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Parc d’Innovation - Bd Gonthier d’Andernach - 67404 Illkirch Cedex - France
Tel : +33 (0)3 88 79 52 00 - fax : +33(0)3 88 66 71 66
www.chiral.fr - e-mail : [email protected]
Novel immobilised polysaccharide-derived CSPs
to enhance success rate in the resolution of enantiomers
P. Franco, D. Nguyen, T. Zhang
Screening strategy for the resolution of enantiomers
Higher success rates can be achieved by: • Screening only LC or SFC with 7 (or more) columns • Screening LC and SFC with a limited number of columns
What is the best choice?
LC SFC
Can we achieve this with a minimum number of columns and efficient screening strategies?
• Resolution of the enantiomeric pair (or isomeric mixture)
• Resolution from impurities
• Short analysis times
• 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 the analytical conditions
Some of the requirements / challenges
• Samples are screened systematically in LC and SFC with a FAST screening
• Systematic screening
• LC on IA, IB and IC (H/IPA, H/EtOH, H/THF, H/DCM, H/AcOEt, ACN and alcohol)
• SFC on IA, IB and IC (MeOH, IPA and EtOH)
How were we handling in the last years?
RO
O
OR
ROO n RO
O
OR
RO
O
n
Amylose-based
carbamates Cellulose-based
carbamates
CHIRALPAK IA CHIRALPAK IB CHIRALPAK IC NH
O CH3
CH3
NH
O CH3
CH3
NH
O Cl
Cl
• Free choice of solvents in the mobile phase to match solubility
• Greater options for chemical stability
• New selectivities (and elution orders)
• Favouring elution kinetics with certain solvents
• Choice in the sample solvent dictated by sample solubility
• Column and method robustness
In which aspects solvent versatility was helping?
T. Zhang et al., J. Chromatogr. A 1191 (2008) 214
New selector – June 2011 CHIRALPAK ID
O
O
O
O O
n
NH
Cl
OONH
NH
O Cl
Cl
CHIRALPAK ID
Based on
amylose tris-(3-chlorophenylcarbamate)
A new selector for the immobilised series!!
CHIRALPAK IA CHIRALPAK IB
N
CH3
H
O CH3
Different substituents New enantiorecognition
N
Cl
H
O Cl
CHIRALPAK IC N
H
O Cl
CHIRALPAK ID
Looking for complementary recognition patterns
HPLC method development
CHIRALPAK ID Complementary properties
Hexane/2-PrOH/DEA 80/20/0.1 (250 x 4.6 mm, 5 µm); 1.0 mL/min; 25 C
min 0 1 2 3 4 5 6 7 min 0 1 2 3 4 5 6 7
min 0 1 2 3 4 5 6 7
CHIRALPAK IA CHIRALPAK IB
CHIRALPAK IC
min 0 1 2 3 4 5 6 7 8 9
CHIRALPAK ID
Isradipine
O
O
O
O
NH
N
O
N
Ex
am
ple
s in
H
PLC
Column dimensions: 250 x 4.6 mm, 5 µm; 1.0 mL/min; 25 C
Mobile phase selection in HPLC Analytical methods
CHIRALPAK IA
min 0 1 2 3 4 5 6 7
ACN/IPA/TFA 95/5
N
O
OH
O
CHIRALPAK IB
Hex/IPA/TFA 80/20/0.1
min 0 1 2 3 4 5 6 7 8 9
O NH
O
OH
O
CHIRALPAK IC
Hex/THF/DEA 85/15/0.1
min 0 2 4 6 8 10
NH
O
O
NO2
O
OOMe
CHIRALPAK ID
MtBE/MeOH/TFA 90/10/0.1
min 2 4 6 8 10 12 14 16 18
OOH
OH O
OH
OMe
HP
LC
Comparison of success rates between immobilized type columns and representative coated type columns for 123 compounds under Hexane/2-PrOH
Example of complementarity CHIRALPAK ID
Success rate with a single mobile phase combination!
Success rate
n-Hexane / 2-PrOH
81.3% 82.1%
89.4%
70%
75%
80%
85%
90%
95%
100%
ADH/ODH /ASH/OJH
IA/IB/IC IA/IB/IC/ID
N
COOC2H5H3COOC
H
NO2
N
H3COOC
O
H
NO2
ON
N
COOC2H5H3COOC
H
ONH2
Cl
n-Hex-MTBE-EtOH-DEA 50:50:1:0.1
0 5 10 15 20 25 30
Minutes
0 5 10 15 20 25 30
Minutes
0 4 8 12 16 20
Minutes
0 4 8 12 16 20
Minutes
n-Hex-THF-DEA 90:10:0.1
n-Hex-THF-DEA 90:10:0.1
n-Hex-IPA-Isopropylamine 80:20:0.1
Nitrendipine Benidipine
Nicardipine Amlodipine
N
H3COOC
O
H
NO2
ON
CHIRALPAK ID (250 x 4.6 mm, 5 µm); 1.0 mL/min; 254 nm; 25 C
Dihydropyridine-type drugs on CHIRALPAK ID
CHIRALPAK ID (250 x 4.6 mm, 5 µm); 1.0 mL/min; 254 nm; 25 C
Phenothiazine-type drugs on CHIRALPAK ID
n-Hex-IPA-DEA 80:20:0.1
0 5 10 15 20 25 30 Minutes
Dimetotiazine
N
S
N
NS
OO
0 2 4 6 8 10 12 14 16 18 20
Minutes
n-Hex-EtOH-DEA 100:0.1:0.1 (0.5mL/min.)
Profenamine
N
S
N
n-Hex-CH2Cl2-EtOH-DEA 95:5:1:0.1
0 2 4 6 8 10 14 18 12 16 20
Minutes
Thioridazine
N
S
N
S
Recognition in RP-mode
0 5 10 15 20 25 30 35 40
Minutes
HCOOH aq.(pH=2.0)/ACN=85/15
O CO2H
2-Phenoxypropionic acid
0 5 10 15 20 25 30
Minutes
Aminoglutethimide
20mM NH4HCO3aq. (pH=9)/ACN=30/70
NH
O ONH2
CHIRALPAK ID (250 x 4.6 mm, 5 µm); 0.5 mL/min; 25 C
Different elution profile and method optimisation
CHIRALPAK IA (4.6 x 150mm)
SFC CO2/ MeOH (0.5% DEA) 80/20
min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min 0 1 2 3 4 5 6 7
SFC CO2/ MeOH (0.5% DEA) 80/20
CHIRALPAK IC (4.6 x 150mm)
HPLC MeOH/DEA 100/0.1
min 0 1 2 3 4 5 6 7 8 9
CHIRALPAK IA (4.6 x 250mm)
min 2 4 6 8 10 12 14
HPLC MeOH/DEA 100/0.1
CHIRALPAK IC (4.6 x 250mm)
Analysis time: 12min
N
N
MeO
MeO
OMe
OMe
Tofisopam • Same enantioselectivity • Reduced column length • Reduced analysis time • No loss in resolution degree
CHIRALPAK IC-3 (4.6 x 150mm)
min 0 1 2 3 4 5 6 7 8
HPLC MeOH/DEA 100/0.1
Analysis time: 8min
Column transfer from 5µm to 3µm
SFC method development
Screening in SFC
CHIRALPAK IA
CHIRALPAK IB
CHIRALPAK IC
CHIRALPAK ID
Mobile phase system
EtOH, MeOH, 2-PrOH
Acetonitrile
Dichloromethane
Ethyl acetate
THF
MtBE
Primary Screen
CHIRALPAK AD-H
CHIRALPAK AS-H
CHIRALPAK AY-H
CHIRALPAK AZ-H
CHIRALCEL OD-H
CHIRALCEL OJ-H
CHIRALCEL OZ-H
CHIRALCEL OX-H
Co-solvents
EtOH, MeOH, 2-PrOH
Acetonitrile
Secondary Screen
Column dimensions: 150 x 4.6 mm, 5 µm; 3.0 mL/min; 25 C
Mobile phase selection in SFC Examples
SFC
CO2 / THF (0.3% TFA) 80/20
min 0 0.5 1 1.5 2 2.5 3
O
COOH
CHIRALPAK IA
CO2 / MeOH (1.0% DEA) 90/10
min 0 0.5 1 1.5 2 2.5 3 3.5
N
N
CHIRALPAK IB
CO2 / ACN (0.3% TFA) 80/20
min 0 1 2 3 4 5
O
OH
COOH
CHIRALPAK IC
CO2 / 2-PrOH (1% DEA) 90/10
min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
N
O N
CHIRALPAK ID
N
O
OH
O
Indoprofen
CHIRALPAK ID Complementary properties
CHIRALPAK IA
min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
CHIRALPAK IB
0 min 0.5 1 1.5 2 2.5 3 3.5 4 4.5
CHIRALPAK IC
min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
CHIRALPAK ID
min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Ex
am
ple
s
in S
FC
30% MeOH (+0.2% TFA) in CO2 (150 x 4.6 mm, 5 µm); 3.0 mL/min; 150 bar
Isocratic or gradient?
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12
Time (min)
*M
eO
H %
(by v
olu
me) MeOH gradient program
* Containing 0.3% DEA for screening of basic compounds
min 0 2 4 6 8 10 12
Cl
Cl
N
N
NOH
Diniconazole
CHIRALPAK IA
Back pressure: 150bar, Flow rate: 3.0ml/min, Temperature: 35°C
CHIRALPAK ID
min 0 2 4 6 8 10 12
N
O
OH
O
Ketorolac
CTE examples in SFC
Back pressure: 150 bar, Flow rate: 3.0ml/min, Temperature: 35
C
Containing 0.2-0.3% DEA for basic compounds
Main co-solvents and their relative eluting strength
MeOH > EtOH > 2-PrOH
MeOH > ACN THF
Time (min)
Co
-so
lve
nt
%
(by v
olu
me
)
0
10
20
30
40
50
60
0 2 4 6 8 10 12
0
10
20
30
40
50
60
0 2 4 6 8 10 12
Time (min)
MeOH-CO2 gradient ACN- or THF-CO2 gradient
Method development - SFC gradient programs
SFC-From gradient to isocratic
CHIRALPAK IB
min 0 0.5 1 1.5 2 2.5 3 3.5
min 0 2 4 6 8 10 12
MeOH-CO2 gradient
CO2 / MeOH (1.0% DEA)
90/10
N
N
Trimipramine
CHIRALPAK IC
min 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
min 0 2 4 6 8 10 12
MeOH-CO2 gradient
CO2 / MeOH (0.3% DEA)
70/30
N
O
OH
N
Carazolol
CHIRALPAK ID
N
N
Bifonazole
THF-CO2 gradient
CO2 / THF (0.3% DEA)
70/30
min 0 2 4 6 8 10 12
min 0 1 2 3 4 5
CHIRALPAK IA
O
2-Ethyl-1-indanone
ACN-CO2 gradient
CO2 / ACN
95/5
min 0 0.5 1 1.5 2 2.5 3 3.5
min 0 2 4 6 8 10 12
SFC-From gradient to isocratic
Conclusions
Screening strategies by LC and SFC
• First objective is fast screen with high success rate
• Different derivatives introduce additional recognition options
• Solvent versatility is necessary to match resolution, solubility and chemical stability
Acknowledgements Team work
All other colleagues at
CHIRAL TECHNOLOGIES and DAICEL
DAICEL Chemical Industries, Ltd.
Tatsushi MURAKAMI
Atsushi OHNISHI
Chiral Technologies Europe
Dr. Tong ZHANG Dung NGUYEN