50 years of size exclusion chromatography
TRANSCRIPT
©2013 Waters Corporation 1
50 Years of Size Exclusion Chromatography (SEC)
Michael O'Leary 1, Baiba Cabovska 1, Edouard Bouvier 1, Bonnie Alden 1,Peter Hancock 2
1 Waters Corp. Milford, MA USA 2 Waters Corp. Manchester UK
Poster Session # 2780
©2013 Waters Corporation 2
50 Years of Size Exclusion Chromatography - Overview
I. History and Overview of SEC
a) System b) Column c) Recent Trends
II. Novel Approach to Modern Polymer Chromatography
a) Speed of Analysis b) Gel Based Columns c) New & Innovative
Polymers
III. Modern Column Materials for Polymer Chromatography
IV. System Requirements for Modern Polymer Chromatography a) Solvent Delivery b) Refractive Index
Detector c) System Dispersion
V. Conclusions
©2013 Waters Corporation 3
Birth of Polymer Chromatography Dow/Waters Collaborations in GPC
1962 - Jim Waters builds prototype low volume, high temperature refractometer for John C. Moore, Dow Chemicals
1963 – Waters exclusive license of US 3,326,875, “ Separation of Large Polymer Molecules in Solution” from Dow Chemicals
1964 – Key paper by Moore – Reduces analysis from days to hours – Coins term “GPC”
Poster Session # 2780
©2013 Waters Corporation 4
Evolution of Hardware - component level evolution
Poster Session # 2780
©2013 Waters Corporation 5
Evolution of Hardware - component level evolution
Poster Session # 2780
©2013 Waters Corporation 6
Evolution of Hardware - component level evolution
Poster Session # 2780
©2013 Waters Corporation 7
Evolution of Hardware - component level evolution
Poster Session # 2780
©2013 Waters Corporation 8
Impact of Particle Size in Size Based Separations
Column Evolution 75 micron to 5 micron particle size
Column: Styragel 300X7.8 mm HR Series, 5,4 2 0.5 Eluent: Tetrahydrofuran Column Temp: 400C Flow Rate: 1 ml/min
Polystyrene Narrow Molecular Weight Standards
106 105 104 103 102
©2013 Waters Corporation 9
GPC:1964 to today Little/no change in Column technology
Primarily polymer-based resins
– Styrene-DVB – Methacrylates
Low resolution technique (“Blobograms”) – Particle size reduction from ~75 micron to ~5 micron – Instrumentation dispersion limitations
The technique of GPC/SEC used in this industry may not have advanced in 20 years, but the economic, competitive and market dynamics of the polymer industry have, driving a need for better information and higher quality data……..faster
Poster Session # 2780
©2013 Waters Corporation 10
Separation Mechanism
– Dissolved polymer sample (a mixture of molecules) passes through a porous gel-based stationary phase
– Macromolecules separate by size
Poster Session # 2780
©2013 Waters Corporation 11
Reminder about GPC Definitions
A Polymer sample is a mixture of large molecules having different chain lengths (molecular weights) but having the same composition
Molecular weight averages (MW) and molecular weight distribution affect the physical properties
These values can be calculated by different techniques but only GPC allows the determination of all of them in a single experiment
Mz niMi3
niMi2 =
∑
∑
Mw
niMi2
niMi =
∑
∑ Mz+1
∑
niMi3 ∑ =
niMi4
Mn
niMi =
∑
∑ ni I
Mn =
Mw
With Mn<Mw<Mz<Mz+1
Poster Session # 2780
©2013 Waters Corporation 12
Recent Trends Polymer Development
Green Chemistry - Decreasing the need for
organic solvents in processes by using water-based chemistry
- Bio-sourced polymers
- Biodegradable polymers
- Lower molecular weight polymers are key to all of these areas
Modern Chemistry - Polymer end-group
functionality has evolved
- Better control of polymerization reactions and achieving desired molecular weight averages and polydispersity
- New catalysts for generating new and innovative polymer structures
©2013 Waters Corporation 13
Polymer Characterization
The resurgence in polymer development requires extensive R&D characterization of these new and innovative polymers
Many techniques are used to characterize polymers – HPLC/GPC – 2-Dimensional LC – Multi-Angle Light Scattering – Viscometry – Spectroscopic techniques (including Mass Spectrometry ) – Thermal Analysis e.g. Rheology, DSC TGA
Gel Permeation Chromatography (GPC) remains the key technique for evaluating the molecular weight distribution of a polymer
©2013 Waters Corporation 14
ACQUITY APC System
Analytical Challenges
ACQUITY© APCTM System
Gel Based Columns
- Styrene DVB and methacrylate based columns are relatively fragile and typically cannot easily be converted from one solvent to another
Speed of Analysis
- Current approaches to reduce analysis time of a GPC assay compromises peak resolution and therefore characterization data quality
Lack of Resolution of Low Molecular Weight
Polymer and Oligomers
- Traditional GPC remains to be a low resolution technique that is inadequate in providing the characterization information required for today’s innovative polymers and building blocks
©2013 Waters Corporation 15
Limitations of High Speed Gel Permeation Chromatography
.
©2013 Waters Corporation 16
Limitations of High Speed Gel Permeation Chromatography
©2013 Waters Corporation 17
Introducing the ACQUITY Advanced Polymer Chromatography (APC) System
Precise solvent
management
Low system dispersion
Compatibility with
challenging solvents
Rigid, solvent-resilient columns
Versatile column
management
Stable refractive
index detection
Flexible detection
techniques
Wide range of APC
standards
©2013 Waters Corporation 18
APC – A Definition
Application technique for the size based separation of polymers in solution using columns packed with sub-
3um rigid, high pore volume hybrid particles combined with a fully optimized low dispersion
ACQUITY system
©2013 Waters Corporation 19
Speed of Analysis
©2013 Waters Corporation 20
Speed of Analysis
©2013 Waters Corporation 21
Speed of Analysis
©2013 Waters Corporation 22
Speed of Analysis
©2013 Waters Corporation 23
Speed of Analysis
©2013 Waters Corporation 24
Replicate Data for Polyvinyl Acetate µR
IU
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
Injection Mz Mw Mn PDI 1 61700 37867 19846 1.908 2 62082 38376 20006 1.918 3 61816 38002 19889 1.911 4 62929 38154 20160 1.893
Ave. 62132 38100 19975 1.907 SD 555 218 141 0.011
%RSD 0.89 0.57 0.70 0.57
0.25% w/v, 20uL injection RI detection 100% THF, 1mL/min ACQUITY APC XT 450 Å, 4.6x150mm 125 Å, 4.6x150mm and 45 Å, 4.6x150mm in series
©2013 Waters Corporation 25
Speed of Analysis - Better Characterization
MV
0
5
10
15
20
25
30
35
Minutes 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26
µRIU
0
2
4
6
8
10
12
14
16
18
20
Minutes 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.7
100K
10K
1K
100K
10K
1K
GPC APC 3 x Styragel 7.8x300mm (4e, 2, 0.5)
3 x APC TMS 4.6x150mm (200,45,45)
©2013 Waters Corporation 26
Speed of Analysis - Better Characterization
MV
0
5
10
15
20
25
30
35
Minutes 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26
µRIU
0
2
4
6
8
10
12
14
16
18
20
Minutes 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.7
100K
10K
1K
100K
10K
1K
GPC APC 3 x Styragel 7.8x300mm (4e, 2, 0.5)
3 x APC TMS 4.6x150mm (200,45,45)
©2013 Waters Corporation 27
Speed of Analysis - Better Characterization
MV
0
5
10
15
20
25
30
35
Minutes 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26
µRIU
0
2
4
6
8
10
12
14
16
18
20
Minutes 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.7
100K
10K
1K
100K
10K
1K
µRIU
0.0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
Minutes 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70
MV
-1.0
0.0
1.0
2.0
3.0
4.0
Minutes 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0
1K polystyrene standard
1K polystyrene standard
GPC APC 3 x Styragel 7.8x300mm (4e, 2, 0.5)
3 x APC TMS 4.6x150mm (200,45,45)
©2013 Waters Corporation 28
More resolution = more points for low molecular weight calibration Better calibration = more accurate data
Log
Mol
Wt
2.40
2.80
3.20
3.60
4.00
4.40
4.80
5.2
5.6
Retention Time 15 16 17 18 19 20 21 22 23 24 25 26 27
Log
Mol
Wt
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
Retention Time 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Alliance GPC system 3 x Styragel 7.8 x 300mm (4e, 2, 0.5) Polystyrene calibration (100K, 10K, 1K)
ACQUITY APC system 3 x APC TMS 4.6 x 150mm (200,45,45) Polystyrene calibration (100K, 10K, 1K)
Speed of Analysis - Better Characterization
©2013 Waters Corporation 29
More calibration points → Better calibration → Better characterization
FASTER calibration → Calibrate in less than 30 minutes, not hours
DAILY calibration, not weekly → Better data consistency and quality
GPC APC
28.00 4.90
Speed of Analysis - Better Characterization
©2013 Waters Corporation 30
Gel Based Columns
THF DMF Toluene
©2013 Waters Corporation 31
Gel Based Columns
THF DMF Toluene
©2013 Waters Corporation 32
Gel Based Columns
THF DMF Toluene
©2013 Waters Corporation 33
Gel Based Columns
One System. One Bank of Columns. Solvent Flexibility.
©2013 Waters Corporation 34
Rigid Hybrid Columns
8227
2
µRIU
-2.00
0.00
2.00
4.00
5842
2
µRIU
-2.00
0.00
2.00
4.00
8170
9
µRIU
-2.00
0.00
2.00
4.00
8136
5
µRIU
-2.00
0.00
2.00
4.00
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
Poly(methyl methacrylate co ethylacrylate) THF
Poly(methyl methacrylate co ethylacrylate) THF
Poly(9,9 di-n-octylfluorenyl 2,7 diyl) Toluene
Poly(bisphenol-A-co epichlorohydrin) DMF before after % change
Mp 82272 81365 0.4Mw 78650 78953 1.5Mn 49383 50110 0.6PDI 1.59 1.58 1.1
poly(methyl methacrylate co ethyl acrylate) in THF
©2013 Waters Corporation 35
New and Innovative Polymers
Polystyrene Standard 510 Mp Alliance 2695/2414
6x150 HSPgel HR1
ACQUITY APC with RI 4.6x150mm; 45Å XT
©2013 Waters Corporation 36
Polymer Growth
Step 1
Step 2
Step 3
Step 4
©2013 Waters Corporation 37
High Resolution of Epoxy µR
IU
-2.00
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
0.5% w/v, 10uL injection RI detection 100% THF, 1mL/min ACQUITY APC XT 200Å, 4.6x150mm and 2 X 45Å 4.6x150mm in series
©2013 Waters Corporation 38
• Five pore sizes
• 45 Å (200 – 5,000) 1.7µm • 125 Å (1,000 – 30,000) 2.5µm • 200 Å (3,000 – 70,000) 2.5µm • 450 Å (20,000 – 400,000) 2.5µm • 900 Å* (Available later this year)
• Two surface chemistries
• Organic - XT • Aqueous - AQ
• Three column lengths
• 30 mm • 75 mm • 150 mm
ACQUITY APC Column Options
©2013 Waters Corporation 39
Hybrid Particle
©2013 Waters Corporation 40
SEM Images: Wide Pore Bridged Ethyl Hybrid
45Å 200Å
450Å 900Å
©2013 Waters Corporation 41
Polysulfone
µRIU
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
Minutes2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
Injection Mz Mw Mn PDI 1 78223 50772 17332 2.929 2 79645 50747 17261 2.940 3 78216 50908 17351 2.934 4 78780 50760 17315 2.932
Ave 78716 50797 17315 2.934 SD 673 75 39 0.005
% RSD 0.86 0.15 0.22 0.16
0.25% w/v, 20uL injection RI detection 100% THF, 1mL/min ACQUITY APC XT 450 Å, 4.6x150mm 125 Å, 4.6x150mm and 45 Å, 4.6x150mm in series
©2013 Waters Corporation 42
LogM
Vt V0
Ve
∆Ve
∆LogM
Flow Rate Precision and MW Accuracy
©2013 Waters Corporation 43
Solvent Flow Precision
• Precise flow essential for precise GPC result
©2013 Waters Corporation 44
Overlay of every 20th injection for 100 injections of Commercial Epoxy Resin
µRIU
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
0.25% w/v, 40uL injection RI detection 100% THF, 1mL/min ACQUITY APC XT 125 Å, 4.6x150mm and 45 Å, 4.6x150mm in series
Injection Mp Mw Mn PDI 20 2743 6143 3870 1.587 40 2753 6160 3884 1.586 60 2754 6156 3884 1.585 80 2745 6146 3870 1.588
100 2746 6151 3878 1.586 % RSD 0.18 0.11 0.18 0.08
©2013 Waters Corporation 45
Bis-Phenol-a Condensation Polymer 4 Replicates in Less Time than Conventional GPC
0.5% w/v, 10uL injection RI detection 100% THF, 1mL/min ACQUITY APC XT 200Å, 4.6x150mm and 2 X 45Å 4.6x150mm in series
Injection Mw Mn PDI 1 3001 1303 2.303 2 3002 1299 2.311 3 3008 1302 2.310 4 3001 1304 2.301
Ave 3003 1302 2.306 SD 3 2 0.005
% RSD 0.11 0.17 0.21
©2013 Waters Corporation 46
Refractive Index (RI) Detector
RI detectors – Among first commercial HPLC detectors – 1960s early 1970s
Measurement based on differential RI (∆n) – need to differentiate RI sample fluid relative to RI reference fluid
Typically referred to as universal detector – Detects all dissolved solutes
o “non-specific”
The higher the specific refractive index increment (dn/dc), the higher the sensitivity ∆n = (dn/dc) · c
©2013 Waters Corporation 47
Factors That Affect RI
Temperature – On average ∆n changes by 450 micro RIU per 1°C
for organic liquids
Pressure – Pressure pulses from solvent delivery system
Composition – Vacuum Degasser – Homogeneous mobile phase
©2013 Waters Corporation 48
ACQUITY Refractive Index Detector Flow Cell
2414 10.3µL
ACQ-RI 1.3µL
1 cm
©2013 Waters Corporation 49
ACQUITY Refractive Index Detector Counter Current Heat Exchanger
HPLC RI ~ 150µL
ACQ-RI < 15µL
©2013 Waters Corporation 50
ACQUITY Refractive Index Detector Results
µRIU
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
Minutes2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70
MV
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
Minutes2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80
ACQUITY APC System with ACQUITY APC XT 4.6 x 150mm (45Å + 45Å + 200Å)
HPLC RI Detector ACQUITY RI Detector
©2013 Waters Corporation 51
ACQUITY Refractive Index Detector Results
µRIU
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
Minutes2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70
MV
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
Minutes2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80
µRIU
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
Minutes3.80 3.85 3.90 3.95 4.00 4.05 4.10 4.15 4.20 4.25 4.30 4.35 4.40 4.45 4.50 4.55 4.60 4.65 4.70
MV
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
Minutes3.80 3.85 3.90 3.95 4.00 4.05 4.10 4.15 4.20 4.25 4.30 4.35 4.40 4.45 4.50 4.55 4.60 4.65 4.70 4.75 4.80
ACQUITY APC System with ACQUITY APC XT 4.6 x 150mm (45Å + 45Å + 200Å)
HPLC RI Detector ACQUITY RI Detector
©2013 Waters Corporation 52
ACQUITY APC System
Analytical Challenges
ACQUITY© APCTM System
Gel Based Columns
- Styrene DVB and methacrylate based columns are relatively fragile and typically cannot easily be converted from one solvent to another
Speed of Analysis
- Current approaches to reduce analysis time of a GPC assay compromises peak resolution and therefore characterization data quality
Lack of Resolution of Low Molecular Weight
Polymer and Oligomers
- Traditional GPC remains to be a low resolution technique that is inadequate in providing the characterization information required for today’s innovative polymers and building blocks
©2013 Waters Corporation 53
Recent Trends Polymer Development
Green Chemistry - Decreasing the need for
organic solvents in processes by using water-based chemistry
- Bio-sourced polymers
- Biodegradable polymers
- Lower molecular weight polymers are key to all of these areas
Modern Chemistry - Polymer end-group
functionality has evolved
- Better control of polymerization reactions and achieving desired molecular weight averages and polydispersity
- New catalysts for generating new and innovative polymer structures
©2013 Waters Corporation 54
Questions?