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
• Optimization summary
Factors Influencing FLASH Purification
• Purity goals
• Yield goals
• Productivity 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 (DCV)– The DCV / DRf relationship– Sample mass effects
• Sample load– Discovery – scale– Development – scale– Use Biotage loading chart
Solvent Selectivity
(From L.R. Snyder, J. Chromatogr., 92, 223 (1974)).
Hexane/EtOAc 100% CH2Cl2
2:1
C
B?
?A
Solvent Front
Origin
?ABC?
Solvent Front
Origin
Solvent Selectivity GroupDiethyl Ether IMethanol IIEthanol II2-Propanol IITetrahydrofuran IIIAcetone VIaEthyl Acetate VIaAcetonitrile VIbDichloromethane VToluene VIIChloroform VIIIHexane ----
Solvent Strength
Solvent Solvent Strength
Methanol .95Ethanol .882-Propanol .82Acetonitrile .65Ethyl Acetate .58Tetrahydrofuran .57Acetone .56Dichloromethane .42Chloroform .40Diethyl Ether .38Toluene .29Hexane .01 Hexane/EtOAc Hexane/CH2Cl2
1:1 1:2
Solvent Front
Origin
Solvent Front
Origin
Calculated Solvent Strength 0.280.30
Solvent Strength Too Strong
• Both target and impurity outside optimal Rf range (0.15 – 0.35)
• Optimized TLC conditions are not optimized FLASH conditions
AB
0 1 2 3 4 5 6 7 8
Column Volumes
1.0 .9 .8 . 7 .6 .5 .4 .3 .2 .1 0
Rf
SOLVENT
FRONT
ORIGIN
A B
}
Optimal Rf range
Optimized Solvent Strength
• Target and impurity within optimal Rf range
• A “weaker” solvent system greatly improves the FLASH separation
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
A
B
1.0 .9 .8 . 7 .6 .5 .4 .3 .2 .1 0
Rf
SOLVENT
FRONT
ORIGIN
A B
}
Optimal Rf range
Determining Loading Capacity
• Compound resolution key to good loading capacity
• TLC data measured in Rf (retention factor)- DRf not a useful term
• Rf values are inversely proportional to FLASH column volumes (CV)
Rf = 1/CV or
CV = 1/Rf• Resolution (DCV) determines load for any size
cartridge:DCV = CV1 - CV2
• FLASH separations and loading capacity governed by DCV, not DRf
The Rf - CV Relationship
• Lower Rf values mean larger CV and DCV values
• Equal changes in Rf (DRf) do not translate to equal changes in CV (DCV)
• Optimal Rf range(0.15 – 0.35)– For compound of interest
with isocratic elution– Maximum resolution– Maximum loading capacity– Minimal solvent
consumption
DCV
CV10.06.75.04.03.32.82.52.22.01.61.4
1.251.111.0
OptimalRange
Rf0.100.150.200.250.300.350.400.450.500.600.700.800.901.00
1.71.00.70.5
0.050.050.050.05
DRf
DCV vs. DRf
• No DRf change with lowering of Rf• Increasing DCV with decreasing Rf• Predict maximum sample loading better with
DCV than DRf
B
A
B
A
B
A
0 1 2 3 4 5 6 7 8 9 10 Column Volumes
0 1 2 3 4 5 6 7 8 9 10
0 1 2 3 4 5 6 7 8 9 10
1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0 1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0 1 .9 .8 .7 .6 .5 .4 .3 .2 .1 0
R f A = .80 Rf B = .67 Rf = .13 RfA= .47 RfB = .34 Rf = .13 RfA = .32 RfB = .18 Rf = .14 CV = 0.08 CV = 0.8 CV = 2.4
A B
Ori
gin
Ori
gin
Ori
gin
A B A B
Optimal Performance
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95
0.00
10.00 0.00
13.33 3.33 0.00
15.00 5.00 1.67 0.00
16.00 6.00 2.67 1.00 0.00
16.67 6.67 3.33 1.67 0.67 0.00 Delta CV Table17.14 7.14 3.81 2.14 1.14 0.48 0.00
17.50 7.50 4.17 2.50 1.50 0.83 0.36 0.00
17.78 7.78 4.44 2.78 1.78 1.11 0.63 0.28 0.00
18.00 8.00 4.67 3.00 2.00 1.33 0.86 0.50 0.22 0.00
18.18 8.18 4.85 3.18 2.18 1.52 1.04 0.68 0.40 0.18 0.00
18.33 8.33 5.00 3.33 2.33 1.67 1.19 0.83 0.56 0.33 0.15 0.00
18.46 8.46 5.13 3.46 2.46 1.79 1.32 0.96 0.68 0.46 0.28 0.13 0.00
18.57 8.57 5.24 3.57 2.57 1.90 1.43 1.07 0.79 0.57 0.39 0.24 0.11 0.00
18.67 8.67 5.33 3.67 2.67 2.00 1.52 1.17 0.89 0.67 0.48 0.33 0.21 0.10 0.00
18.75 8.75 5.42 3.75 2.75 2.08 1.61 1.25 0.97 0.75 0.57 0.42 0.29 0.18 0.08 0.00
18.82 8.82 5.49 3.82 2.82 2.16 1.68 1.32 1.05 0.82 0.64 0.49 0.36 0.25 0.16 0.07 0.00
18.89 8.89 5.56 3.89 2.89 2.22 1.75 1.39 1.11 0.89 0.71 0.56 0.43 0.32 0.22 0.14 0.07 0.00
18.95 8.95 5.61 3.95 2.95 2.28 1.80 1.45 1.17 0.95 0.77 0.61 0.49 0.38 0.28 0.20 0.12 0.06 0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
Rf1Rf2
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 (DCV)– Larger DCV = 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
• Generic gradient designed to elute compound of interest last
• Use steeper gradient to elute more retained compounds
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 1CVFinal %B – Twice polar concentration of TLC system over 10 CV, hold 2 CV
TLC Scouting
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
• Set load based on Biotage DCV/cartridge chart
• Use flow rate = cartridge diameter
TLC to Gradient Example
• TLC 80:20 Hexane:Ethylacetate (20% EtOAc)– Segment 1, Initial Segment 5% EtOAc– Segment 2, Increase from 5% to 40% EtOAc over
10CV’s– Segment 3, Hold for 2CV’s at 40% EtOAc– Segment 4, If required increase to 100% EtOAc over
3CV’s• 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
Origin
2
3
1
Solvent Front1.00.90.80.70.60.50.40.30.20.10.0
Rf
Sample components1-Nitronaphthalene2-Nitroaniline4-Nitroaniline
Solvent systemHexane/EtOAc 8:2
Gradient Impact on Separation
23
1
10
%B
100%
5%CV
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/minLoad: 50 mg# CV: 10
Linear
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 mLFlow rate: 13 mL/minLoad: 50 mg# CV: 15
2
31 Step
15
Isocratic
%B
75%
20%
0%
100%
10CV
Step
Cartridge: FLASH 12+S (12 x 75 mm)Eluent: Hexane/EtOAc 80:20, isocraticFlow rate: 13 mL/minLoad: 50 mg# CV: 30
2
3
Legend1. 1-Nitronaphthalene2. 2-Nitroaniline3. 4-Nitroaniline
1
20 3010CV
Isocratic
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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