preparative chiral chromatography what can go wrong … · 2020-03-30 · preparative chiral...

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PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong and how to solve it M. Schaeffer, T. Zhang, D. Robin, J.M. Heym, D. Colantuono, J. Lee, S. Khattabi, P. Franco

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Page 1: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

PREPARATIVECHIRAL CHROMATOGRAPHY

What can go wrong and how to solve it

M. Schaeffer, T. Zhang, D. Robin, J.M. Heym, D. Colantuono, J. Lee, S. Khattabi, P. Franco

Page 2: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Summary

1) Preparative chiral chromatography

FeaturesPotential

2) What can go wrong? How to solve it?

SampleChromatographic methodChromatographic systemProduct recovery

3) Conclusions

Page 3: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Preparative chiral chromatography

Easily scalablefrom the analytical study

SemiprepBatch

PreparativeBatch

SMB

MT+

1 MT

100 kg

10 kg

1 kg

g

1mg

4,99

7,51

0 2 4 6 8 10 12 14 16 18 20

Retention Time (min)

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

Abso

rban

ce (

AU)

Analytical injection

Loading study

Page 4: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Preparative chiral chromatography

09/01/200700:05:36

09/01/200700:52:44

09/01/200700:29:10

-205.0

256.0

717.0

1178.0

1639.0

2100.0

CycliqueCyclique signal UV (PIC04002:ao_F_mes_UV_DETECTOR) signal UV (PIC04002:ao_F_mes_UV_DETECTOR)(09/01/2007 00:05:36) -17.1 mv(09/01/2007 00:05:36) -17.1 mv

-42.4 mv (0 jours, 00:47:09)(09/01/2007 00:52:44) -59.5 mv(09/01/2007 00:52:44) -59.5 mv -42.4 mv (0 jours, 00:47:09)

Different to achiral chromatography

Isolation of two components, not purification from a crudeUses Chiral

Stationary Phases (CSP) Can separate racemates,

diastereomers and atropisomers

Batch and Continuous Systems (SMB)

Yield is ca. 90%

Specification is ≥ 98% e.e.

Page 5: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Preparative chiral chromatography

Several systems and modes possible

Batch LC Batch SFC

SMB

To be chosen based on recognition and scale

Page 6: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Preparative chiral chromatography

What can go wrong?

&

How to solve it?

Page 7: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

What can go wrong?

… Observing the different elements…

… to find the potential solution.

SAMPLE

METHOD

SYSTEM RECOVERY

Page 8: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

What can go wrong?Some examples

Not exhaustive list

SAMPLESolubilityImpuritiesStability

METHODRobustness TemperatureConditioning

Recycling

SYSTEMPacking

Static Electricity

RECOVERYResidual solventSample VolatilityFoaming

Page 9: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

SAMPLESolubilityImpuritiesStability

METHODRobustness TemperatureConditioning

Recycling

SYSTEMPacking

Static Electricity

RECOVERYResidual SolventSample VolatilityFoaming

Page 10: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Sample solubility

Potential causes of insolubility:• Nature of the sample• Presence of impurities

Potential associated problems:• Frit blockage, with or without associate higher pressure• Perturbance of the separation and method instability• Precipitation of the sample in the chromatographic system or tanks• Need for a very large sample volume to be injected

Potential ways to solve the problem:• Look for alternative mobile phase combinations• Apply thorough filtration of the sample or even preliminary chromatographic step

• Choose a different molecule in the synthetic route or a more soluble derivative• Inject sample in solvent different to mobile phase• Thermostat the feed solution• Try selective precipitation/crystallisation of selected components prior to

chromatography

Page 11: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Sample solubility

n-heptane / THF 60/40

Rs = 2.0 – α = 2.1

min0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

250

300

350

400

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\23090008.D) 2

.853

4.2

44

5.8

23

n-heptane / methyl-THF 40/60

Rs = 3.5 – α = 4.5

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

50

100

150

200

250

300

350

400

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\24090008.D)

2.9

85

4.1

35

8.1

57

Two improvements:

• Larger resolution

• Lower n-heptane content, which may enhance solubility

Optimising conditions in method development

Our best investment!!

CHIRALPAK IC – 20 µm

Page 12: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Sample solubility

THIS IS NOT A CLEAR SOLUTION…

The importance of filtration

Peak distorsion in SFC due to dirty frits

New injection after frit replacement

M i n u t e s

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

mA

U

- 1 6

- 1 4

- 1 2

- 1 0

- 8

- 6

- 4

- 2

0

2

mA

U

- 1 6

- 1 4

- 1 2

- 1 0

- 8

- 6

- 4

- 2

0

2K - 2 5 0 1

R e t e n t i o n T i m e

M i n u t e s

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 3 8 4 0

mA

U

- 7 . 5

- 5 . 0

- 2 . 5

0 . 0

2 . 5

5 . 0

7 . 5

1 0 . 0

1 2 . 5

mA

U

- 7 . 5

- 5 . 0

- 2 . 5

0 . 0

2 . 5

5 . 0

7 . 5

1 0 . 0

1 2 . 5

K - 2 5 0 1R e t e n t i o n T i m e

… and leads to dirty frits

Page 13: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Presence of impurities in the sample

It is possible to work in the presence of impurities, but they may get absorbed in the CSP

Stack injections will be more difficult

Backflush or washing steps with stronger solvents are possible

Page 14: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

TroubleshootingContaminants

Blocked Frits: 5 cm SFC columns

When the inlet frit is blocked, there is a high pressure differential across it; it may deform…

Page 15: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Sample solubility

Injecting in a solvent different from mobile phase

min0 1 2 3 4 5 6 7min0 1 2 3 4 5 6 7

NH

CH3

Prenylamine

CHIRALPAK IAHexane/THF/DEA 85/15/0.1

Injection in100% THF

Injection inHexane/THF 85/15

Ideally injection should be done in mobile phase (or close composition)…

There are several risks associated to such a practice:

• Strong peak distorsion due to sample solvent

• Changes in the separation due to solvent front

• Higher risks of sample precipitation due to different composition

• Difficulties for solvent recycling

• Process instability with continuous systems

Page 16: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Sample solubility

Injecting in a solvent different from mobile phase

CHIRALPAK IC(250 x 30 mm)

CO2/EtOH 70/30 120 ml/min, 25°C

Analytical injection Injection in EtOH/DCM 90/10 – 2ml - 116 mg

23/03/200914:32:21

23/03/200914:42:23

23/03/200914:37:22

-25.0

22.0

69.0

116.0

163.0

210.0

CycliqueCyclique signal UV (PIC04002:ao_F_mes_UV_DETECTOR) signal UV (PIC04002:ao_F_mes_UV_DETECTOR)(23/03/2009 14:32:21) -1.7 mv(23/03/2009 14:32:21) -1.7 mv

-0.2 mv (0 jours, 00:10:01)(23/03/2009 14:42:23) -1.9 mv(23/03/2009 14:42:23) -1.9 mv -0.2 mv (0 jours, 00:10:01)

23/03/200916:07:12

23/03/200916:17:15

23/03/200916:12:14

-25.0

102.0

229.0

356.0

483.0

610.0

CycliqueCyclique signal UV (PIC04002:ao_F_mes_UV_DETECTOR) signal UV (PIC04002:ao_F_mes_UV_DETECTOR)(23/03/2009 16:07:12) -0.7 mv(23/03/2009 16:07:12) -0.7 mv

3.0 mv (0 jours, 00:10:03)(23/03/2009 16:17:15) 2.3 mv(23/03/2009 16:17:15) 2.3 mv 3.0 mv (0 jours, 00:10:03)

No perturbation of the separation

DCM is less polar than alcohols or THF(not compatible with all columns)

Solubility in EtOH < 2 g/LSolubility in EtOH/DCM 90/10 = 58 g/L

Page 17: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Compound stability

min2 4 6 8 10

4.36

8

6.58

2

min0 2 4 6 8 10

4.46

3

6.10

8

CHIRALPAK IA

MeOH / ACN80/20

A case study

0

3.34

0

3.63

2

3.92

5

4.33

1

6.55

4

1st eluted enantiomerafter 12 h at 55°C

DEGRADATION AND RACEMISATION

Compound with glutarimide moiety

min0 1 2 3 4 5 6 7 8 9

5.425

6.614

min1 2 3 4 5 6 7 8 9

5.519

6.529

min0 1 2 3 4 5 6 7 8 9

5.344

min0 1 2 3 4 5 6 7 8 9

6.635

CHIRALPAK IC

ACN / DCM90/10

FULLY STABLE CONDITIONSIn certain cases, degradation and/or

racemisation can be controlled with lower evaporation temperature

Page 18: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

SAMPLESolubilityImpuritiesStability

METHODRobustness TemperatureConditioning

Recycling

SYSTEMPacking

Static Electricity

RECOVERYResidual SolventSample VolatilityFoaming

Page 19: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Robustness of chromatographic method

min0 5 10 15 20 25

mAU

0

20

40

60

80

100

120

140

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\17090045.D)

8.3

30

19.

962

100% ACN

Run >20 min

Rs=5.0 – α=3.5

Startingconditions

A case study

Final conditions should consider method robustness

and solvent recovery

min0 5 10 15 20 25

mAU

0

50

100

150

200

250

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\22090009.D)

3.0

11 3

.321

5.0

20

8.2

93

ACN / EtOAc 40/60

Rs=2.9 – α=2.6

min0 5 10 15 20 25

mAU

0

50

100

150

200

250

300

350

400

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\21090004.D)

3.4

85 3

.967

5.1

21

8.0

07

ACN / MeOH 90/10

Rs=3.2 – α=2.8

min0 5 10 15 20 25

mAU

0

50

100

150

200

250

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\21090024.D)

3.4

06 3

.713

5.3

50

8.6

35

ACN / THF 90/10

Rs=2.8 – α=2.4

TargetRun time < 9 min

CHIRALPAK IC – 20 µm

Page 20: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

min0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

250

300

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\23090004.D)

2.9

11 3

.275

6.5

85

8.8

30

CHIRALPAK IA

n-heptane / THF 60/40

min0 2 4 6 8 10 12 14 16 18

mAU

0

25

50

75

100

125

150

175

200

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\23090005.D)

2.9

11 3

.284

8.1

78

Racemate

Target enantiomer

min0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

250

300

350

400

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\23090008.D)

2.8

53

4.2

44

5.8

23

min0 2 4 6 8 10 12 14 16 18

mAU

0

100

200

300

400

DAD1 D, Sig=270,4 Ref=off (X:\ANALYTIC\09_2009\0909_13\DATA\NC\23090009.D)

2.8

54

3.9

89

Target enantiomer

eluted first

Optimising conditions in method development

CHIRALPAK IC

n-heptane / THF 60/40

Suitability of chromatographic method

Page 21: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Temperature effects

• Column : CHIRALPAK IA 5 µm LC – 250 x 50 mm• Eluent : n-Heptane / 2-PrOH 90/10• Flow rate : 120 ml/min • Trans-stilbene oxide injections

• Column Temperature : 30°C

• Eluent Temperature : 30°C

• Column temperature : ambient

• Solvent coming from external storage: in winter!!

Page 22: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

CSP conditioning

Initial separation found after the screening on the analytical column

Column used for standard screening

with different solvents including alcohols

• Column : CHIRALPAK IA 5 µm • Eluent : n-Heptane / DCM• Flow rate : 1 ml/min • Temperature : 25°C

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mA

U

0

200

400

600

800

1000

1200

1400

mA

U

0

200

400

600

800

1000

1200

1400

1: 230 nm, 4 nm STAB T0 CE09021 - IA - 40-60 HEPT-DCM-Rep2

Retention Time

Page 23: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

CSP conditioning

• Column : CHIRALPAK IA 5 µm • Eluent : n-Heptane / DCM• Flow rate : 20 ml/min • Temperature : 25°C

Transfer on the LC preparative column

+1% EtOH in the mobile phase

Page 24: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

CSP conditioning - Mobile phase composition

Influence of water in certain separations

S. Svensson et al., J. Chromatogr. A 839 (1999) 23

together with:

K. Balmér et al., J. Chromatogr. A 592 (1992) 331

Influence of water in the separation of metoprolol analogues

Page 25: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Method optimisationThe use of additives

CHIRALPAK AD-H

(250 x 4.6 mm)3 ml/min, 25°CP outlet 150 bar

20% Isopropanol

20% Isopropanol+1% Diethylamine

(in co-solvent)

20% Isopropanol+1% Butylamine

(in co-solvent)

Page 26: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Mobile phase recycling

Some thoughts:

• Solvent recycling will get more challenging when increasing number of components

(i.e. heptane/ethanol/methanol)

• Having mobile phases with relatively different boiling components

(i.e. DCM and heptane)

• Working close to the azeotrope composition, when possible, can help

• Product carryover should be controlled in the recycled solvent

Page 27: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

SAMPLESolubilityImpuritiesStability

METHODRobustness TemperatureConditioning

Recycling

SYSTEMPacking

Static Electricity

RECOVERYResidual SolventSample VolatilityFoaming

Page 28: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Column packing

Essential parameter

either in batch

or continuous chromatography

Retention times tr1 and tr2 (TSO)

0123456789

10

06068-02 06068-03 06068-04 06068-05 06068-07 06068-08 06068-09 06068-10

column

time

(min

)

tr1 before

tr2 before

tr1 after

tr2 after

Efficiency values N1 and N2 (TSO)

0

500

1000

1500

2000

2500

3000

06068-02 06068-03 06068-04 06068-05 06068-07 06068-08 06068-09 06068-10

column

effic

ien

cy

N1 before

N2 before

N1 after

N2 after

Need of well packed columns and homogenous sets, with clean frits

Page 29: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Electrostatic energy

Separation of glutethimide on CHIRALPAK IAMini-SMB study in 100% ethyl acetate

The operation in pure ethyl acetate producedelectrostatic energy!!

It was necessary to add additional earth contact points in the mini-SMB system

Page 30: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Electrostatic energy

L. Miller and M. Juza, J. Chromatogr. A 1094 (2005) 165-168

Consequences of the use of solvent mixtures with high alkane content

Page 31: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

SAMPLESolubilityImpuritiesStability

METHODRobustness TemperatureConditioning

Recycling

SYSTEMPacking

Static Electricity

RECOVERYResidual SolventSample VolatilityFoaming

Page 32: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Product recovery

Residual solvent removal:• Exhaustive drying, without compromising stability• Azeotropic distillation with a different solvent

• (i.e. THF removal with MeOH, ethyl acetate with acetone)

Removal of solvent stabiliser:• The case of BHT in stabilised THF

Sample volatility:• Adjustment of evaporation temperature and vacuum• Choice of suitable chromatographic solvent, if possible• Avoid complete evaporation and ship in selected solvent

Foam formation:• Product to be recuperated from evaporator more frequently• Frequent replacement of evaporator filter cartridge

Page 33: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Preparative chiral chromatography

Don’t take me wrong!!!

Preparative chromatography is a very reliable technique.

A number of industrial processes demonstrate this statement.

However, we are working very often in a developmental environment…

… at this point, we have limited information about the molecules and processes.

In all cases, our best investment would be having a proper method development.

Page 34: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

Conclusions

We will always have better chances with a solid basis

• Selectivity• Loadability• Stability in the operating conditions• Availability (analytical and bulk)• Batch-to-batch reproducibility

CSP

• Fit for purpose• Properly maintainedEQUIPMENT

• Loadability• Solubility in mobile phase• Viscosity of mobile phase• Temperature• Stability in operating conditions• No interference with sample impurities• Solvent recycling• Repeatability

METHOD

Page 35: PREPARATIVE CHIRAL CHROMATOGRAPHY What can go wrong … · 2020-03-30 · Preparative chiral chromatography Easily scalable from the analytical study Semiprep Batch Preparative Batch

PREPARATIVE CHIRAL SEPARATIONS

Our Vendor seminar today at 12:15 h

… more details