SFC Säulen für analytische und preparative Anwendungen · •Calculate the other from (L/d p)...

©2016 Waters Corporation 1

SFC Säulen für analytische und

preparative Anwendungen

Technology Symposium

Vienna, 25th October 2016

DI Verena Schmid


CHIRAL SEPARATIONS ACQUITY UPC2 Trefoil™


ACHIRAL SEPARATIONS Viridis Columns


New Torus™ 5 µm

Analytical and OBD™ Preparative

Achiral SFC Columns

October 2016


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


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


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


Factors that control effective

scale-up in SFC


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


Scaling up


“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


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


Outline

Introduction






Conclusion


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


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


Caffeine, Carbamazepine, Uracil, Hydrocortisone, Prednisolone, and Sulfanilamide

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 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


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


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


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


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


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


Step by Step.


Effect of Lower Density


Density Profiles


Use of Average Density


Use of Average Density

ABPR = Automatic Back Pressure Regulator


Impact of Flow Rate


Regarding Particle Size & Prep Practice


Professional Scale Up


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


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)

http://www.waters.com/waters/promotionDetail.htm?id=134771024&locale=en_US

http://www.waters.com/waters/promotionDetail.htm?id=134771024&locale=en_US

https://www.youtube.com/playlist?list=PL6yA4jv5tA-k5q6M391V0_yuFesh_eywJ




Acknowledgements

Waters Corporation

– Abhijit Tarafder

– Jason Hill

– Tom Swann

– Steve Collier


THANK YOU FOR YOUR ATTANTION!

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SFC Säulen für analytische und preparative Anwendungen · •Calculate the other from (L/d p)...

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