preparative chiral chromatography what can go wrong … · 2020-03-30 · preparative chiral...
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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
Summary
1) Preparative chiral chromatography
FeaturesPotential
2) What can go wrong? How to solve it?
SampleChromatographic methodChromatographic systemProduct recovery
3) Conclusions
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
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.
Preparative chiral chromatography
Several systems and modes possible
Batch LC Batch SFC
SMB
To be chosen based on recognition and scale
Preparative chiral chromatography
What can go wrong?
&
How to solve it?
What can go wrong?
… Observing the different elements…
… to find the potential solution.
SAMPLE
METHOD
SYSTEM RECOVERY
What can go wrong?Some examples
Not exhaustive list
SAMPLESolubilityImpuritiesStability
METHODRobustness TemperatureConditioning
Recycling
SYSTEMPacking
Static Electricity
RECOVERYResidual solventSample VolatilityFoaming
SAMPLESolubilityImpuritiesStability
METHODRobustness TemperatureConditioning
Recycling
SYSTEMPacking
Static Electricity
RECOVERYResidual SolventSample VolatilityFoaming
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
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
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
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
TroubleshootingContaminants
Blocked Frits: 5 cm SFC columns
When the inlet frit is blocked, there is a high pressure differential across it; it may deform…
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
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
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
SAMPLESolubilityImpuritiesStability
METHODRobustness TemperatureConditioning
Recycling
SYSTEMPacking
Static Electricity
RECOVERYResidual SolventSample VolatilityFoaming
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
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
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!!
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
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
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
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)
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
SAMPLESolubilityImpuritiesStability
METHODRobustness TemperatureConditioning
Recycling
SYSTEMPacking
Static Electricity
RECOVERYResidual SolventSample VolatilityFoaming
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
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
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
SAMPLESolubilityImpuritiesStability
METHODRobustness TemperatureConditioning
Recycling
SYSTEMPacking
Static Electricity
RECOVERYResidual SolventSample VolatilityFoaming
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
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.
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
PREPARATIVE CHIRAL SEPARATIONS
Our Vendor seminar today at 12:15 h
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