Convergence Chromatography - Turning SFC into a robust ... · ©2014 Waters Corporation 4...
Transcript of 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
©2014 Waters Corporation 2
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.
©2014 Waters Corporation 3
Evolution of Separation Technology
Gas Chromatography Convergence Chromatography
GC
Capillary GC
HPLC
UPLC
SFC
UPC2
Liquid Chromatography
©2014 Waters Corporation 4
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
©2014 Waters Corporation 5
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
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Driving UPC2 performance
–Management of supercritical fluids
–Selectivity that can be addressed
–Innovative chemistries
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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
©2014 Waters Corporation 8
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
©2014 Waters Corporation 9
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
©2014 Waters Corporation 10
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
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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
©2014 Waters Corporation 12
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
©2014 Waters Corporation 13
UPC2 Configuration for MS
UV Detector
Make-Up Pump
Convergence Manager MS
©2014 Waters Corporation 14
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
©2014 Waters Corporation 15
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
©2014 Waters Corporation 16
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.
©2014 Waters Corporation 17
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)
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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
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Oligomeric Materials
©2014 Waters Corporation 20
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
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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
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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”
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UPC2 Polymer Separations:- Condensation co-polymer synthesis
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
©2014 Waters Corporation 24
UPC2 Polymer Separations:- Condensation co-polymer synthesis
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
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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)
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UPC2 Polymer Separations:- Poly [Phenylglycidyl ether–co-formaldehyde]
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
©2014 Waters Corporation 27
UPC2 Polymer Separations:- Poly [Phenylglycidyl ether–co-formaldehyde]
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
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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