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