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©2012 Waters Corporation 1
Acquity UPSFC
Granada, 23 Febrero 2012
Alberto Méndez Applications Specialist
©2012 Waters Corporation 2
Remember Smaller Particles - Enabler of Productivity
©2012 Waters Corporation 3
SFC Column Efficiency Curves
2.5 µm XBridge™ HILIC, 3.0x50 mm
5.0 µm XBridge™ HILIC, 3.0x50 mm
3.5 µm XBridge™ HILIC, 3.0x50 mm
1.7 µm ACQUITY BEH HILIC, 3.0x50 mm
Isocratic Conditions CO2/Methanol 85:15 130 bar outlet pressure
©2012 Waters Corporation 4
Resolution Comparison
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
AU
0.00
0.05
0.10
0.15
0.20
0.25
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
1.7 µm
5 µm 3.0x100 mm Column 2 mL/minute CO2/Methanol Gradient
©2012 Waters Corporation 5
Effect of System Volume
0.46
4
0.51
3
0.62
0
0.64
6
0.70
0
0.82
6
0.85
50.
870
0.90
9
0.94
3
1.03
9
1.07
3
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.47
1
0.53
9
0.65
20.
670
0.89
8
0.92
60.
946
0.98
5
1.01
9
AU
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10
3.0x100 mm, 1.7 µm, at 3 mL/minute, CO2/MeOH gradient
non-optimized
optimized
©2012 Waters Corporation 6
Effect of System Volume
0.62
0
0.64
6
AU
0.00
0.02
0.04
0.06
0.65
2
0.67
0
AU
-0.010
0.000
0.010
0.020
0.030
0.040
0.050
Minutes0.590 0.600 0.610 0.620 0.630 0.640 0.650 0.660 0.670 0.680 0.690
non-optimized
optimized
©2012 Waters Corporation 7
ACQUITY UPSFC™ Design Objectives
Develop an Analytical SFC System based upon proven UPLC® technology
Purposeful Innovation: – Peltier cooling of BSM pump heads – Accurate, linear and precise injections – CO2 management and pressure control – Reduced system volume – Detection sensitivity
Meaningful Impact: – Total solution – Reduced Chiral/Achiral separation times – Reduced cost per injection – System validation
©2012 Waters Corporation 8
ACQUITY UPSFC™ Design Objectives
Column Manager CM-A
SFC PDA Detector
SFC Manager
Sample Manager - FL
SFC BSM Pump
©2012 Waters Corporation 9
18 Compounds Mixture 6 Overlaid Injections
3-B
enzo
ylpy
ridin
e - 1
.187
Cho
leca
lcife
rol -
1.4
77
6-H
ydro
xyfla
vano
ne -
1.76
6
4-N
itroa
nilin
e - 2
.179
Caf
fein
e - 2
.308
Theo
phyl
line
- 2.7
70
Car
bam
azep
ine
- 3.3
89
Est
radi
ol -
3.59
4 4,4'
-Bip
heno
l - 3
.695
Cor
tison
e - 4
.285
Pre
dnis
one
- 4.5
92
Ace
tam
inop
hen
- 4.8
56
Sul
fisox
azol
e - 5
.014
Hyd
roco
rtiso
ne -
5.29
2
Sul
fam
ethi
zole
- 5.
487
Pre
dnis
olon
e - 5
.647
Sul
faph
enaz
ole
- 5.8
15
4-A
min
ophe
nyl S
ulfo
ne -
7.18
5
AU
0.00
0.02
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
0.32
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
Consistent peak widths High resolution <0.4% RSD in RT
2.1 x 150 mm Viridis Hybrid 1.7 µm @ 1.20 mL/min @ 45 °C A=Carbon Dioxide B=Methanol UV 230 nm (compensated 400-500 nm) 2 µL injection Gradient 2% B to 16% B over 7 minutes
©2012 Waters Corporation 10
Pea
k1 -
2.53
9
AU
0.00
0.02
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
0.32
0.34
0.36
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
1.5% MeOH
Pea
k1 -
4.44
1
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Pea
k1 -
1.4
46
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
2.5% MeOH
Pea
k1 -
1.80
6
AU
0.00
0.02
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
0.32
0.34
0.36
0.38
0.40
0.42
0.44
0.46
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
2.0% MeOH
Mobile phase strength is a non-linear function of modifier concentration
The first small additions of modifier have a large effect on retention
1.0% MeOH
Precision at Low % Co-Solvent
Viridis Hybrid 2.1x150 mm @ 1 mL/min
©2012 Waters Corporation 11
1-Couma
rin - 0.8
40
2-Flavon
e - 1.116
3-Caffein
e - 1.247
4-Thymin
e - 1.584
5-Papave
rine - 1.6
64
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
Minutes0.00 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 1.40 1.50 1.60 1.70 1.80 1.90 2.00
1-Coumar
in - 0.788
2-Flavone
- 1.072
3-Caffeine
- 1.209
4-Thymin
e - 1.559
5-Papave
rine - 1.64
1
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
Minutes0.00 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 1.40 1.50 1.60 1.70 1.80 1.90 2.00
Gradient Performance
RT %RSD: <0.15 Area %RSD: <0.67
1-20% B back to 1% B
RT %RSD: <0.34 Area %RSD: <0.69
1.5-20% B back to 1.5% B
Viridis® BEH 2-Ethylpyridine, 3 x 100 mm, 1.7 µm 2.00 mL/min, 55 °C, 1958 psi (135 Bar) Injection Volume = 3 µL, 10 repeated injections PDA 280 nm compensated (500-600 nm) 20 Hz, TC = 0.2 sec
©2012 Waters Corporation 12
Linearity - 1-10µL injections AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0.100
0.110
0.120
0.130
0.140
0.150
0.160
Minutes1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60
Peak 1 - Coumarin Peak 2 - Flavone Peak 3 - Caffeine Peak 4 - Thymine Peak 5 - Papaverine
Column 4.6 x 150 mm Viridis Silica 2-EP, 5 um; Flow Rate : 3.00 mL/min; Column Temp = 55°C,A=CO2 B=Methanol 5-20% B Grad over 5 min followed by a return to 5% B over 1 min; ABPR = 1958 psi (135 Bar) PDA 280 nm compensated (500-600 nm) 20 Hz, TC = 0.2 sec
©2012 Waters Corporation 13
Linearity - 1-10µL injections
Coumarin: y = 43495x - 4326.9 R² = 0.9999 Flavone: y = 28758x - 2908 R² = 0.9999
Caffeine: y = 52213x - 5212.3 R² = 0.9999
Papaverine: y = 14608x - 2097.1 R² = 0.9994 Thymine: y = 26624x - 2774.5 R² = 0.9999
0
100000
200000
300000
400000
500000
600000
0 2 4 6 8 10
Are
a C
ou
nts
uL Injected
Coumarin
Flavone
Caffeine
Thymine
Papaverine
Linear (Coumarin)
Linear (Flavone)
Linear (Caffeine)
Linear (Thymine)
Linear (Papaverine)
©2012 Waters Corporation 14
ABPR Performance: “FINE” Control
0.54
8
1.24
3
2.85
9
4.65
6
0.55
3
1.25
9
2.90
6
4.73
4
0.55
1
1.25
2
2.88
4
4.70
0
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
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 4.20 4.40 4.60 4.80 5.00
ABPR @ 1980 psi ABPR @ 2000 psi ABPR @ 2020 psi
©2012 Waters Corporation 15
(Cathode)
(Anode)
(Glass board)
Organic Light Emitting Diode (OLED))
OLED Separation and Impurity Profiling
©2012 Waters Corporation 16
OLED Degradation Investigation with LC/UV/MS
TCTA
Flrpic
α-NHP
BAlq
Flrpic Structural Isomers
Normal Phase LC Separation
©2012 Waters Corporation 17
Standards Flrpic
1.01
9
1.36
0
1.46
3
1.61
9
1.93
5
2.74
6
3.62
2
4.12
4
AU
0.00
0.03
0.06
0.09
0.12
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Flrpic
N
FF
N
F
F
Ir
N
F
F
N
F
F
Ir
N
F
F
N
F
F
Flrpic Isomer
N
F
Ir
N
F
F
N
F
F
Flrpic -F
N
F
Ir
N
F
N
F
F
Flrpic -2F
Flrpic -3F
N
F
Ir
N
F
N
F
©2012 Waters Corporation 18
Standards and Spectra Flrpic and MS ES+ m/z Values
1.00
3
1.34
51.
448
1.60
01.
643
1.92
0
2.73
0
3.60
4
4.10
2
Inte
nsity
1.8x107
3.6x107
5.4x107
7.2x107
9.0x107
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
1.345 Peak 2 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune
743.93
745.90
Inte
nsity
0.0
9.0x105
1.8x106
2.7x106
3.6x106
m/z734.40 737.10 739.80 742.50 745.20 747.90 750.60 753.30 756.00 758.70
1.446 Peak 3 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune
743.97
745.91
Inte
nsity
0.0
1.2x106
2.4x106
3.6x106
4.8x106
m/z738.00 740.00 742.00 744.00 746.00 748.00 750.00 752.00 754.00
1.601 Peak 4 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune
743.90
745.90
746.97
Inte
nsity
0
1x106
2x106
3x106
4x106
m/z739.20 741.30 743.40 745.50 747.60 749.70 751.80 753.90 756.00
1.922 Peak 6 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune
761.97
763.93
765.08
Inte
nsity
0.0
7.0x106
1.4x107
2.1x107
2.8x107
m/z757.80 759.60 761.40 763.20 765.00 766.80 768.60 770.40 772.20
2.731 Peak 7 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune
743.96
745.95
747.05
Inte
nsity
0.0
6.0x106
1.2x107
1.8x107
2.4x107
m/z738.00 740.00 742.00 744.00 746.00 748.00 750.00 752.00 754.00 756.00
3.605 Peak 8 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune
725.97
727.94
729.01
Inte
nsity
0.0
1.6x106
3.2x106
4.8x106
6.4x106
m/z722.40 723.80 725.20 726.60 728.00 729.40 730.80 732.20 733.60 735.00
4.101 Peak 9 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune
804.01
805.99
806.96
Inte
nsity
0.0
1.2x106
2.4x106
3.6x106
4.8x106
m/z802.80 803.70 804.60 805.50 806.40 807.30 808.20 809.10 810.00
1.004 Peak 1 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune
761.95
763.92
765.01
Inte
nsity
0.0
7.0x106
1.4x107
2.1x107
2.8x107
m/z759.60 760.80 762.00 763.20 764.40 765.60 766.80 768.00 769.20
m/z=763.9 m/z=763.9 m/z=746.0 m/z=727.9 m/z=706.0
m/z=745.9 m/z=745.9 m/z=763.9
ES+
©2012 Waters Corporation 19
Comparison MS/UV Flrpic Mixture
1.01
9
1.36
0
1.46
3
1.61
9
1.93
5
2.74
6
3.62
2
4.12
5
AU
0.00
0.06
0.12
0.18
0.24
1.00
3
1.34
5
1.44
8
1.59
8
1.92
0
2.73
0
3.60
4
4.10
2Inte
nsity
1.8x107
3.6x107
5.4x107
7.2x107
9.0x107
1.91
9
2.73
1
4.10
4Inte
nsity
0.0
6.0x106
1.2x107
1.8x107
2.4x107
Minutes0.00 0.55 1.10 1.65 2.20 2.75 3.30 3.85 4.40 4.95
Conditions: 3.0 x 100mm Viridis BEH 2-EP, 1.7µm; OLED Mixture prepared at 0.1mg/mL in THF; Inj. Volume:2 µL; Temperature:60°C; ABPR: 130 bars; Flow: 2mL/min, CO2, MeOH (ammonium formate) 10 to 25% gradient
©2012 Waters Corporation 20
Major Constituents in OLED Mixture
1.00
9
1.42
1
1.92
0
2.02
8
2.47
1
AU
0.000
0.025
0.050
0.075
0.100
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Flrpic Area = 0.4% Rt = 0.1%
TCTA Area = 1.1% Rt = 0.1%
Balq (a) Area = 1.1% Rt = 0.1%
α-NHP Area = 0.4% Rt = 0.1%
*Minor impurities of 1% or lower yielded values typical of impurity analysis: Area %RSD < 5% Rt %RSD < 0.2%
©2012 Waters Corporation 21
There are hundreds of FSV formulations on the market
Vitamin A Only
Vitamins A + D
Vitamin E
Vitamin K
Vitamins K + D
Vitamin D Only
Fat Soluble Vitamins
©2012 Waters Corporation 22
1.20
71.
304
Retin
yl A
ceta
te -
1.79
71.
938
2.44
0
3.21
8Re
tinol
Pal
mita
te -
3.35
4
Retin
ol -
3.82
6
4.64
4
5.80
7
6.37
6
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
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
Vitamin A Forms
©2012 Waters Corporation 23
0.29
7 0.41
1
0.79
6
0.94
3
1.15
51.
207
1.30
4
1.51
61.
586 1.
797
1.93
8
2.15
3 2.44
0
2.88
6
3.02
6
3.21
83.
268
4.05
54.
144
4.39
14.
493
4.64
44.
701
4.93
75.
064
5.45
8
5.67
9
5.80
7
6.01
0
6.17
7
6.37
6
6.69
3
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
Vitamin A Forms (Zoom)
©2012 Waters Corporation 24
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Fat Soluble Vitamins
Red - MS TIC (ESi+) Blue - UV at 317 nm Black - UV at 262 nm
©2012 Waters Corporation 25
Chiral Method Development with MS
Speeding up chiral method development can be achieved using Mass Spectrometry
Thanks to the selectivity of MS detection, several chiral mixtures can be injected together and the chiral resolution is confirmed using the MS data
©2012 Waters Corporation 26
IC, 4.6 x 150mm, 5 µm 3 ml/min CO2 / Methanol 5-45% - 4 min 45% - 1 min 45-5% - 0.5 min 5% - 0.5 min 40 °C, 120 bar Oxfendazole & Pantoprazole sodium hydrate (0.2 mg/mL)
Chiral Method Development with MS
©2012 Waters Corporation 27
Conclusions
SFC is a highly orthogonal analytical tool, compatible with many detection techniques, and applicable in any field
Technique of choice for normal phase methods replacement – uses up to 80% less solvent, low solvent cost and disposal
UPSFC™ , designed with proven UPLC® technology, provides measurable increases in productivity for UV, MS and ELSD based work flows
UPSFC™ capability with LC familiarity
Complete solution for SFC analysis including chemistry, software, service, and support
©2012 Waters Corporation 28
Thank You for Your Attention and…
Many thanks to: -Michael D Jones -Andy Aubin -Magdalena Abad -Rui Chen -Stéphane Dubant -Isabelle Durieux