Convergence Chromatography - Turning SFC into a robust ... · ©2014 Waters Corporation 4...

©2014 Waters Corporation 1

Convergence Chromatography - Turning SFC into a robust, routine separation technique

for the polymer chromatographer

Oliver Burt, Business Development Manager


UltraPerformance Convergence Chromatography

Convergence Chromatography is a category of separation science that provides orthogonal and increased separation power, compared to liquid or gas chromatography, to solve separation challenges. UltraPerformance Convergence Chromatography ™ [UPC2®] is a holistically designed chromatographic system that utilizes liquid CO2 as a mobile phase to leverage the chromatographic principles and selectivity of normal phase chromatography while providing the ease-of-use of reversed-phase LC.


Evolution of Separation Technology

Gas Chromatography Convergence Chromatography

GC

Capillary GC

HPLC

UPLC

SFC

UPC2

Liquid Chromatography


Understanding Convergence Chromatography

Chromatographic technique similar to HPLC – Instead of mobile phase A being aqueous, it is replaced with CO2

Mobile phase is supercritical fluid + one or more co-solvents – CO2 is the most common supercritical fluid – MeOH is the most common co-solvent

Gives normal phase like selectivity

Substance Critical Temp oC Critical Pressure (bar)

Comments

Carbon Dioxide 31 74 Physical state easily changed

Water 374 221 Extreme conditions needed

Methanol 240 80 Extreme temperature needed

Ammonia 132 111 Highly corrosive

Freon 96 49 Environmentally unfriendly

Nitrous Oxide 37 73 Oxidizing agent


Lack of robustness

– Shifting retention times – Low accuracy for partial loop injections – Unstable modifier delivery at low percentages of co-solvent (< 5%)

Lack of instrument performance – Insufficient instrumentation reliability (pumping system, injection

mechanism, backpressure regulator) – Large system dispersion and dwell volumes prevented adoption of

smaller particles and high throughput analysis

Low sensitivity – High detector and pump noise – Refractive index effects of CO2

Historical challenges for SFC


Driving UPC2 performance

–Management of supercritical fluids

–Selectivity that can be addressed

–Innovative chemistries


What are the benefits of using SFC based separations?

Deliver Orthogonal separations – Different relative retention helps ensure full characterization – Check method specificity by comparison to a second

procedure – Reveal “hidden” impurity or degradation peaks – Increase confidence in characterization of complex samples

Simplify the workflow with UPC2

– Combine multiple techniques (LC & GC into CC) – Access robust normal phase separations – Eliminate solvent exchange steps for organic extracts

Deal with compound Similarity challenges – Chiral Separations (enantiomers & diastereomers) – Positional isomers (differ in location of functional groups)

SIMPLICITY

SIMILARITY

ORTHOGONALITY

Built upon proven UPLC® Technology – Quantifiable increase in productivity

Robust, reliable and reproducible – Modernization of SFC-based technology,

making this technique a viable analytical separations tool


ACQUITY UPC2 Flow Path and Components

Inject valve

Auxiliary Inject valve

Column Manager

PDA detector

Back Pressure Regulator (Dynamic and Static)

Waste Modifier CO2 Supply CO2

Pump Modifier

Pump

mixer Thermo-electric heat exchanger

Make-up Pump

Mass Spec

Splitter


Addressing the Challenges of SFC through Innovation: ACQUITY UPC2 Binary Solvent Manager

Volumetric density control so precise it models mass flow Improved density control = improved solvating

strength control = controlled elution times

Exceptional precision and accuracy for

controlled mobile phase composition Accurately and precisely blends liquid CO2 and

desired modifier (organic), even at compositions < 5%

Method development flexibility Select from 1 of 4 modifiers with integrated solvent

select valve


Auxiliary injection valve to maintain pressure of system and physical state of CO2

– Enables repeatable and reproducible partial loop injections

– Specially designed rotor and stator to accommodate supercritical CO2

Ability to accurately and precisely deliver

partial loop injections in a reproducible fashion – 0.1 – 50 µL in 0.1 µL increments – Precision < 1% RSD

Exceptionally low carryover (<0.005%) with

strong and weak needle wash options

Exceptional injection linearity – >0.999 R2

Addressing the Challenges of SFC through Innovation: ACQUITY UPC2 Sample Manager


Innovative two-stage dynamic and static back pressure regulator for improved density control – Decreased baseline noise and retention

control

Contains active back pressure regulator

[ABPR] to maintain desired CO2 pressure – Zirconia needle valve accurately and

precisely controls mobile phase density to fine tune methods

Heated static cartridge BPR

– Improved thermal management – Mitigates valve freezing due to endothermic

behavior of CO2 liquid-to-gas phase change

Addressing the Challenges of SFC through Innovation: ACQUITY UPC2 Convergence Manager


Photo Diode Array (PDA)

High strength silica lens improves low UV energy – Maximize sensitivity – Compensates for differences in RI effects between CO2 and

modifier – Reduces baseline noise

Thermal management of optics bench

– Exceptional baseline stability – Mitigates RI effects

Low dispersion stainless steel TaperSlit flow cell

– Ensures high sensitivity while maintaining optimal spectral performance

– Low dispersion to accommodate narrow peak widths – 10 mm path length to maximize sensitivity – 6,000 psi compatibility

Addressing the Challenges of SFC through Innovation: ACQUITY UPC2 Optical Detection


UPC2 Configuration for MS

UV Detector

Make-Up Pump

Convergence Manager MS


What Drives UPC2 Performance? - Addressable Selectivity

Solvent Pentane, Hexane, Heptane

Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8

Reversed-Phase

Selectivity Space

Normal Phase Selectivity

Space


Convergence Chromatography Selectivity Space

What Drives UPC2 Performance? - Addressable Selectivity

Solvent Pentane, Hexane,

Heptane

Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8

Wea

k Str

ong

Supercritical CO2

Organic Modifier


The Advent of Convergence Chromatography

Convergence Chromatography

SFC UltraPerformance Convergence Chromatography

Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland

UltraPerformance Convergence Chromatography is the result of significant technological advancements in Supercritical Fluid Chromatography instrumentation and chemistry design while providing exceptional increases in available selectivity.


What Drives UPC2 Performance? - Innovative Chemistries

ACQUITY UPC2™ BEH 2-EP • Good retention, peak shape, and selectivity • Lipids, steroids, pesticides

ACQUITY UPC2 BEH

• Heightened interaction with polar groups such as phospholipids • OLED’s, polymer additives, pesticides

ACQUITY UPC2 CSH Fluoro-Phenyl • Good retention of weak bases • Alternate elution for acidic and neutral compounds • Vitamin D metabolites, steroids, natural products

ACQUITY UPC2 HSS C18 SB • Reversed-phase-like selectivity • Fat soluble vitamins, lipids (free fatty acids)

Scalable to larger particle sizes ACQUITY UPC2/Viridis® BEH (1.7, 3.5 and 5 µm) ACQUITY UPC2/Viridis BEH 2-EP (1.7, 3.5 and 5 µm) ACQUITY UPC2/Viridis CSH Fluoro-phenyl (1.7, 3.5 and 5 µm) ACQUITY UPC2 HSS C18 SB (1.8 and 3.5 µm)


The Key Benefits of UPC²

Simplify the workflow with UPC2

– Combine multiple techniques (LC & GC into CC) – Access robust normal phase separations – Eliminate solvent exchange steps for organic extracts

Deal with compound Similarity challenges – Chiral separations (enantiomers & diastereomers) – Positional isomers (differ in location of functional groups)

Deliver Orthogonal separations

– Different relative retention helps ensure full characterization – Check method specificity by comparison to a second

procedure – Reveal “hidden” impurity or degradation peaks – Increase confidence in characterization of complex samples

SIMPLICITY

SIMILARITY

ORTHOGONALITY


Oligomeric Materials


Sample Set Id: 1462 SampleName: Triton X-100 Date Acquired: 6/21/2012 8:54:56 AM EDT Injection Id: 1522

Peak1 -

0.2

71

Peak2 -

0.4

55

Peak3 -

0.5

74

Peak4 -

0.6

62

Peak5 -

0.7

33

Peak6 -

0.7

92

Peak7 -

0.8

41

Peak8 -

0.8

84

Peak9 -

0.9

21

Peak10 -

0.9

54

Peak11 -

0.9

85

Peak12 -

1.0

13

Peak13 -

1.0

41

Peak14 -

1.0

67

Peak15 -

1.0

93

Peak16 -

1.1

19

Peak17 -

1.1

58

Peak18 -

1.1

88

Peak19 -

1.2

17

Peak20 -

1.2

48

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

Minutes0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50

Triton X 100

UPC2 Polymer Separations:- Triton X-100

• CO2 and Methanol gradient @ 40°C • 75s to elute all components • Approx. 20 oligomers separated and detected

1.5 mins


Column Name: SampleName: triton h/e 65 deg 3-20 gr 20 mni Date Acquired: 8/16/2012 8:44:13 AM EDT Instrument Method Id: 8156 Injection Id: 8174

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

UPC2 Polymer Separations:- Triton X-100

Optimise separation for resolution (20 mins run time) See significant fine structure Ability to detect and monitor minor components and by-products


UPC2 Polymer Separations:- Condensation co-polymer synthesis

Bisphenol A and formaldehyde condensation co-polymer

HO OHOH

OH

H2C

HO

OH

CH2

Formaldehyde

Bisphenol A

Resin “Trimer”



AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00

Unknown m/z 227

Dimer m/z 467

Trimer m/z 707

UPC2 with UV and MS detection



AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50

Combined - SQ 1: MS Scan 1: 200.00-2000.00 ES-, Centroid, CV=Tune

227.0

271.0

Inten

sity

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

m/z200.00 250.00 300.00 350.00 400.00

Sample Mass spectrum

Bisphenol-A standard

Mass spectrum


UPC2 Polymer Separations:- Poly [Phenylglycidyl ether–co-formaldehyde]

Potentially complex mixture of oligomers depending on where linkage occurs between the units.

3 Potential dimers are shown below

O

O

O

O

O O

O

O

O

O

O

O

Phenylglycidyl ether

+ Formaldehyde

Dimers

(1)

(2)

(3)



Dim

er 1

Trim

er 1

Dim

er 2

D

imer

3

Trim

er 2

Tr

imer

3

Trim

er 5

Tr

imer

4

Trim

er 6

Trim

er 7

UPC2 with UV detection



Inte

nsity

0.0

5.0x107

1.0x108

1.5x108

2.0x108

Minutes0.00 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 15.00 16.00 17.00

Dimers m/z 312

Trimers m/z 474 Tetramers

m/z 636

m/z 404 m/z

402

O

O

O

O

O

O

O

O

m/z 404 m/z 402

UPC2 with MS detection


Conclusion

Convergence Chromatography makes SFC type separations – Routine – Reproducible – Accessible

3 critical separation mechanisms now accessible – Size Exclusion – Reverse Phase – Normal Phase

Orthogonal techniques and 2D will give us more information about polymer samples

SIMPLICITY

SIMILARITY

ORTHOGONALITY

Comprehensive 2D Separations

Convergence Chromatography - Turning SFC into a robust ... · ©2014 Waters Corporation 4...

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