPC.
GPC/SEC
Molecular Weight Molecular Size Information
Technique
Conventional (RI or Relative to standards used for No Molecular weight distribution,
UV) calibration concentration
Viscometry More accurate from Universal Yes, Conformation, branching.
Calibration
hydrodynamic Works with copolymers
radius (Rh).
Light Scattering Absolute determination Yes, Radius of Conformation, branching.
Gyration (Rg)
directly.
Triple Absolute determination Yes, Rg and Rh, The ultimate configuration
directly. for comprehensive
polymer characterisation
Summary
Consider you choice of solvent carefully for the
Solvent Selection type of sample, conditions, & columns required
for analysis.
Sample & Instrument Use appropriate concentrations & inj. volumes
based on your sample’s MW & check your
Considerations system parameters to also optimize your analysis
Look to make the appropriate selection based on
GPC Column Selection expected MW, but also be sure to ask ‘what is it
that I want or need for my analysis?
Concentration type detectors for Conventional
Detectors GPC or look to Multi detector SEC to get
additional information for your polymer
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GPC/SEC Resources
Brochure Agilent GPC/SEC solutions - comprehensively better polymer analysis 5990-8844EN
Chemicals and energy applications 5991-2517EN
Pharma applications 5991-2519EN
Food applications 5991-2029EN
Engineering polymers 5990-6970EN
Application Compendia Polyolefin analysis 5990-6971EN
Analysis of elastomers by GPC/SEC 5990-6866EN
Biodegradable polymers 5990-6920EN
Low molecular weight resins and prepolymers 5990-6845EN
Excipient analysis 5990-7771EN
Analysis of food additives by GPC/SEC 5990-8634EN
Introduction to GPC/SEC 5990-6969EN
Primers A guide to multi-detector GPC 5990-7196EN
Calibrating GPC columns – a guide to best practice 5991-2720EN
An Automated System for Cleanup of Environmental Samples 5991-5321EN
1260 Infinity GPC/SEC System 5990-9920EN
1260 Infinity Multi-Detector GPC/SEC System 5990-9921EN
Agilent 1290 Infinity II Refractive Index Detector (Micro) 5991-6561EN
Product Guides Agilent PL-GPC 220 5990-9926EN
Agilent GPC/SEC Software 5991-0478EN
Aqueous and polar GPC/SEC columns 5990-7995EN
Organic GPC/SEC columns 5990-7994EN
GPC/SEC standards 5990-7996EN
Select Guide Quick guide for selecting columns and standards for GPC/SEC 5990-6868EN
Quick Reference Guide Agilent 1260 Infinity GPC/SEC System supplies 5990-9947EN
Agilent PL-GPC 220 System supplies 5990-9946EN
Flyers Agilent EnviroPrep GPC columns 5991-1588EN
Agilent Fast GPC columns 5991-2785EN
Wall Chart Achieve more with the Polymer Analysis People 5991-3802EN
Download at www.agilent.com/chem/GPCresources
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THANK YOU FOR ATTENDING
ANY QUESTIONS??
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