Flash Optimization 5-6-08
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Simple, Efficient FLASH Optimization Jeff Horsman
Outline
• Factors influencing FLASH purification • Optimizing isocratic purification • Optimizing gradient purification
Factors Influencing FLASH Purification
• Purity goals • Yield goals
Influencing Purification Goals • TLC – Surface chemistry – Resolution (DCV) – Elution solvents
• Sample solvent • Impurities – Excess starting materials – Synthetic by-products
• Elution conditions – Isocratic – Gradient
Optimizing Isocratic Purification Use TLC to determine: • Optimal solvent conditions – Solvent selectivity – Solvent strength
• Sample load factors – Resolution (CV) – The CV / Rf relationship – Sample mass effects
• Sample load – Discovery – scale – Development – scale – Use Biotage loading chart
Solvent Selectivity Solvent Selectivity Group Diethyl Ether I Methanol II Ethanol II 2-Propanol II Tetrahydrofuran III Acetone VIa Ethyl Acetate VIa Acetonitrile VIb Dichloromethane V Toluene VII Chloroform VIII (From L.R. Snyder, J. Chromatogr., 92, 223
Solvent
Solvent
? A B C ?
C B ?
? A
Origi
Hexane/EtOAc CH2Cl2
Origi
100%
Solvent Strength Solvent
Solvent Strength Solvent
Methanol .95 Ethanol .88 2-Propanol .82 Acetonitrile .65 Ethyl Acetate .58 Tetrahydrofuran .57 Acetone .56 Dichloromethane.42 Chloroform .40 Diethyl Ether .38 Toluene .29 Hexane .01
Origi
Solvent
Origi
Hexane/EtOAc Hexane/CH2Cl2 Calculated Solvent Strength
0.30
0.28
Solvent Strength Too Strong Optimal Rf
B
}
A
S OF LR VO EN NT T
1.0 .9 .1 0
AB
.8
.7
.6
R
.5
O R I G I N
.4
.3
.2
0
1
2
3
4
5
Column
• Both target and impurity outside optimal Rf range (0.15 – 0.35)
6
Optimized Solvent Strength
}
Optimal Rf
S O L V E N T
A B
A B
F R O N T
1.0 .9 .1 0
.8
.7
.6
R
.5
O R I G I N
.4
.3
.2
0
1
2
3
4
5
6
7
Column
• Target and impurity within optimal Rf range • A “weaker” solvent system greatly improves
8
Determining Loading Capacity • Compound resolution key to good loading capacity • TLC data measured in Rf (retention factor) - Rf not a useful term
• Rf values are inversely proportional to FLASH column volumes (CV)
Rf = 1/CV or CV = 1/Rf • Resolution (CV) determines load for any size cartridge: CV = CV1 - CV2 • FLASH separations and loading capacity governed by CV, not Rf
The Rf - CV Relationship Rf
Optimal Range
0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.60 0.70 0.80
CV Rf
0.05 0.05 0.05 0.05
10.0 6.7 5.0 4.0 3.3 2.8 2.5 2.2 2.0 1.6 1.4 1.25
CV
1.7 1.0
• Lower Rf values mean larger CV and CV values • Equal changes in Rf (Rf) do not translate to equal changes in CV (CV) • Optimal Rf range(0.15 – 0.35) – For compound of interest with isocratic elution – Maximum resolution – Maximum loading capacity – Minimal solvent consumption
CV vs. Rf
1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0
0
RfA= .47
9
1
10
RfB = .34
1 .9 .8 .7 .6 .5 .4 .3 .2 .1
Rf = .13
RfA = .32 B
B A
A
A
0
Origin
1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0
Rf = .13
R f A = .80 Rf B = .67 .18 Rf = .14 B
A
Origin
A Origin
A
2
3
4
5
6
7
8
0
9
1
10
2
3
4
Column
5
6
7
8
0
9
1
10
2
3
4
5
6
• No Rf change with lowering of Rf • Increasing CV with decreasing Rf • Predict maximum sample loading better with
7
8
RfB =
Optimal Performance Rf
0
0
0
0
.05 0
0 .00 10.0
0
0
13.3
3
0
0 .20 0
15.0 0 16.0
5 .00 6
1 .67 2
0 .00 1
0
0
16.6
6
3
1
0
0
0 .35 0
17.1 4 17.5
7 .14 7
3 .81 4
2 .14 2
1 .14 1
0 .48 0
0 .00 0
0
0
17.7
7
4
2
1
1
0
0
0
0 .50 0
18.0 0 18.1
8 .00 8
4 .67 4
3 .00 3
2 .00 2
1 .33 1
0 .86 1
0 .50 0
0 .22 0
0 .00 0
0
0
18.3
8
5
3
2
1
1
0
0
0
0
0
0 .65 0
18.4 6 18.5
8 .46 8
5 .13 5
3 .46 3
2 .46 2
1 .79 1
1 .32 1
0 .96 1
0 .68 0
0 .46 0
0 .28 0
0
18.6
8
5
3
2
2
1
1
0
0
0
0 .80 0
18.7 5 18.8
8 .75 8
5 .42 5
3 .75 3
2 .75 2
2 .08 2
1 .61 1
1 .25 1
0 .97 1
0 .75 0
0 .57 0
0
18.8
8
5
3
2
2
1
1
1
0
0 .95
18.9 5
8 .95
5 .61
3 .95
2 .95
2 .28
1 .80
1 .45
1 .17
0 .95
Rf
20
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0 .13 0
0 .00 0
0
0
0
0
0
0 .42 0
0 .29 0
0 .18 0
0
0
0
0 .77
0 .61
0 .49
0
0 .08 0
0 .00 0
0
0
0
0
0
0
0 .38
0 .28
0 .20
0 .12
0 .06
0
Delta CV
0 .00
Why use Column Volumes • Easy scale-up – Optimization work on TLC – Quick and cost effective – Test many solvent systems at the same time – Direct scale up to any cartridge size
– Direct relationship between cartridge sizes – Using CV is independent of flowrate – Scale up based on flowrate requires column diameter ratio calculations
Sample Load Factors • Resolution (CV) – Larger CV = larger loads
• Mass ratios – Beware of overload – Total loadable mass based on amount of crude, not amount of product
• Required purity – Higher purity requirements = lower loads, lower yields
• Required yield – Higher yield requirements = lower purity
• Cartridge size – Larger cartridges = larger loads
Optimizing Gradient Purification
TLC Scouting TLC: Use binary solvent mixture to develop TLC method – Rf~ 0.4 for target component
FLASH Gradient: Initial %B - Use ¼ of the polar solvent composition from TLC 1CV Final %B – Twice polar concentration of TLC system over 10 CV, hold 2 CV
• Generic gradient designed to elute compound of interest last • Use steeper gradient to elute more retained
Optimizing Gradient Purification • Always TLC Sample Biotage Algorithms set gradient according to the following rules – Measure Rf – Try for compound of interest Rf = 0.4 – Gradient conditions – Initial = ¼ polar solvent concentration from TLC – Final = Twice polar solvent concentration from TLC – Segment 1 = 1 CV @ initial conditions – Segment 2 = 10 CV, Initial to Final conditions – Segment 3 = 2 CV @ final conditions – Segment 4 = 3CV Final conditions to 100% polar solvent (may not be required)
• Difficult samples are no problem
TLC to Gradient Example • TLC 80:20 Hexane:Ethylacetate (20% EtOAc) – Segment 1, – Segment 2, 10CV’s – Segment 3, – Segment 4, over 3CV’s
Initial Segment 5% EtOAc Increase from 5% to 40% EtOAc over Hold for 2CV’s at 40% EtOAc If required increase to 100% EtOAc
• Above example is for initial work – If the same or similar sample is run again vary slightly based on earlier separation – Remove segment 3 or 4 if not required – Use 8CV’s instead of 10 for main gradient
Case Study Nitro-organics
Solvent Front
1 2 3 Origin
1.0 0.9 0.8 0.7 0.6 0.5 Rf 0.4 0.3 0.2 0.1 0.0
Sample components 1-Nitronaphthalene 2-Nitroaniline 4-Nitroaniline Solvent system Hexane/EtOAc 8:2
Gradient Impact on Separation Isocratic Legend 1. 1-Nitronaphthalene 2. 2-Nitroaniline 3. 4-Nitroaniline
1 2
Cartridge: FLASH 12+S (12 x 75 mm) Eluent: Hexane/EtOAc 80:20, isocratic Flow rate: 13 mL/min Load: 50 mg # CV: 30
3 CV
10
20
30
Linear 100%
1 2
3
%B
10
CV
5%
Step 1
3
100%
Step
75%
2
%B
Isocratic CV
20% 10
15
0%
Cartridge: FLASH 12+S (12 x 75 mm) Eluent: A) Hexane B) EtOAc Gradient: Linear, 5%B to 100%B in 60 mL (10 CV) Flow rate: 13 mL/min Load: 50 mg # CV: 10
Cartridge: FLASH 12+S (12 x 75 mm) Eluent: A) Hexane B) EtOAc Gradient: Step 1 - 20%B for 60 mL Step 2 - 75%B for 30 mL Flow rate: 13 mL/min Load: 50 mg # CV: 15
FLASH Optimization Summary • Optimize solvent systems for maximum separation performance – Adjust selectivity first – Adjust solvent strength for Rf between 0.15 0.35 (CV = 6 - 3) for isocratic elution – Adjust solvent strength for Rf = 0.4 (CV = 2.5) for gradient elution
• Calculate CV and CV from Rf data • Use Biotage loading charts for initial load
Outline
• Factors influencing FLASH purification • Optimizing isocratic purification • Optimizing gradient purification
Factors Influencing FLASH Purification
• Purity goals • Yield goals
Influencing Purification Goals • TLC – Surface chemistry – Resolution (DCV) – Elution solvents
• Sample solvent • Impurities – Excess starting materials – Synthetic by-products
• Elution conditions – Isocratic – Gradient
Optimizing Isocratic Purification Use TLC to determine: • Optimal solvent conditions – Solvent selectivity – Solvent strength
• Sample load factors – Resolution (CV) – The CV / Rf relationship – Sample mass effects
• Sample load – Discovery – scale – Development – scale – Use Biotage loading chart
Solvent Selectivity Solvent Selectivity Group Diethyl Ether I Methanol II Ethanol II 2-Propanol II Tetrahydrofuran III Acetone VIa Ethyl Acetate VIa Acetonitrile VIb Dichloromethane V Toluene VII Chloroform VIII (From L.R. Snyder, J. Chromatogr., 92, 223
Solvent
Solvent
? A B C ?
C B ?
? A
Origi
Hexane/EtOAc CH2Cl2
Origi
100%
Solvent Strength Solvent
Solvent Strength Solvent
Methanol .95 Ethanol .88 2-Propanol .82 Acetonitrile .65 Ethyl Acetate .58 Tetrahydrofuran .57 Acetone .56 Dichloromethane.42 Chloroform .40 Diethyl Ether .38 Toluene .29 Hexane .01
Origi
Solvent
Origi
Hexane/EtOAc Hexane/CH2Cl2 Calculated Solvent Strength
0.30
0.28
Solvent Strength Too Strong Optimal Rf
B
}
A
S OF LR VO EN NT T
1.0 .9 .1 0
AB
.8
.7
.6
R
.5
O R I G I N
.4
.3
.2
0
1
2
3
4
5
Column
• Both target and impurity outside optimal Rf range (0.15 – 0.35)
6
Optimized Solvent Strength
}
Optimal Rf
S O L V E N T
A B
A B
F R O N T
1.0 .9 .1 0
.8
.7
.6
R
.5
O R I G I N
.4
.3
.2
0
1
2
3
4
5
6
7
Column
• Target and impurity within optimal Rf range • A “weaker” solvent system greatly improves
8
Determining Loading Capacity • Compound resolution key to good loading capacity • TLC data measured in Rf (retention factor) - Rf not a useful term
• Rf values are inversely proportional to FLASH column volumes (CV)
Rf = 1/CV or CV = 1/Rf • Resolution (CV) determines load for any size cartridge: CV = CV1 - CV2 • FLASH separations and loading capacity governed by CV, not Rf
The Rf - CV Relationship Rf
Optimal Range
0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.60 0.70 0.80
CV Rf
0.05 0.05 0.05 0.05
10.0 6.7 5.0 4.0 3.3 2.8 2.5 2.2 2.0 1.6 1.4 1.25
CV
1.7 1.0
• Lower Rf values mean larger CV and CV values • Equal changes in Rf (Rf) do not translate to equal changes in CV (CV) • Optimal Rf range(0.15 – 0.35) – For compound of interest with isocratic elution – Maximum resolution – Maximum loading capacity – Minimal solvent consumption
CV vs. Rf
1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0
0
RfA= .47
9
1
10
RfB = .34
1 .9 .8 .7 .6 .5 .4 .3 .2 .1
Rf = .13
RfA = .32 B
B A
A
A
0
Origin
1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0
Rf = .13
R f A = .80 Rf B = .67 .18 Rf = .14 B
A
Origin
A Origin
A
2
3
4
5
6
7
8
0
9
1
10
2
3
4
Column
5
6
7
8
0
9
1
10
2
3
4
5
6
• No Rf change with lowering of Rf • Increasing CV with decreasing Rf • Predict maximum sample loading better with
7
8
RfB =
Optimal Performance Rf
0
0
0
0
.05 0
0 .00 10.0
0
0
13.3
3
0
0 .20 0
15.0 0 16.0
5 .00 6
1 .67 2
0 .00 1
0
0
16.6
6
3
1
0
0
0 .35 0
17.1 4 17.5
7 .14 7
3 .81 4
2 .14 2
1 .14 1
0 .48 0
0 .00 0
0
0
17.7
7
4
2
1
1
0
0
0
0 .50 0
18.0 0 18.1
8 .00 8
4 .67 4
3 .00 3
2 .00 2
1 .33 1
0 .86 1
0 .50 0
0 .22 0
0 .00 0
0
0
18.3
8
5
3
2
1
1
0
0
0
0
0
0 .65 0
18.4 6 18.5
8 .46 8
5 .13 5
3 .46 3
2 .46 2
1 .79 1
1 .32 1
0 .96 1
0 .68 0
0 .46 0
0 .28 0
0
18.6
8
5
3
2
2
1
1
0
0
0
0 .80 0
18.7 5 18.8
8 .75 8
5 .42 5
3 .75 3
2 .75 2
2 .08 2
1 .61 1
1 .25 1
0 .97 1
0 .75 0
0 .57 0
0
18.8
8
5
3
2
2
1
1
1
0
0 .95
18.9 5
8 .95
5 .61
3 .95
2 .95
2 .28
1 .80
1 .45
1 .17
0 .95
Rf
20
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0 .13 0
0 .00 0
0
0
0
0
0
0 .42 0
0 .29 0
0 .18 0
0
0
0
0 .77
0 .61
0 .49
0
0 .08 0
0 .00 0
0
0
0
0
0
0
0 .38
0 .28
0 .20
0 .12
0 .06
0
Delta CV
0 .00
Why use Column Volumes • Easy scale-up – Optimization work on TLC – Quick and cost effective – Test many solvent systems at the same time – Direct scale up to any cartridge size
– Direct relationship between cartridge sizes – Using CV is independent of flowrate – Scale up based on flowrate requires column diameter ratio calculations
Sample Load Factors • Resolution (CV) – Larger CV = larger loads
• Mass ratios – Beware of overload – Total loadable mass based on amount of crude, not amount of product
• Required purity – Higher purity requirements = lower loads, lower yields
• Required yield – Higher yield requirements = lower purity
• Cartridge size – Larger cartridges = larger loads
Optimizing Gradient Purification
TLC Scouting TLC: Use binary solvent mixture to develop TLC method – Rf~ 0.4 for target component
FLASH Gradient: Initial %B - Use ¼ of the polar solvent composition from TLC 1CV Final %B – Twice polar concentration of TLC system over 10 CV, hold 2 CV
• Generic gradient designed to elute compound of interest last • Use steeper gradient to elute more retained
Optimizing Gradient Purification • Always TLC Sample Biotage Algorithms set gradient according to the following rules – Measure Rf – Try for compound of interest Rf = 0.4 – Gradient conditions – Initial = ¼ polar solvent concentration from TLC – Final = Twice polar solvent concentration from TLC – Segment 1 = 1 CV @ initial conditions – Segment 2 = 10 CV, Initial to Final conditions – Segment 3 = 2 CV @ final conditions – Segment 4 = 3CV Final conditions to 100% polar solvent (may not be required)
• Difficult samples are no problem
TLC to Gradient Example • TLC 80:20 Hexane:Ethylacetate (20% EtOAc) – Segment 1, – Segment 2, 10CV’s – Segment 3, – Segment 4, over 3CV’s
Initial Segment 5% EtOAc Increase from 5% to 40% EtOAc over Hold for 2CV’s at 40% EtOAc If required increase to 100% EtOAc
• Above example is for initial work – If the same or similar sample is run again vary slightly based on earlier separation – Remove segment 3 or 4 if not required – Use 8CV’s instead of 10 for main gradient
Case Study Nitro-organics
Solvent Front
1 2 3 Origin
1.0 0.9 0.8 0.7 0.6 0.5 Rf 0.4 0.3 0.2 0.1 0.0
Sample components 1-Nitronaphthalene 2-Nitroaniline 4-Nitroaniline Solvent system Hexane/EtOAc 8:2
Gradient Impact on Separation Isocratic Legend 1. 1-Nitronaphthalene 2. 2-Nitroaniline 3. 4-Nitroaniline
1 2
Cartridge: FLASH 12+S (12 x 75 mm) Eluent: Hexane/EtOAc 80:20, isocratic Flow rate: 13 mL/min Load: 50 mg # CV: 30
3 CV
10
20
30
Linear 100%
1 2
3
%B
10
CV
5%
Step 1
3
100%
Step
75%
2
%B
Isocratic CV
20% 10
15
0%
Cartridge: FLASH 12+S (12 x 75 mm) Eluent: A) Hexane B) EtOAc Gradient: Linear, 5%B to 100%B in 60 mL (10 CV) Flow rate: 13 mL/min Load: 50 mg # CV: 10
Cartridge: FLASH 12+S (12 x 75 mm) Eluent: A) Hexane B) EtOAc Gradient: Step 1 - 20%B for 60 mL Step 2 - 75%B for 30 mL Flow rate: 13 mL/min Load: 50 mg # CV: 15
FLASH Optimization Summary • Optimize solvent systems for maximum separation performance – Adjust selectivity first – Adjust solvent strength for Rf between 0.15 0.35 (CV = 6 - 3) for isocratic elution – Adjust solvent strength for Rf = 0.4 (CV = 2.5) for gradient elution
• Calculate CV and CV from Rf data • Use Biotage loading charts for initial load
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