Supercritical Fluid Chromatography
Screening Approach
for Chiral Separations
T. Zhang, D. Colantuono, B. Freer, P. Franco
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 : cte@chiral.fr
Outline
•
•
Finding an approach for Small and Medium Scale separations
•
SFC or LC
•
SFC: which columns and mobile phases
•
Immobilised vs Coated Columns
•
Use of Additives
•
Troubleshooting in SFC
CTE has an analytical method development & custom separation laboratory
This is what we do:
First decision
Racemic mixture
(diastereomeric mixture)
to be analysed or separated
CHOICE OF TECHNOLOGY
HPLC
SFC
Screening strategy at Small/Medium Scale
LC and SFC
High success rates can be achieved by:
•
Screening in LC or SFC with multiple columns ( >7)
•
Screening LC and SFC with a limited number of columns
At Small Scale we use SFC and LC
Fast primary screen - maximum hits
Secondary Screen - difficult compounds
Success rate 2008
Success rate 2009
Success rate
2008+2009
SFC+LC
99
99
99
SFC
86
81
83
LC
85
76
82
Success rate at Small Scale
Screening options
Polysaccharide-derived CSPs
OR
O
RO
CSP
RO
OR
Amylose-based
Cellulose-based
O
O
RO
n
Nature
CSP
-R
O
RO
n
Nature
-R
CH3
CH3
CHIRALPAK AD-H
Coated
H
N
CHIRALCEL OD-H
CHIRALPAK AS-H
Coated
CHIRALPAK AY-H
Coated
Coated
(S)
CHIRALCEL OJ-H
O
O
CHIRALPAK IA
Immobilised
CH3
Cl
Coated
CH3
O
H
N
CHIRALCEL OZ-H
CHIRALPAK IB
Coated
Immobilised
O
CH3
Cl
CH3
Cl
CHIRALPAK IC
CH3
H
N
O
Cl
CH3
H
N
O
CH3
CH3
H3C
H
N
O
O
CH3
H
N
CHIRALPAK AZ-H
Coated
CH3
O
H
N
H
N
Immobilised
H
N
O
Cl
New selector – June 2011
CHIRALPAK ID
CHIRALPAK ID
H
N
O
Cl
O
Cl
O
n
O
N
H
O
O
O
O
HN
Cl
Based on
amylose tris-(3-chlorophenylcarbamate)
A new selector
for the immobilised series!!
Screening in SFC
Primary Screen
Secondary Screen
CHIRALPAK IA
CHIRALPAK IB
CHIRALPAK IC
CHIRALPAK ID
CHIRALPAK AD-H
CHIRALPAK AS-H
CHIRALPAK AY-H
CHIRALPAK AZ-H
Mobile phase system
CHIRALCEL OD-H
CHIRALCEL OJ-H
CHIRALCEL OZ-H
EtOH, MeOH, 2-PrOH
Acetonitrile
Co-solvents
Dichloromethane
Ethyl acetate
THF
MtBE
EtOH, MeOH, 2-PrOH
Acetonitrile
Small Scale method development
•
Samples are screened systematically in LC and SFC with a FAST screening
•
Systematic screening (only 5 µm)
•
•
LC on IA, IB, IC and ID (H/IPA, H/EtOH, H/THF, H/DCM, H/AcOEt, ACN and alcohol)
SFC on IA, IB, IC and ID (MeOH, IPA and EtOH)
•
Coated phases are only screened if no efficient separation
If separation on IA or IB present, but not enough a quick check on AD-H/OD-H
QN/QD are also screened for acids if no efficient separation
•
Short stability test of the sample (with racemate) in the MP
•
Prep systems involved: 2 SP-LC and 2 SFC
•
•
SFC screening in practice
CO2 / MeOH + 1%DEA
80/20
CO2 / IPA + 1%DEA
80/20
CO2 / EtOH + 1%DEA
80/20
1400
1600
1600
1300
3
4
1200
3
1100
2.71
1400
1400
2.68
1200
1000
2.68
1200
900
1000
1000
800
IA
4
5
700
4
600
800
800
3.34
3.68
3.05
600
500
600
1
2 3
400
400
300
1
200
1.01
1
2
400
1.02
1.091.32
2
200
2.45
1.02
1.29
200
100
0
0
0
1
2
3
4
5
6
7
8
9
10
0
0
1
2
3
4
5
6
7
8
9
0
10
1
2
3
4
5
6
7
8
9
10
3
4
5
6
7
8
9
10
3
4
5
6
7
8
9
10
1500
1300
1400
1400
1200
1300
1200
1100
IB
1300
4
1100
4
1200
2.5
1000
2.7
1100
900
1000
900
800
900
800
2
700
2.08
4
2.45
1000
700
3
600
2.32
600
800
3
700
2.09
600
500
500
500
400
400
400
300
200
1
3
1.0
2.36
300
1
200
1.06
2
100
1
2
3
4
5
6
7
8
9
1.34
0
0
1
2
3
4
5
6
7
8
9
10
1200
0
1
2
1800
1100
1200
1100
1600
5
1400
2.67
1000
4
4
1000
900
3.18
3.15
900
800
800
IC
1.04
0
10
1300
1
200
100
0
0
300
2
1.56
100
1200
700
1000
700
3
600
2.99
500
600
5
500
800
3.47
400
600
400
1
300
2 3
400
1.07
2 3
2
1.23
1.44
1.84
200
100
100
0
0
0
1
2
3
4
5
6
7
8
9
10
Also:
THF mixtures
MtBE mixtures
DCM mixtures
Ethyl acetate mixtures …
4
1.07
1.33 1.67
1.18
200
1.06
200
1
300
1
0
0
1
2
3
4
5
6
7
8
9
10
0
1
2
CHIRALPAK® IA
1600
4
CO2 / 2-PrOH + 1% DEA
1400
2.7 min
1200
2.68
80/20
1000
5
800
3.68
600
3.7 min
1
2 3
400
1.02
1.091.32
200
0
0
1
2
3
4
5
6
7
8
9
10
basic molecule
SFC screening in practice
CHIRALPAK IA
O
CHIRALPAK IC
O
H
O
N
C H3
N
S
OM e
N
M eO
C H3
Omeprazole
300
250
CO2 / ethyl acetate 80/20
200
150
T=35°C
Flow rate: 5.0ml/min.
100
50
CHIRALPAK IC
0
H
N
-50
1
2
3
4
5
6
7
8
9
10
H3C
CO2 / THF (+1%DEA) 70/30
O
N
CH3
T=30°C
Flow rate: 3.0ml/min.
CO2 / THF / MeOH 70/28.5/1.5
T=35°C
Flow rate: 4.0ml/min.
Isocratic or gradient?
L. Miller et al., J. Chromatogr. B 875 (2008) 230-236
1.
2.
3.
Combination of LC and SFC
Initial screening in gradient mode
Final isocratic method
Literature example
Isocratic or gradient?
*MeOH % (by volume)
CTE examples
in SFC
45
MeOH gradient program
40
35
30
25
20
15
10
5
0
0
2
4
6
8
10
12
Time (min)
Back pressure: 150bar, Flow rate: 3.0ml/min,
Temperature: 35°C
* Containing 0.3% DEA for screening of basic compounds
Cl
O
Cl
N
N
N
N
OH
O
OH
Ketorolac
Diniconazole
0
2
4
6
8
CHIRALPAK IA
10
12
0
2
4
6
8
min
CHIRALPAK ID
10
12
min
Method optimisation
The use of additives
CHIRALPAK AD-H
20% Isopropanol
20% Isopropanol
+1% Diethylamine
20% Isopropanol
+1% Butylamine
(250 x 4.6 mm)
3 ml/min, 25°C
P outlet 150 bar
Why use immobilised CSPs?
•
Choice in the sample solvent dictated by sample solubility.
•
Free choice of solvents in the mobile phase
•
Greater options for chemical stability
•
New selectivities
•
Use of DCM in sample solvent GREATLY improves solubility,
loadability and SPEED
Solubility issue – Injection in DCM in SFC
DCM is less polar than alcohols
•
•
•
•
•
•
Column : CHIRALPAK IC 5 µm SFC – 25 x 3 cm
Eluent : 70/30 CO2 / EtOH
Flow rate : 120 ml/min
Temperature : 25°C
P outlet : 150 bar
Solubility in EtOH < 2 g/L → injection in EtOH/DCM 90/10
new solubility = 58 g/L
signal UV (PIC04002:ao_F_mes_UV_DETECTOR)
(23/03/2009 14:32:21) -1.7 mv
(23/03/2009 14:42:23) -1.9 mv
Cyclique
signal UV (PIC04002:ao_F_mes_UV_DETECTOR)
(23/03/2009 16:07:12) -0.7 mv
(23/03/2009 16:17:15) 2.3 mv
-0.2 mv (0 jours, 00:10:01)
210.0
610.0
163.0
483.0
116.0
356.0
69.0
229.0
22.0
102.0
-25.0
Cyclique
3.0 mv (0 jours, 00:10:03)
-25.0
23/03/2009
14:32:21
23/03/2009
14:37:22
Analytical injection
23/03/2009
14:42:23
23/03/2009
16:07:12
23/03/2009
16:12:14
23/03/2009
16:17:15
Injection in EtOH/DCM 90/10 – 2ml - 116 mg
No perturbation of the separation
CSP distribution at Small/Medium Scale
CTE data
OD-I
3%
AS-H
1%
OZ
1%
OD-H
1%
AD/AD-H
25%
IC 5µm
45%
IA 5µm
24%
Screening with few CSPs
combining LC and SFC
New chiral selectors
Primary and Secondary screen
Different substituents
New enantiorecognition
CHIRALPAK IA
CHIRALPAK IB
CHIRALCEL OZ
CHIRALPAK AZ
CH3
Cl
H
H
N
N
O
CH3
CH3
O
CHIRALPAK IC
CHIRALPAK AY
Cl
H
H3C
N
O
H
Cl
CHIRALPAK ID
H
N
O
Cl
N
O
Cl
Complementary resolutions
3.0 ml/min, BPr150bar, 30% MeOH (+0.2% TFA) in CO2
O
N
O
OH
Indoprofen
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
min
0
0.5
1
CHIRALPAK IA
0
0.5
1
1.5
2
2.5
3
CHIRALPAK IC
1.5
2
2.5
3
3.5
4
4.5
min
3.5
4
4.5
min
CHIRALPAK IB
3.5
4
4.5
min
0
0.5
1
1.5
2
2.5
3
CHIRALPAK ID
Moving to semipreparative
or
preparative scale
Using different particles sizes in SFC
Adjustment of the needs
650
600
COOH
1
550
3 µm
500
450
2.65
2
400
4.65
350
300
F
250
200
150
550
100
50
500
0
450
1
Flurbiprofen
2.51
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
5 µm
6
400
2
350
4.45
300
250
200
150
100
50
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
350
20 µm
300
CHIRALPAK® AD-type
1
250
2.69
2
200
4.15
CO2 / MeOH 90/10
3 ml/min, 30°C
150
100
50
Columns 150 x 4.6 mm
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
Stack injections
Production level
•
•
•
•
•
•
Column : CHIRALPAK AD-H SFC – 25 x 3 cm
Eluent : 80/20 CO2/MeOH
Flow rate : 120 ml/min
Temperature : 25°C
P outlet : 150 bar
Loading : 200 mg
signal UV (PIC04002:ao_F_mes_UV_DETECTOR)
(08/01/2007 22:08:40) 1476.3 mv
(09/01/2007 02:49:40) 76.0 mv
Cyclique
-1400.3 mv (0 jours, 04:41:01)
2100.0
1639.0
1178.0
717.0
256.0
-205.0
08/01/2007
22:08:40
signal UV (PIC04002:ao_F_mes_UV_DETECTOR)
(03/01/2007 16:07:25) -1.5 mv
(03/01/2007 16:16:16) 14.8 mv
Cyclique
signal UV (PIC04002:ao_F_mes_UV_DETECTOR)
(09/01/2007 00:05:36) -17.1 mv
(09/01/2007 00:52:44) -59.5 mv
16.3 mv (0 jours, 00:08:51)
2500.0
2100.0
1989.0
1639.0
1478.0
1178.0
967.0
717.0
456.0
256.0
-55.0
09/01/2007
00:29:10
09/01/2007
02:49:40
Cyclique
-42.4 mv (0 jours, 00:47:09)
-205.0
03/01/2007
16:07:25
03/01/2007
16:11:50
Injection 200 mg – run time 10 min
03/01/2007
16:16:16
09/01/2007
00:05:36
09/01/2007
00:29:10
09/01/2007
00:52:44
Stack Injections 200 mg every 6 min
Troubleshooting
Dirty frits
First injection
K -2 5 0 1
2
2
R e te n ti o n T i m e
-2
-4
-4
-6
-6
-8
-8
mAU
0
-2
mAU
0
-1 0
-1 0
-1 2
-1 2
-1 4
-1 4
-1 6
-1 6
0
2
4
6
8
10
12
14
16
18
20
22
24
M in u t e s
Second injection - new inlet frit
K -2 5 0 1
1 2.5
1 0.0
1 0.0
7.5
7 .5
5.0
5 .0
2.5
2 .5
0.0
0 .0
-2 . 5
-2 . 5
-5 . 0
-5 . 0
-7 . 5
-7 . 5
0
2
4
6
8
10
12
14
16
18
20
M in u t e s
22
24
26
28
30
32
34
36
38
40
mAU
mAU
R e te n ti o n T i m e
1 2.5
Conclusions
Small and Medium Scale chromatography
by SFC
•
SFC is a fast technique - so use a fast screen
•
Primary screen in SFC alone will give you an 85% success rate overnight!
(99% by SFC and HPLC)
•
Extending primary screen will give you 90% - 95%
•
•
Secondary Screen will give you ca. 100%
Using immobilised CSPs and DCM will make prep FASTER