Finding the best separationfor enantiomeric mixtures

Chiral Technologies EuropeIllkirch Cedex – France

Chiral Technologies, Inc.West Chester, PA

Analytical Method Development Goals

Resolution of the enantiomeric pair

Resolution from impurities

Short analysis time

Suitable elution order

Compatibility of the sample media with mobile phase and column

Low LOD/LOQ

Reproducibility and robustness of the method

Stability of the sample under analytical conditions

Method Development Overview for Polysaccharide-derived CSPs

Racemic mixture

HPLC SFC

Organic mobile phases

Water compatible

mobile phases

CHOICE OF SEPARATION MODE

CHOICE OF TECHNOLOGY

Choice of LC and SFC Technique

Recognition behavior

Timing and productivity

Solubility considerations

Stability issues

Amount of mixture to be separated

Solvent volume to be evaporated

Equipment availability09/01/2007

00:05:36

09/01/2007

00:52:44

09/01/2007

00:29:10

-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)

Choice of LC and SFC Technique

SFC Advantages of Small Scale Separations:

Resolution of the enantiomeric pair

Less solvent to be evaporated

Single solvent

Short retention times

Enhanced solubility in certain cases

Less perturbance when injecting in different solvent

HPLC method development in organic mobile phases

Cellulose-based

CHIRALCEL OD-H (Coated)

CHIRALCEL OJ-H (Coated)

CHIRALCEL OZ-H (Coated)

CHIRALPAK IB (Immobilised)

CHIRALPAK IC (Immobilised)

Polysaccharide-derived Columns

Amylose-based

CHIRALPAK AD-H (Coated)

CHIRALPAK AS-H (Coated)

CHIRALPAK AZ-H (Coated)

CHIRALPAK AY-H (Coated)

CHIRALPAK IA (Immobilised)

NH

O CH3

CH3

CH3NH

O

(S)

NH

O

CH3

Cl

NH

O Cl

CH3

NH

O CH3

CH3

NH

O CH3

CH3

NH

O Cl

CH3

NH

O CH3

CH3

NH

O Cl

Cl

OCH3

Primary Screening

Immobilised

CHIRALPAK IA CHIRALPAK IB CHIRALPAK IC

Coated

CHIRALPAK AD-H CHIRALCEL OD-H CHIRALPAK AS-H CHIRALCEL OJ-H

Primary Screening

Primary Screening

Immobilised

Alkane / EtOH Alkane / 2-PrOH MtBE mixtures Dichloromethane

(or THF) mixtures

Coated

Alkane / EtOH Alkane / 2-PrOH EtOH / MeOH Acetonitrile

IA

IB

IC

Hept/EtOH/DEA

Hept/IPA/DEA

Hept/THF/DEA

Hept/DCM/DEA

Minutes

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

mAU

-50

0

50

100

150

200

250

300

350

400

450

mAU

-50

0

50

100

150

200

250

300

350

400

450

1: 290 nm, 4 nmCE070128 - IA - Heptane-EtOH - 70-30-Rep2

Retention Time

Minutes

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

mAU

0

50

100

150

200

250

300

mAU

0

50

100

150

200

250

300

1: 290 nm, 4 nmCE070128 - IA - Heptane-IPA - 70-30-Rep2

Retention Time

Minutes

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

mAU

-2,5

0,0

2,5

5,0

7,5

10,0

12,5

15,0

17,5

20,0

22,5

25,0

mAU

-2,5

0,0

2,5

5,0

7,5

10,0

12,5

15,0

17,5

20,0

22,5

25,0

1: 290 nm, 4 nmCE07128 - IA - Hept-THF 90-10-Rep2

Retention Time

Minutes

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

mAU

-5

0

5

10

15

20

25

30

mAU

-5

0

5

10

15

20

25

301: 290 nm, 4 nm

CE07128 - IA - Hept-DCM 80-20-Rep2Retention Time

Minutes

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

mAU

-50

0

50

100

150

200

250

300

350

400

450

mAU

-50

0

50

100

150

200

250

300

350

400

450

1: 290 nm, 4 nmCE070128 - IB - Heptane-EtOH - 70-30-Rep2

Retention Time

Minutes

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

mAU

-25

0

25

50

75

100

125

150

175

200

225

250

275

mAU

-25

0

25

50

75

100

125

150

175

200

225

250

2751: 290 nm, 4 nmCE070128 - IB - Heptane-IPA - 70-30-Rep2

Retention Time

Separation Retention time > 30 min

Separation Retention time > 30 min

Minutes

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

mAU

-50

0

50

100

150

200

250

300

350

400

450

mAU

-50

0

50

100

150

200

250

300

350

400

450

1: 290 nm, 4 nmCE070128 - IC - Heptane-EtOH - 70-30-Rep2

Retention Time

Minutes

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

mAU

-25

0

25

50

75

100

125

150

175

200

225

250

mAU

-25

0

25

50

75

100

125

150

175

200

225

2501: 290 nm, 4 nmCE070128 - IC - Heptane-IPA - 70-30-Rep2

Retention Time

Minutes

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

mAU

-2,5

0,0

2,5

5,0

7,5

10,0

12,5

15,0

17,5

20,0

22,5

25,0

mAU

-2,5

0,0

2,5

5,0

7,5

10,0

12,5

15,0

17,5

20,0

22,5

25,0

1: 290 nm, 4 nmCE07128 - IC - Hept-THF 90-10-Rep2

Retention Time

Minutes

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

mAU

-10

0

10

20

30

40

50

mAU

-10

0

10

20

30

40

50

1: 290 nm, 4 nmCE07128 - IC - Hept-DCM 80-20-Rep2

Retention Time

Basic Molecule – Analytical Scale

70/30/0.1 70/30/0.1 90/10/0.1 80/20/0.1

Minutes

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

mA

U

-25

0

25

50

75

100

125

150

175

200

225

250

275

mA

U

-25

0

25

50

75

100

125

150

175

200

225

250

2751: 290 nm, 4 nmCE070128 - IB - Heptane-IPA - 70-30-Rep2

Retention Time

CHIRALPAK IBn-heptane/2-PrOH/DEA

70/30/0.1

12 min

7 min

Basic Molecule – Analytical Scale

(D)

min0 2.5 5 7.5 10 12.5 15 17.5

(L)

min0 2.5 5 7.5 10 12.5 15 17.5

(D)

(L)

min

0 2.5

5 7.5

10

12.5

15 17.5

(D)

(L)

FMOC-D,L-Leucine

O

O

NH

OHO

Complementary Properties of CSPs

CHIRALPAK IA

CHIRALPAK IB

CHIRALPAK IC

n-hexane/2-PrOH/TFA90/10/0.1

Complementary Properties of CSPs

Advantages

Specific resolutions Changes in enantiomeric elution order Different elution of impurities

SFC method development in organic mobile phases

Compatible Co-Solvents in SFC

Coated CSPs

Ethanol Methanol 2-Propanol Acetonitrile Other alcohols

Immobilised CSPs

Ethanol Methanol 2-Propanol Acetonitrile Other alcohols

Compatible Co-Solvents in SFC

Extended Solvent Range

MtBE Ethyl acetate THF Dichloromethane Chloroform 1,4-dioxane Acetone DMSO or DMF (as injection solvents)

0 200 400 600 800 1000 1200 (sec)

CO2 / MeOH 90/10T=25°CFlow rate: 3.0ml/min.

CHIRALPAK IA

CO2 / THF (+1%DEA) 70/30T=30°CFlow rate: 3.0ml/min.

1 2 3 4 5 6 7 8 9 10

-50

0

50

100

150

200

250

300

CHIRALPAK IC

CSPs in SFC: Analytical Scale

CSPs in SFC: Analytical Scale

All the advantages of the SFC applications on polysaccharide-derived CSPs

Possibility of eluting and/or injecting in different solvents (DCM, THF, …) when using the immobilised CSPs

HPLC method development in water compatible mobile phases

Water Compatible Mobile Phases

Identify the compound nature

Acidic Neutral Basic

HCOOHpH 2.0

40% ACN60% MeOH

H2O

40% ACN60% MeOH

20mM NH4HCO3

pH 9.0

40% ACN60% MeOH

Aqueous Solution

Organic modifier

CHIRALPAK IA

furoinmin0 2 4 6 8

MeOH/H2O55:45

Separations RP-Mode

O O

OH

O

min0 2 4 6 8

CHIRALPAK IC

2,3-dibenzoyl-DL-tartaric acid

HCOOH aq. (pH2.0)

ACN: 55%

Separations RP-Mode

O

O

H HO

OOH

O

OH

O

Faster screening and enhanced resolution:

3-µm CSPs

Hexane-EtOH 80:20 (+0.1% AE)Flow rate: 5.0 ml/min

DCM-MeOH 98:2 (+0.1% AE)Flow rate: 5.0 ml/min

MeOH 100% Flow rate: 5.0 ml/min

NN

OH

Cl Cl

40 seconds

N

N CH3

OH

Cl

O2N

30 seconds

N

N

O

OHO

20 seconds

Fast Analysis: CHIRALPAK IA-3 (4.6 x 50 mm)

Looking for new separations:different chiral selectors

CHIRALCEL OD

CHIRALPAK AD

CHIRALCEL OZ

CHIRALPAK AZ

N

Cl

H

O Cl

CHIRALPAK IC CHIRALPAK AY

N

CH3

H

O CH3

N

Cl

CH3

H

O

N

H

O

CH3

Cl

Different Substituents; New Enantiorecognition

CH3

N

O

Cl

CH3OCH3

Metolachlor

CHIRALPAK IA CHIRALPAK IB CHIRALPAK IC CHIRALPAK IA CHIRALPAK IB CHIRALPAK IC

Are New Selectors Needed?

Hexane / EtOH 95:5

1ml/min, 25°C(4.6 x 250 mm)

Metolachlor

CHIRALPAK AS-H CHIRALPAK AD-H CHIRALCEL OD-H CHIRALCEL OJ-H

Hexane / EtOH 95:5

1ml/min, 25°C(4.6 x 250 mm)

Are New Selectors Needed?

CH3

N

O

Cl

CH3OCH3

CH3

N

O

Cl

CH3OCH3

Metolachlor

CHIRALPAK AY-H

Hexane / EtOH 95:5

1ml/min, 25°C(4.6 x 250 mm)

Are New Selectors Needed?

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