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Finding the best separation for enantiomeric mixtures
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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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