Part III – Polar Compound Retention Supercritical Fluid ... · Part III – Polar Compound...
Transcript of Part III – Polar Compound Retention Supercritical Fluid ... · Part III – Polar Compound...
©2015 Waters Corporation 1
Part III – Polar Compound Retention
Supercritical Fluid Chromatography (SFC)
Waters Meet-The-Experts Webinar December 2, 2016
Isabelle François, PhD, UPC²/SFC & Strategic Separations Business Development Europe & India
©2015 Waters Corporation 2
Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
Polarity limits of chromatographic techniques
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Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
Polarity limits of chromatographic techniques
WebinarPart 1
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Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
Polarity limits of chromatographic techniques
WebinarPart 2
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Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
Polarity limits of chromatographic techniques
WebinarToday!
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Fundamentals on supercritical fluid chromatography (SFC)
Practical aspects
– How does an SFC instrument work? – Parameters which influence separation
Application examples with focus on:
– Retention for polar compounds – Orthogonality in comparison to reversed phase liquid chromatography (RPLC)
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Separation Technology Overview
Gas Chromatography
Liquid Chromatography
Supercritical Fluid Chromatography
Separation achieved by a temperature gradient
•High efficiency [N] • Virtually no limitation to column length
•Limited selectivity [α]
• Limited stationary phase options
Separation achieved by a solvent gradient
•Limited efficiency [N] • Limited due to pressure drop across column
• High selectivity [α] • Different modes: reversed-phase, normal-phase, SEC, IEX, affinity, ion pair, HILIC, GPC…etc.
Separation achieved by density/solvent gradient
•High efficiency [N] • Low viscosity allows longer columns and/or smaller particles
•High selectivity [α]
• Wide variety of stationary phase and mobile phase (modifier / additive) • Pressure and temperature as additional parameters to alter selectivity
GC
LC
SFC
©2015 Waters Corporation 8
History of SFC
• Introduced by Klesper et al. in J. Org. Chem. ‘High Pressure Gas Chromatography Above Critical
Temperatures’ • Initially performed on capillary columns
• First capillary SFC available on the market (Hewlett-Packard) based on work of Berger and Gere • CO2 only – density gradients • GC-like with applications limited to non polar compounds
• SFC is involved in the ‘green chemistry’ global effort • Acceleration of instrumental developments • 2012 – Waters introduces UPC²
• Extended to packed column SFC with improved and optimized instrumentation
• Introduction of polar modifier to CO2 • LC-like with development of applications for more polar
compounds
©2015 Waters Corporation 9
Advantages of a Supercritical Fluid in Chromatography
Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland
©2015 Waters Corporation 10
Why CO2 ?
Chromatographic technique similar to HPLC – Was introduced to replace NP -> unpolar solvent had to be mimicked by
supercritical fluid -> CO2 has very low dipole
Mobile phase is supercritical fluid + one or more co-solvents – CO2 is miscible with all organic solvents – CO2 is the most common supercritical fluid – MeOH is the most common co-solvent
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
©2015 Waters Corporation 11
Why CO2 ?
CO2 reaches supercritical state at 31.1°C and 73.8 bar – Its physical state can be easily manipulated
CO2 is non toxic, non flammable
CO2 is chemically pure, stable and non-polar solvent
CO2 is compatible with LC detectors
CO2 is as a Green Solvent
– Environmentally neutral: • Recovered e.g. from industrial and fermentation plants
– Avoids the production of CO2 that would have been generated from disposal of the solvents it replaces
– Less time and energy are used to evaporate fractions to get to pure analytes as CO2 is a gas at room temperature
Interesting for prep scale!
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CO2 Phase Diagram Critical point of CO2 Tc = 31.1°C Pc = 73.8 bar
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CO2 Phase Diagram Critical point of CO2 Tc = 31.1°C Pc = 73.8 bar
But ... Almost all modern SFC is not supercritical!
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Convergence Chromatography
Supercritical Fluid Chromatography
UPC²
SFC
Super Fluid Chromatography
Enhanced Fluidity Chromatography
Science Fiction Chromatography
Separations Facilitated by Carbon dioxide Caroline West @ HPLC2015 SFC short course
Unified Chromatography
SFC - What’s in a name?
©2015 Waters Corporation 15
What is SFC? Supercritical Fluid Chromatography? Convergence Chromatography?
SFC is like HPLC except the primary mobile phase is supercritical CO2 instead of an aqueous solution – CO2 is much less polar than water (similar to heptane) – Elution order is reversed (non-polar compounds elute first) – Organic solvent is used as a strong solvent (e.g. methanol, ethanol) - Modifier – Typical conditions are:
o Gradients go to typically 40-50% of modifier o Setting of back pressure is required o Flow rates are typically high in comparison to (UP)LC
• 1.5 – 2 mL/min for 3 mm id x 1.7 µm dp columns • Up to 4 mL/min for 4.6 mm id x 5 µm dp columns
©2015 Waters Corporation 16
• Column: Polar - SiO2, diol … • Mobile phase: (A) Heptane; (B) IPA Mostly isocratic • No MS compatibility
NPLC SFC RPLC
SFC Comparison with LC
• Column: Apolar – C18 • Mobile phase: (A) H2O; (B) MeOH Mostly gradient • MS compatibility
• Column: Polar - SiO2, diol, EP … Apolar – Cyano, C18 ... • Mobile phase: (A) CO2 ; (B) MeOH Mostly gradient • MS compatibility
• P and T as finetuning parameters – density control
©2015 Waters Corporation 17
SFC Comparison with LC
SFC
Like NPLC
Can provide identical separations but with ... – Better reproducibility – Fully MS compatible – Much faster — no long equilibration times – Minimal use of toxic organic solvents – Amenable to gradients (not only isocratic)
Orthogonal to RPLC
Typical RP applications can also be done but ... – SFC provides different/orthogonal separation – Similar reproducibility and robustness as UPLC – Fast analyses and equilibration – Ideal for scale up
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Similar to UPLC – from hardware as well as operational standpoint, both fully MS compatible
ACQUITY UPC² ACQUITY UPLC Xevo TQD
SFC Instrumentation
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ACQUITY UPC² ACQUITY UPLC Xevo TQD
Similar to UPLC – from hardware as well as operational standpoint, both fully MS compatible
SFC Instrumentation
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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
How does it work? What is different? UPC² - Instrumentation
©2015 Waters Corporation 21 21
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
How does it work? What is different? UPC² - Instrumentation
©2015 Waters Corporation 22 22
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
How does it work? What is different? UPC² - Instrumentation
©2015 Waters Corporation 23
Flow rate
Additives
Diluent
Co-solvent/ modifier
Additives
Injection solvent Temperature
Back pressure
Flow rate
SFC Column
UPC² method development
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Flow rate
Diluent
SFC-MS
Make-up solvent
Additives Flow rate
Interface
UPC²-MS method development
©2015 Waters Corporation 25
Stationary phases
2-ethylpyridine
Triazole
Amide
Silica
Propanediol
Aminopropyl
Cyanopropyl
PGC
Pentafluorophenyl
Phenylpropyl
Phenylhexyl
C4
C8
C18
RP-HPLC
SFC
Hig
h Lo
w
Polarity in SFC
NP-HPLC & HILIC
Solvents
Alkanes
Toluene
Dichloromethane
Chloroform
Acetone
Dioxane
THF
Ethyl acetate
DMSO
Acetonitrile
Isopropanol
Ethanol
Methanol
Water
Solv
ent
Stro
ng
Solvent strength with Supercritical
CO2
NP-HPLC
RP-HPLC & HILIC
UPC² method development
©2015 Waters Corporation 26
Binary Solvent Manager: – Mobile phase: o A: Supercritical CO2 o B: Methanol (MeOH)
– Flow rate: 1.8 mL/min – End time: 7 min – Gradient: see table
Column: – ACQUITY UPC² BEH 100 mm L x 3 mm i.d. x 1.7 µm dp – T = 40°C
Sample manager: – Strong wash: IPA – Weak wash: MeOH – Loop volume: 10 µL – Injection volume: 1 µL
Convergence manager: – P (outlet, APBR): 130 bar PDA: – Acquisition rate: 10 or 20 Hz
UPC² method development Typical method
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– Column Selection o Stationary phase (interaction type)
• Achiral • Chiral
– Mobile Phase o Composition and type modifier o Isocratic/Gradient Mode o Additive o Flow rate
– Solute Properties – Pressure/Temperature – Density
A. Tarafder and I. François – ISC2016 – SFC Short Course
UPC² method development Parameters
©2015 Waters Corporation 28
– Column Selection o Stationary phase (interaction type)
• Achiral • Chiral
– Mobile Phase o Composition and type modifier o Isocratic/Gradient Mode o Additive o Flow rate
– Solute Properties – Pressure/Temperature – Density
UPC² method development Parameters
©2015 Waters Corporation 29
Column has most influence on the separation SFC holds high orthogonality within the technology itself
: CSH PFP
AU
0.000
0.012
0.024
0.036
0.048
: HSS C18 SB
AU
0.000
0.012
0.024
0.036
0.048
: BEH HILIC
AU
0.000
0.012
0.024
0.036
0.048
: 2-EP
AU
0.000
0.012
0.024
0.036
0.048
Minutes0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00
ACQUITY UPC2 BEH 2-EP 1.7 µm
ACQUITY UPC2 BEH 1.7 µm
A B C(1,2)
D
G H
9
F
A B C
D
G H
9
F
A B
C D
G H
9 F
A B C
D G
H*
9
F
Unknown [M+H] = 266
Unknown [M+H] = 272
*Imp “H” is a broad peak under the unknown
Unknown [M+H] = 226
Unknown [M+H] = 226
ACQUITY UPC2 CSH Fluoro-Phenyl 1.7 µm
ACQUITY UPC2 HSS C18 SB 1.7 µm
UPC² method development Column
Identical methods and modifier, just changing the column!
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©2015 Waters Corporation 31
©2015 Waters Corporation 32
©2015 Waters Corporation 33
C. West et al., The Column 10 (2014) 2-8
Column #17
Column #26
Column #40
Different selectivity
1
2 3 4
5
0 1 2 3 4
analysis time (min)
UPC² method development Column
©2015 Waters Corporation 34
S. Khater et al. J. Chromatogr.A 1319 (2013) 148-159
CO2-MeOH 90:10 (v/v) 25°C 150 bar 3 mL/min
BEH silica HSS C18 SB
CSH Fluoro-Phenyl
BEH Shield RP18
BEH 2-EP
e
s
a -b
v
UPC² method development Column
©2015 Waters Corporation 35
Torus DIOL • high density diol Torus 2-PIC • 2-picolylamine selector Torus DEA • diethylamine selector Torus 1-AA • aminoanthracene selector
O SiO
OO
OHOH
O SiO
OO
OHN
O SiO
OO
OHNH
O SiO
OO
OHNH
N
US 6,686035 US 7,223,473 Others patent pending
UPC² method development Column – Torus Column Technology
©2015 Waters Corporation 36
BZD 1. Diazepam 2. Midazolam 3. Flunitrazepam 4. Lormetazepam 5. Flurazepam 6. Alprazolam 7. Triazolam 8. Clorazepate 9. Bromazepam 10. Nitrazepam 11. Clonazepam 12. Oxazepam 13. Lorazepam 14. Clozapine 15. Olanzapine
N
N R2
R3 R4
R5
R1
150 x 4.6mm, 5µm 30bar
2 1 3
4
5 + 6 7
8 9
10 + 11
12 13 + 14
15 Pc = 63
450 x 4.6mm, 5µm
2 1 3
4
6 7 8 9 11 12
13 14
80bar
5 10 15
Pc = 108
Analytical conditions : CO2-MeOH gradient mode, 4mL/min ; PrincetonSFC 2EP 150 x 4.6mm, 5µm; Oven temp @ 40 C ; BPR @ 150bar ; UV @ 220nm
Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
Reduced mobile phase viscosity and low pressure drops allow easy serial coupling and efficiency increase
UPC² method development Column length
©2015 Waters Corporation 37
Data courtesy of Davy Guillarme, Alexandre Grand-Guillaume Perrenoud, Jean-Luc Veuthey LCAP University of Geneva, Switzerland
Implementation of sub 2 µm particles aids in separation like in UHPLC Pressure increase is mild due to reduced viscosity of mobile phase From practitioner’s standpoint: go as fast as the instrument allows you! Van
Deemter is flat at elevated flow rates
UPC² method development Column particle size
©2015 Waters Corporation 38
– Column Selection o Stationary phase (interaction type)
• Achiral • Chiral
– Mobile Phase o Composition and type modifier o Isocratic/Gradient Mode o Additive o Flow rate
– Solute Properties – Pressure/Temperature – Density
UPC² method development Parameters
©2015 Waters Corporation 39
C. West, Current Anal. Chem. 10,1 (2014) 99-120
0 20 40 60 80 100
Proportion of research articles where modifier is used (%)
MeOH
EtOH
iPrOH
nPrOH
ACN
Other
UPC² method development Mobile phase
Methanol is the most widely applied modifier in SFC today
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Minutes 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
AU
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0.30
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0.20
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0.20
AU
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0.05
0.10
17% MeOH
15% MeOH
13% MeOH
11% MeOH
9% MeOH
7% MeOH
3.0 mL/min; 50°C; 1800 psi backpressure; 3.0x150 mm BEH 5 µm; ACQUITY UPC2
UPC² method development Mobile phase – isocratic mode
©2015 Waters Corporation 41
2 25% MeOH over 2.5min
2 35% MeOH over 2.5min
2 45% MeOH over 2.5min
3x100mm 1.7µm Torus Diol column
AUAU
AU
Time0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50
0.0
5.0e-2
1.0e-1
1.5e-1
0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.500.0
5.0e-2
1.0e-1
1.5e-1
0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.500.0
5.0e-2
1.0e-1
1.5e-1
Increasing co-solvent percentage in gradient methods Reduces elution and analysis time Provides peak sharpening and higher sensitivity In contrast to NPLC, gradients can be easily applied in SFC!!
UPC² method development Mobile phase – gradient mode
©2015 Waters Corporation 42
15
14 5 9
1312 3 6
1110
164 2
8
7
AU
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Minutes1.00 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
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63 11
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2
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Modifier MeOH (0.1% HCOOH)
Modifier MeOH/ACN 1/1 (0.1% HCOOH)
Courtesy of David Clicq, UCB, Belgium
3x100 mmx1.7µm ACQUITY UPC² BEH
UPC² method development Mobile phase – solvent selection
©2015 Waters Corporation 43
Evaporates quickly from bottle
43
– As in UPLC, additives are added to enhance peak shape – Typical SFC additives and concentrations are:
Additive Concentration
Basic NH4OH 0.1%
Ammoniumacetate / formate 10mM
Triethylamine 0.1%
Acidic Formic, acidic 0.1%
TFA 0.5%
Neutral Water 2%
Not compatible with MS
Cannot be too high as phase demixing might occur
UPC² method development Mobile phase additives
©2015 Waters Corporation 44 Grand-Guillaume Perrenoud et al., J. Chromatogr. A, 1262 (2012) 205
BEH 2-EP
N N N
SiO
SiO
SiO
Without additives With 20 mM NH4OH
0 < pKa < 4
6 < pKa < 8
8 < pKa < 11
Be careful with column resistance to strong bases !
UPC² method development Mobile phase additives
©2015 Waters Corporation 45
– Column Selection o Stationary phase (interaction type)
• Achiral • Chiral
– Mobile Phase o Composition and type modifier o Isocratic/Gradient Mode o Additive o Flow rate
– Solute Properties – Pressure/Temperature – Density
UPC² method development Parameters
©2015 Waters Corporation 46
AU
AU
Minutes 1.4 1.5 1.6 1.7 1.8 2.1 2.2 1.3
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Butylparaben dissolved in diluents listed at right
Injection volumes:
0.5 (black), 1.0 (red), 1.5 (blue), 2.0 (green), 2.5 (light blue), 3.0 (pink), and 4.0 µL (brown).
AU
0.0
0.1
0.2
0.3
0.4
0.5
0.6 A
30/70 heptane/THF
B
Isopropanol
C
Methanol
Notice, there is some peak distortion for injection volumes of 0.5 µL!
Conditions Column: BEH 2-EP 5 µm 2.1x150 mm Method: 97/3 CO2/MeOH; 2 mL/min; 40°C; 2000 psi ABPR; 254 nm (350-450 nm compensation)
2.0 1.9
– Injection in weak solvent ensures optimal injection conditions
– For SFC, best are CO2-like solvents
UPC² method development Injection solvent
©2015 Waters Corporation 47
– Injection conditions in SFC are important – BUT ... Practicality is too!
– Solubility – Ease in sample prep – Small volumes of ‘polar’ solvents can be injected (even traces of
water) – Initial conditions of chromatographic separation can be adjusted
to force focusing at the head of the analytical column
UPC² method development Injection solvent
L. Novakova et al., Anal. Chim. Acta 853 (2015) 647-659
Doping agents in urine samples
©2015 Waters Corporation 48
– Column Selection o Stationary phase (interaction type)
• Achiral • Chiral
– Mobile Phase o Composition and type modifier o Isocratic/Gradient Mode o Additive o Flow rate
– Solute Properties – Pressure/Temperature – Density
UPC² method development Parameters
©2015 Waters Corporation 49
Courtesy of Eric Lesellier, University of Orléans, France
– Although pressure and temperature have significant impact on density, these parameters are typically only used for finetuning
– Both parameters have most impact in the low modifier regime and less at higher modifier percentages
– Typically, higher pressures and lower temperatures reduce analysis times and ensure peak sharpening
– Temperature has higher impact on density but pressure is quicker to change
UPC² method development Pressure / temperature
©2015 Waters Corporation 50
AU
0.110
0.120
0.130
0.140
0.150
0.160
0.170
0.180
Minutes9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
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
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
110 Bar
AU
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0.40
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Minutes7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00
AU
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
Minutes6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50
Original: 130 Bar AU
0.090
0.100
0.110
0.120
0.130
0.140
0.150
0.160
0.170
0.180
0.190
Minutes9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80
150 Bar
AU
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Minutes6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50
AU
0.110
0.120
0.130
0.140
0.150
0.160
0.170
0.180
Minutes9.10 9.20 9.30 9.40 9.50 9.60 9.70 9.80 9.90 10.00 10.10 10.20 10.30 1
Courtesy of David Clicq, UCB, Belgium
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
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
©2015 Waters Corporation 51
AU
0.00
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0.40
0.50
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
40°C
50°C
Courtesy of David Clicq, UCB, Belgium
UPC² method development Temperature
©2015 Waters Corporation 52
©2015 Waters Corporation 53 T. Berger, B. Berger, SFC 2012, Brussels, Belgium
SFC Applications
Status before UPC² launch in 2012 ...
©2015 Waters Corporation 54
Courtesy of A. Grand-Guillaume Perrenoud, D. Guillarme, Pr J-L. Veuthey, University of Geneva
UPC² Applications Status today and beyond …
©2015 Waters Corporation 55
Normal phase
Compounds with no retention in RPLC Compounds degrading
in H2O Orthogonal in
comparison to C18
Future scale-up
Vitamins
Simplified sample prep
Chiral = no-brainer Structural Isomers
UPC² Applications
Alternative to HILIC
Easy MS coupling!
©2015 Waters Corporation 56
Compounds with no retention in RPLC
Compounds degrading in H2O
UPC² Applications
Alternative to HILIC
Might be challenging to find alternative, more retentive RP column
As aqueous mobile phases are used, compounds might degrade at injection
HILIC is great to achieve higher retention, but column regeneration times are typically quite long
UPC² delivers instantly a different separation and many columns to select
UPC² uses non-aqueous mobile phases. When MeOH causes hydrolysis too, ACN can be used as modifier
UPC² is characterized by extremely fast separations and column regeneration times
UPC² is a great orthogonal tool to have in your laboratory when addressing challenging RP separations!
©2015 Waters Corporation 57
Separation of isomers
UPLC – 1 peak
UPC² – 2 peaks
UPLC – low retention + tailing
UPC² - higher retention + sharp peaks
Compounds with no retention in RPLC
Courtesy of Arjen Gerssen, Rikilt, Netherlands
Structural Isomers
Problematic compounds
in UPLC
Easy MS coupling!
©2015 Waters Corporation 58
Adenosine
Uracil
Sulfapyridine
Log DpH3 of -0.7
Log DpH3 of 0.0 Log DpH3 of -2.1
SFC hybrid silica HILIC bare silica
at pH 6
Very good retention of hydrophilic compounds in SFC.
Complementary selectivity.
RPLC at pH 9
SFC hybrid silica
HILIC bare silica at pH 6
RPLC at pH 9
SFC hybrid silica
HILIC bare silica at pH 6
RPLC at pH 9
2.0 mL of ACN
HILIC SFC 1.2 mL of
MeOH
Compounds with no retention in RPLC
Alternative to HILIC
Slide courtesy of D. Guillarme, University of Geneva J. Sep. Sci. 36 (2013) 3141-3151
©2015 Waters Corporation 59 Courtesy of Jason Hill, Waters Corporation
LSU
0.00
120.00
240.00
360.00
480.00
Minutes 0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60 4.00
1.7µm, 3 x 100mm 2PIC 1.4mL/min 35% MeOH 90C 3500psi ABPR ELSD detection
Alternative to HILIC
1. Fructose 2. Glucose 3. Sucrose 4.Lactose 5. Maltose
1
2
3
4 5
Food Sugar Mix
©2015 Waters Corporation 60
Courtesy of Michal Holcapek, University of Pardubice, Czech Republic
Workflow simplification
Lipidomics
Alternative to HILIC
Normal phase
©2015 Waters Corporation 61
Courtesy of Michal Holcapek, University of Pardubice, Czech Republic
Workflow simplification
Lipidomics
Alternative to HILIC
Normal phase
©2015 Waters Corporation 62
• Separation of 30 polar and nonpolar lipid class standards within 6 min!
Nonpolar lipids
Polar lipids
• Conditions: column UPC² ACQUITY BEH (100x3 mm, 1.7 µm, Waters), positive-ion ESI-MS, 60°C, modifier MeOH/water (99:1) + 30 mM ammonium acetate, flow 1.9 mL/min, ABPR 1800 psi, standards of lipid classes
M. Lísa, M. Holčapek, Anal. Chem. 87 (2015) 7187
UHPSFC/ESI-MS of All Lipid Classes
©2015 Waters Corporation 63
Analyte
1. Vit A Acetate
2. Vit E Acetate
3. Vit K1
4. Vit A Palmitate
5. Vit E Tocopherol
6. Lycopene
7. Vit E Succinate
8. β-Carotene
9. Vit D3
10. Lutein Simultaneous analysis of fat-soluble vitamins and carotenoids in 10 minutes
Analyte LC mode Time
Vit A NP 12 min
Vit E NP 30 min
Lycopene NP 10 min
β-Carotene NP 10 min
Vit D3 NP 20 min
Vit K1 RP 12 min
Lutein RP 10 min
7 HPLC methods with total time ≈ 2 hours
SFC
HPLC
Simplified sample prep
Easy MS coupling!
Replacing Multiple LC Methods
©2015 Waters Corporation 64
Vitamins in Food - Sample Workflow for HPLC
Infant Formula sample
Add D2 Int. Std. & Saponify
Hexane extraction
NPLC Analysis
RPLC Analysis Vitamin E
Vitamin A
Evaporate
Redissolve in MeOH
Evaporate
Redissolve in iPrOH
Semi-prep RPLC purification
Collect peaks
Evaporate
Redissolve in hexane NPLC Analysis
Vitamin D3
10 sample prep steps 3 analytical methods
3 instruments
Simplified sample prep
Easy MS coupling!
©2015 Waters Corporation 65
3 sample prep steps 1 analytical method
1 instrument
Infant Formula sample
Add D2 Int. Std. & Saponify
Hexane extraction
Concentrate
SFC Analysis
SFC Analysis
Vitamin E
Vitamins A & D3
Vitamins in Food - Sample Workflow for HPLC
Simplified sample prep
Easy MS coupling!
©2015 Waters Corporation 66
Orthogonal in comparison to C18
©2015 Waters Corporation 67
15
14 5 9
1312 3 6
1110
164 2
8
7
AU
0.00
0.20
0.40
0.60
Minutes1.00 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
Courtesy of David Clicq, UCB, Belgium
Pharma sample UCB on ACQUITY UPLC BEH C18 – 210 nm
Pharma sample UCB on ACQUITY UPC² BEH – 210 nm
Orthogonal in comparison to C18 Application at UCB – Comparison of current UPLC method
with UPC²
©2015 Waters Corporation 68
Courtesy of Alex Brien, Reach Separations, UK
Orthogonal in comparison to C18
Application at Reach Separations, UK Pharma CRO
UPLC
ACQUITY UPLC BEH C18
UPC²
ACQUITY TORUS 2-PIC
ADDITIONAL PEAK IN UPC²!
©2015 Waters Corporation 69
GC
LC
polarity
Log
MW
SFC
Which gap ? Application gap No Speed gap Yes Detection gap Yes ‘Mildness’ gap Yes Sustainability gap Yes
Hans-Gerd Janssen Unilever and professor @ University of Amsterdam, NL
Courtesy of Hans-Gerd Janssen, Unilever, Netherlands
UPC² Bridging the gap between LC and GC
©2015 Waters Corporation 70
Compound coverage in SFC!!
SFC does not have many unique applications. Chiral separation is (the only?) one. But SFC has many applications it can do ‘better’. SFC is faster, SFC is greener, Fine tuning is easier, etc. SFC can provide a frame of reference for GC: Do these labile compounds degrade? Are these adsorptive compounds lost? SFC has a very broad compound coverage.
Courtesy of Hans-Gerd Janssen, Unilever, Netherlands
UPC² Bridging the gap between LC and GC
©2015 Waters Corporation 71
SFC: broad distribution; LC: three series; GC: three series
Food emulsifier by NPLC, NP-SFC and GC
E3
E7
E7
SFC-MS
LC-MS
GC-MS
Compound coverage in SFC!
BPR gradient
1% modifier
BPR constant
Modifier gradient
Courtesy of Hans-Gerd Janssen, Unilever, Netherlands
UPC² Bridging the gap between LC and GC
©2015 Waters Corporation 72
Reduce Solvent Costs
Green Chemistry
Orthogo-nality
Increase Speed &
Throughput
Enantiomer isomer
Separations
Issues with Range of Polarity
Structural isomer
separations
Simplify Workflow
CONCLUSIONS
SFC / UPC²
©2015 Waters Corporation 73
Thank You!
You for your attention!
Acknowledgements
• Davy Guillarme, Alexandre Grand-Guillaume Perrenoud, Jean-Luc Veutthey, University of Geneva, Switzerland • Caroline West and Eric Lesellier, ICOA, France • David Clicq, UCB, Belgium • Hans-Gerd Janssen, Unilever, Netherlands • Alex Brien, Reach Separations, UK • Lucie Novakova, Charles University, Czech Republic • Michal Holcapek, University of Pardubice, Czech Republic • Arjen Gerssen, Rikilt, Netherlands