Post on 27-Mar-2020
©2016 Waters Corporation 1
SFC Säulen für analytische und
preparative Anwendungen
Technology Symposium
Vienna, 25th October 2016
DI Verena Schmid
©2016 Waters Corporation 2
CHIRAL SEPARATIONS ACQUITY UPC2 Trefoil™
©2016 Waters Corporation 4
ACHIRAL SEPARATIONS Viridis Columns
©2016 Waters Corporation 5
New Torus™ 5 µm
Analytical and OBD™ Preparative
Achiral SFC Columns
October 2016
©2016 Waters Corporation 6
Torus Columns
A new particle technology designed specifically for SFC
Novel, two-stage bonding process yielding high density ligands
The second stage imparts the unique selectivity for each phase
– new interactions with analytes
US 6,686,035
US 7,223,473
Others patent pending
1.7 & 5 µm BEH Particles
High Density Bonding
O Si
O
O
O
OH
N
H
N
O Si
O
O
O
OH
N
H
O Si
O
O
O
OH
N
O Si
O
O
O
OH
OH
Torus 2-PIC
Torus DEA
Torus DIOL
Torus 1-AA
©2016 Waters Corporation 7
Torus™ Columns
Highlights
– Scale-up from 1.7 µm analytical to 5 µm preparative scale
– 4 innovative chemistries for SFC Applications
o 1.7 µm and 5 µm particles
– Excellent peak shape
– Added selectivity – wide range of compounds
– Improved Robustness
©2016 Waters Corporation 8
Time-0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
AU
-2.0e-3
0.0
2.0e-3
4.0e-3
6.0e-3
8.0e-3
1.0e-2
1.2e-2
Goldenseal_Extract-01_2016-09-14_19x150mm-1w014a-05_MeOH AmOH_02_2PIC_800µL 2: Diode Array 220
Range: 6.672e-1
1.05
1.99
1.64 2.882.28 2.48
3.02
3.84
4.024.32
1
3 4
Scale from Torus Analytical to Achiral Preparative SFC Columns Torus 2-PIC 1.7 µm to 5 µm Scale-up
Goldenseal Extract: 1. Canadine 2. Hydrastine 3. Isocorypalmine 4. Methyl Hydrastine 5. Berberine
AU
0.000
0.028
0.056
0.084
0.112
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.000
0.028
0.056
0.084
0.112
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00
1 4
3
Analytical SFC Column:
Torus 2-PIC 1.7 µm
3.0 x 50 mm
Analytical SFC Column:
Torus 2-PIC 5 µm
3.0 x 50 mm
Preparative SFC Column:
Torus 2-PIC 5 µm
19 x 150 mm
2 5
1 4
3
2 5
2 5
Analytical SFC Conditions
System: ACQUITY UPC2 with PDA
Columns: Torus 2-PIC 1.7 µm 3.0 x 50 mm
Torus 2-PIC 5 µm 3.0 x 50 mm
Co-Solvent: 20mM Ammonium Hydroxide in Methanol
Flow Rate: 1.2 mL/min
Gradient: 5 to 50% B in 2.85 min, hold at 50%
Column Temp: 30°C
Detection: UV @ 220 nm
ABPR Setting: 1625 (1.7 µm)
2250 (5 µm)
Injection Volume: 2.0 µL
Preparative SFC Conditions
System: SFC Prep 100q with PDA
Column: Torus 2-PIC 5 µm 19 x 150 mm
Co-Solvent: 20mM Ammonium Hydroxide in Methanol
Flow Rate: 100 mL/min
Gradient: 5 to 50% B in 5.14 min
Column Temp: 30°C
ABPR Setting: 120 bar (1740 psi)
Detection: UV @ 220nm
Injection Volume: 1.0 mL
©2016 Waters Corporation 9
Factors that control effective
scale-up in SFC
©2016 Waters Corporation 10
Outline
Introduction
– Importance of scale-up
– Different approaches
A rule-based approach for SFC
– Why LC approach may not always work in SFC
– Solution – an additional step in LC rule
Conclusion
©2016 Waters Corporation 11
Scaling up
©2016 Waters Corporation 12
“L/dp” rule for HPLC systems
Designing prep column dimensions and particle size
Stationary phase
• Select the same chemistry
Column specs
• Choose either L or dp
• Calculate the other from (L/dp)anal = (L/dp)prep
Linear velocity (u)
• Calculate u from (u x dp)anal = (u x dp)prep
Flow rate
• Calculate Q from, Q=u x A
L = column length, dp = particle size, u = linear velocity,
A = column void cross-section
©2016 Waters Corporation 13
Works for LC Method Transfer
AU
0.00
0.01
0.02
0.03
0.04
0.05
0.60 1.00 1.40 1.80 2.20 2.60 3.00 3.40 3.75
AU
0.00
0.01
0.02
0.03
0.04
0.05
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
XBridge C18 5 µm, 4.6 x 150 mm
XBridge C18 2.5 µm, 3.0 x 75 mm
ACQUITY BEH C18 1.7 µm, 2.1 x 50 mm
1485 psi
5555 psi
10433 psi
AU
0.00
0.01
0.02
0.03
0.04
0.05
Minutes 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
Courtesy of Jon Turner – April 2015
©2016 Waters Corporation 14
Outline
Introduction
– Importance of scale-up
– Different approaches
A rule-based approach for SFC
– Why LC approach may not always work in SFC
– Solution – an additional step in LC rule
Conclusion
©2016 Waters Corporation 15
Outline
Introduction
– Importance of scale-up
– Different approaches
A rule-based approach for SFC
– Why LC approach may not always work in SFC
– Solution – an additional step in LC rule
Conclusion
©2016 Waters Corporation 16
Effect of Pressure Difference on LC scale-up
LC scale-up – minimum effect from pressure changes
Nearly incompressible fluid
uAnal > uPrep
DP
Column length
DP
Column length
Dr
Column length
Dr
Column length
Analytical Prep
Analytical
Prep
Matching density profile
©2016 Waters Corporation 17
Effect of Pressure Difference on SFC scale-up
SFC scale-up – effect from pressure changes
Compressible fluid
uAnal > uPrep
DP
Column length
DP
Column length
Dr
Column length
Dr
Column length
Analytical Prep
Analytical Prep
Different density profiles
©2016 Waters Corporation 18
Caffeine, Carbamazepine, Uracil, Hydrocortisone, Prednisolone, and Sulfanilamide
AU
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0.15
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0.25
0.30
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
1
2,3
6
4
5
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
1
2
6
4
5
3
4.50 5.00
Minutes
Higher Density
Mobile Phase
Lower Density
Mobile Phase
Different density = different thermodynamic interactions = different retentions
2.1 x 150 mm 5µm
BEH 2-EP,
1.4 mL/min
ABPR = 103.5 bar
Temp: 40°C
What it means?
To apply LC scaling
rules,
address the density
difference 1.7 µm
5 µm
Effect of Density Changes
2.1 x 150 mm 1.7µm
BEH 2-EP,
1.4 mL/min
ABPR = 103.5 bar
Temp: 40°C
C. Hudalla et. al. Waters Application Note. 720004818en
©2016 Waters Corporation 19
Addressing Density Difference
Ideal situation – matching density profiles
Simpler approach - matching average density
Analytical column
Dr
Column length
Prep column
Dr
Column length
Analytical column Dr
Column length
Prep column Dr
Column length
Average density
Average density
©2016 Waters Corporation 20
Addressing Density Difference
SFC System Scheme
Any changes upstream of ABPR can change
chromatography!
Set: ABPR
B
Column
Injector Detector
C
O2
ABPR = Automatic Back Pressure Regulator
©2016 Waters Corporation 21
How to manipulate Ave P?
Assuming linear pressure drop – Calculate average pressure
Manipulating ABPR pressure, match average pressure
Analytical column
DP
Column length
Prep column
DP
Column length
Analytical column
DP
Column length
Prep column
DP
Column length
Increased ABPR pressure
System P ABPR P
System P ABPR P
Ave P
Ave P
©2016 Waters Corporation 22
Simplified - manipulate Ave P!
Sensor = pump outlet Sensor = regulator inlet
Back pressure
regulator
Assume linear pressure drop across the entire range
Implementable
Take simple average pressure
across the system
SystemP
ABPRP
2
ABPRSystem
Average
PPP
©2016 Waters Corporation 23
Scale-up Steps for SFC
Stationary phase
• Select the same chemistry
Column specs
• Choose either L or dp
• Calculate the other from (L/dp)anal = (L/dp)prep
Linear velocity (u)
• Calculate u from (u x dp)anal = (u x dp)prep
Flow rate
• Calculate Q from, Q=u x A
Average Density
• Adjust ABPR to ensure same average pressure
L = column length, dp = particle size, u = linear velocity,
A = column void cross-section
©2016 Waters Corporation 24
Step by Step.
©2016 Waters Corporation 25
Effect of Lower Density
©2016 Waters Corporation 26
Effect of Lower Density
©2016 Waters Corporation 27
Density Profiles
©2016 Waters Corporation 28
Use of Average Density
©2016 Waters Corporation 29
Use of Average Density
ABPR = Automatic Back Pressure Regulator
©2016 Waters Corporation 30
Impact of Flow Rate
©2016 Waters Corporation 31
Impact of Flow Rate
©2016 Waters Corporation 32
Regarding Particle Size & Prep Practice
©2016 Waters Corporation 33
Regarding Particle Size & Prep Practice
©2016 Waters Corporation 34
Professional Scale Up
©2016 Waters Corporation 35
Conclusion
A simplified method-transfer rule in SFC is developed
Average pressures in SFC and UPSFC systems should be the
same
Applicable over a wide range of standard conditions used in
SFC/UPSFC operations
©2016 Waters Corporation 36
Links and Literature
upc2.waters.com
All Application Notes:
http://www.waters.com/waters/promotionDetail.htm?id=13477
1024&locale=en_US
YouTube Channel:
https://www.youtube.com/playlist?list=PL6yA4jv5tA-
k5q6M391V0_yuFesh_eywJ
UPC2 Strategy for Scaling from Analytical to Preparative
SFC Separations (LiteraturNr. 720004818EN)
©2016 Waters Corporation 37
Acknowledgements
Waters Corporation
– Abhijit Tarafder
– Jason Hill
– Tom Swann
– Steve Collier
©2016 Waters Corporation 38
THANK YOU FOR YOUR ATTANTION!
QUESTIONS?!