SFC Säulen für analytische und preparative Anwendungen

SFC Säulen für analytische und preparative Anwendungen Technology Symposium Vienna, 25 th October 2016 DI Verena Schmid 2016 Waters Corporation 1

CHIRAL SEPARATIONS ACQUITY UPC 2 Trefoil 2016 Waters Corporation 2

ACHIRAL SEPARATIONS Viridis Columns 2016 Waters Corporation 4

New Torus 5 µm Analytical and OBD Preparative Achiral SFC Columns October 2016 2016 Waters Corporation 5

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 O O Si O O OH H N N Torus 2-PIC High Density Bonding O O Si O O OH N Torus DEA 1.7 & 5 µm BEH Particles O O Si O O OH OH Torus DIOL O O Si O O OH H N Torus 1-AA US 6,686,035 US 7,223,473 Others patent pending 2016 Waters Corporation 6

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 7

AU AU AU Scale from Torus Analytical to Achiral Preparative SFC Columns Torus 2-PIC 1.7 µm to 5 µm Scale-up 0.112 0.084 0.056 0.028 0.000 0.112 0.084 0.056 0.028 0.000 0.00 0.50 1.00 1.50 2.00 2.50 3.00 Minutes 1 2 5 1 4 3 2 5 3 4 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 Analytical SFC Conditions System: ACQUITY UPC 2 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 0.00 0.50 1.00 1.50 2.00 2.50 3.00 Minutes Goldenseal_Extract-01_2016-09-14_19x150mm-1w014a-05_MeOH AmOH_02_2PIC_800µL 1.2e-2 1.0e-2 8.0e-3 6.0e-3 4.0e-3 2.0e-3 0.0 1 1.05 2 1.99 3 4 1.64 2.88 3.02 2.28 2.48 2: Diode Array 220 Range: 6.672e-1-2.0e-3-0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5 3.84 4.02 4.32 Preparative SFC Column: Torus 2-PIC 5 µm 19 x 150 mm Time 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 Goldenseal Extract: 1. Canadine 2. Hydrastine 3. Isocorypalmine 4. Methyl Hydrastine 5. Berberine 2016 Waters Corporation 8

Factors that control effective scale-up in SFC 2016 Waters Corporation 9

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 10

Scaling up 2016 Waters Corporation 11

L/d p rule for HPLC systems Designing prep column dimensions and particle size Stationary phase Select the same chemistry Column specs Choose either L or d p Calculate the other from (L/d p ) anal = (L/d p ) prep Linear velocity (u) Calculate u from (u x d p ) anal = (u x d p ) prep Flow rate Calculate Q from, Q=u x A L = column length, d p = particle size, u = linear velocity, A = column void cross-section 2016 Waters Corporation 12

AU AU AU Works for LC Method Transfer 0.05 0.04 0.03 0.02 0.01 0.00 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 0.05 0.04 XBridge C18 5 µm, 4.6 x 150 mm 1485 psi XBridge C18 2.5 µm, 3.0 x 75 mm 0.03 0.02 0.01 0.00 0.05 0.04 0.03 0.02 0.01 0.00 0.60 1.00 1.40 1.80 2.20 2.60 3.00 3.40 3.75 ACQUITY BEH C18 1.7 µm, 2.1 x 50 mm 5555 psi 10433 psi 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 Minutes Courtesy of Jon Turner April 2015 2016 Waters Corporation 13

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 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

Effect of Pressure Difference on LC scale-up LC scale-up minimum effect from pressure changes u Anal > u Prep DP Analytical DP Prep Column length Column length Nearly incompressible fluid Matching density profile Analytical Dr Dr Prep Column length Column length 2016 Waters Corporation 16

Effect of Pressure Difference on SFC scale-up SFC scale-up effect from pressure changes u Anal > u Prep DP Analytical DP Prep Column length Column length Compressible fluid Different density profiles Dr Analytical Dr Prep Column length Column length 2016 Waters Corporation 17

AU AU Effect of Density Changes 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0.30 0.25 0.20 0.15 0.10 0.05 0.00 1 2,3 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 1 Different density = different thermodynamic interactions = different retentions 2 3 4 Minutes address the density 6 5 Lower Density Mobile Phase What it means? To apply LC scaling rules, Higher Density Mobile Phase 4 5 5 µm difference 1.7 µm 6 2.1 x 150 mm 5µm BEH 2-EP, 1.4 ml/min ABPR = 103.5 bar Temp: 40 C 2.1 x 150 mm 1.7µm BEH 2-EP, 1.4 ml/min ABPR = 103.5 bar Temp: 40 C 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Caffeine, Carbamazepine, Uracil, Hydrocortisone, Prednisolone, and Sulfanilamide C. Hudalla et. al. Waters Application Note. 720004818en 2016 Waters Corporation 18

Addressing Density Difference Ideal situation matching density profiles Analytical column Prep column Dr Dr Column length Column length Simpler approach - matching average density Dr Analytical column Dr Prep column Average density Average density Column length Column length 2016 Waters Corporation 19

Addressing Density Difference SFC System Scheme Any changes upstream of ABPR can change chromatography! CO 2 Injector Column Detector Set: ABPR B ABPR = Automatic Back Pressure Regulator 2016 Waters Corporation 20

How to manipulate Ave P? Assuming linear pressure drop Calculate average pressure Analytical column Prep column System P ABPR P System P ABPR P Ave P DP DP Ave P Column length Column length Manipulating ABPR pressure, match average pressure DP Analytical column DP Prep column Increased ABPR pressure Column length Column length 2016 Waters Corporation 21

Simplified - manipulate Ave P! P System P Average P System 2 P ABPR P ABPR Assume linear pressure drop across the entire range Sensor = pump outlet Sensor = regulator inlet Take simple average pressure across the system Implementable Back pressure regulator 2016 Waters Corporation 22

Scale-up Steps for SFC Stationary phase Select the same chemistry Column specs Choose either L or d p Calculate the other from (L/d p ) anal = (L/d p ) prep Linear velocity (u) Calculate u from (u x d p ) anal = (u x d p ) prep Calculate Q from, Q=u x A Flow rate Average Density Adjust ABPR to ensure same average pressure L = column length, d p = particle size, u = linear velocity, A = column void cross-section 2016 Waters Corporation 23

Step by Step. 2016 Waters Corporation 24

Effect of Lower Density 2016 Waters Corporation 25

Effect of Lower Density 2016 Waters Corporation 26

Density Profiles 2016 Waters Corporation 27

Use of Average Density 2016 Waters Corporation 28

Use of Average Density ABPR = Automatic Back Pressure Regulator 2016 Waters Corporation 29

Impact of Flow Rate 2016 Waters Corporation 30

Impact of Flow Rate 2016 Waters Corporation 31

Regarding Particle Size & Prep Practice 2016 Waters Corporation 32

Regarding Particle Size & Prep Practice 2016 Waters Corporation 33

Professional Scale Up 2016 Waters Corporation 34

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 35

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=pl6ya4jv5tak5q6m391v0_yufesh_eywj UPC2 Strategy for Scaling from Analytical to Preparative SFC Separations (LiteraturNr. 720004818EN) 2016 Waters Corporation 36

Acknowledgements Waters Corporation Abhijit Tarafder Jason Hill Tom Swann Steve Collier 2016 Waters Corporation 37

THANK YOU FOR YOUR ATTANTION! QUESTIONS?! 2016 Waters Corporation 38