50 years of size exclusion chromatography

©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


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


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”



Evolution of Hardware - component level evolution



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


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



Separation Mechanism

– Dissolved polymer sample (a mixture of molecules) passes through a porous gel-based stationary phase

– Macromolecules separate by size



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



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


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


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


Limitations of High Speed Gel Permeation Chromatography

.


Limitations of High Speed Gel Permeation Chromatography


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


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


Speed of Analysis


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


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)



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


3 x APC TMS 4.6x150mm (200,45,45)



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


3 x APC TMS 4.6x150mm (200,45,45)


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)



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



Gel Based Columns

THF DMF Toluene


Gel Based Columns

One System. One Bank of Columns. Solvent Flexibility.


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


New and Innovative Polymers

Polystyrene Standard 510 Mp Alliance 2695/2414

6x150 HSPgel HR1

ACQUITY APC with RI 4.6x150mm; 45Å XT


Polymer Growth

Step 1

Step 2

Step 3

Step 4


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


• 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


Hybrid Particle


SEM Images: Wide Pore Bridged Ethyl Hybrid

45Å 200Å

450Å 900Å


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


LogM

Vt V0

Ve

∆Ve

∆LogM

Flow Rate Precision and MW Accuracy


Solvent Flow Precision

• Precise flow essential for precise GPC result


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


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


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


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


ACQUITY Refractive Index Detector Flow Cell

2414 10.3µL

ACQ-RI 1.3µL

1 cm


ACQUITY Refractive Index Detector Counter Current Heat Exchanger

HPLC RI ~ 150µL

ACQ-RI < 15µL


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


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


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


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


Questions?

50 years of size exclusion chromatography

Science

Transcript of 50 years of size exclusion chromatography