This presentation discusses the history of size exclusion chromatography from gel permeation to modern day columns, and introduces the next evolution in polymer chromatography, the ACQUITY Advanced Polymer Chromatography system.

1. ©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

2. ©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

3. ©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

4. ©2013 Waters Corporation 4
Evolution of Hardware
- component level evolution
Poster Session # 2780

5. ©2013 Waters Corporation 5
Evolution of Hardware
- component level evolution
Poster Session # 2780

6. ©2013 Waters Corporation 6
Evolution of Hardware
- component level evolution
Poster Session # 2780

7. ©2013 Waters Corporation 7
Evolution of Hardware
- component level evolution
Poster Session # 2780

8. ©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: 400
C
Flow Rate: 1 ml/min
Polystyrene Narrow Molecular Weight Standards
106 105 104 103 102

9. ©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

10. ©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

11. ©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

12. ©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

13. ©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

14. ©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

15. ©2013 Waters Corporation 15
Limitations of High Speed
Gel Permeation Chromatography
.

16. ©2013 Waters Corporation 16
Limitations of High Speed
Gel Permeation Chromatography

17. ©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

18. ©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

19. ©2013 Waters Corporation 19
Speed of Analysis

20. ©2013 Waters Corporation 20
Speed of Analysis

21. ©2013 Waters Corporation 21
Speed of Analysis

22. ©2013 Waters Corporation 22
Speed of Analysis

23. ©2013 Waters Corporation 23
Speed of Analysis

24. ©2013 Waters Corporation 24
Replicate Data for Polyvinyl Acetate
µRIU
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
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 4.60 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

25. ©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)

26. ©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)

27. ©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)

28. ©2013 Waters Corporation 28
More resolution = more
points for low molecular
weight calibration
Better calibration =
more accurate data
LogMolWt
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
LogMolWt
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

29. ©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

30. ©2013 Waters Corporation 30
Gel Based Columns
THF DMF Toluene

31. ©2013 Waters Corporation 31
Gel Based Columns
THF DMF Toluene

32. ©2013 Waters Corporation 32
Gel Based Columns
THF DMF Toluene

33. ©2013 Waters Corporation 33
Gel Based Columns
One System.
One Bank of Columns.
Solvent Flexibility.

34. ©2013 Waters Corporation 34
Rigid Hybrid Columns
82272
µRIU
-2.00
0.00
2.00
4.00
58422
µRIU
-2.00
0.00
2.00
4.00
81709
µRIU
-2.00
0.00
2.00
4.00
81365
µRIU
-2.00
0.00
2.00
4.00
Minutes
0.00 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)
DMFbefore after % change
Mp 82272 81365 0.4
Mw 78650 78953 1.5
Mn 49383 50110 0.6
PDI 1.59 1.58 1.1
poly(methyl methacrylate co ethyl acrylate)
in THF

35. ©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

36. ©2013 Waters Corporation 36
Polymer Growth
Step 1
Step 2
Step 3
Step 4

37. ©2013 Waters Corporation 37
High Resolution of EpoxyµRIU
-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
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 4.60 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

38. ©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

39. ©2013 Waters Corporation 39
Hybrid Particle

40. ©2013 Waters Corporation 40
SEM Images: Wide Pore
Bridged Ethyl Hybrid
45Å 200Å
450Å 900Å

41. ©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
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 4.60 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

42. ©2013 Waters Corporation 42
LogM
VtV0
Ve
∆Ve
∆LogM
Flow Rate Precision and MW Accuracy

43. ©2013 Waters Corporation 43
Solvent Flow Precision
• Precise flow essential for precise GPC result

44. ©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
Minutes
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
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

45. ©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

46. ©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

47. ©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

48. ©2013 Waters Corporation 48
ACQUITY Refractive Index Detector
Flow Cell
2414
10.3µL
ACQ-RI
1.3µL
1 cm

49. ©2013 Waters Corporation 49
ACQUITY Refractive Index Detector
Counter Current Heat Exchanger
HPLC RI ~
150µL
ACQ-RI < 15µL

50. ©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
Minutes
2.20 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
Minutes
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 4.80
ACQUITY APC System with ACQUITY APC XT 4.6 x 150mm (45Å + 45Å + 200Å)
HPLC RI Detector ACQUITY RI Detector

51. ©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
Minutes
2.20 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
Minutes
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 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
Minutes
3.80 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
Minutes
3.80 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

52. ©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

53. ©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

54. ©2013 Waters Corporation 54
Questions?