Natural Products Pres 12-7-08

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Developing a Purification Strategy for Natural Product Extracts Jeff Horsman Commercial Product Manager Phone: 434 825 0058 E-mail: [email protected]

Natural Product Extraction & Isolation • Natural Products are usually extracts from – Plant products – Leaves – Fruits or seeds – Roots

– Fermentation – Fungi – Mycelium – Cell cultures

Typical Plant products • Fresh or dried biomass – Liquidized or ground with solvents to produce biomass slurry

• Biomass slurry separated by filtration or centrifugation – Organic water immiscible solvents – Evaporated to give concentrated extracts

– Aqueous Filtrate/centrate – Product capture step onto solid media – Product extracted into water immiscible solvent

– Solids – Extracted with water miscible solvents

Typical Fermentation products • Whole broth solvent extractions – Water immiscible solvent extracts – Evaporated to give concentrated extracts

• Whole broth separated by filtration or centrifugation – Filtrate/centrate – Water immiscible solvent extracts – Capture steps onto solid media

– Separated solids – Extracted with water miscible solvents

Typical Extraction Protocols • Aqueous extracts – Products retrieved by capture step onto solid media – SDVB resins, HP-20 – Reversed phase media, C18

• Organic solvent extracts – Water miscible high polarity – alcohols

– Water miscible low to medium polarity – Acetone

– Water immiscible

Typical initial Work-up Procedures • Water miscible solvents, direct purification – Concentrated to dryness – Purified by chromatography

– Biotage Snap silica – Absorb extract onto silica – Separate on silica using step gradients through elutropic series, hexane/ethylacetate/methanol

– Biotage Snap C18 – Absorb extract onto C18 silica – Separate on C18 using gradients, step or continuous, increasing in polarity strength, water/methanol

Typical initial Work-up Procedures • Aqueous or water miscible solvents, indirect purification – Diluted with water for capture step – Styrene divinyl benzene (SDVB) resins, HP-20 etc. – Reversed phase media, C18, C8 etc.

– SDVB resins eluted – Water washed – Eluted with step gradients increasing in organic solvent •

Water/alcohol, or water/acetonitrile or water/acetone

– Reversed phase media eluted – Water washed – Eluted with gradients (step or continuous)

Typical initial Work-up Procedures • Water immiscible organic solvents – Evaporated to concentrated extracts

• Direct purification – Chromatography, normal phase or reverse phase – Dried onto silica or C18 to aid column loading – Liquid loaded onto chromatography columns

• Indirect purification – Triturated with other solvents to remove gross impurities – Hexane or di-ethyl ether to remove non-polar oils

– Chromatography, normal phase or reverse phase – Dried onto silica or C18 to aid column loading – Liquid loaded onto chromatography columns

How Can Biotage Help, downstream processing • Isolute products – Phase separation tubes – Separates DCM (or other halogenated solvents) from aqueous, DCM flows through Aqueous retained

– HMN water absorbing media – Traps aqueous allowing organic solvents to flow through, good for non-halogenated water immiscible solvents

– Filter cartridges – Plain Frits – Pre-filled with Celite filter aid

• Available as SPE tube or 96-well plate formats – SPE formats low numbers higher volumes – Typically more manual interaction

– 96-well formats high numbers low volumes – Suitable for automation

How Can Biotage Help, downstream processing • Isolute manifold racks, Vac-Master or Gravity Rack – Vacuum assisted manifold or simple gravity fed – Available as 10 or 20 position – Ideal for – Simple filtration – Capture of organic phase

• Filter into 20 or 30mL vials ideal for evaporation directly on Biotage V-10 high speed evaporator – Sample evaporation to dryness – Pre-absorb onto solid media for

How Can Biotage Help, purification • Chromatography systems – Isolera automated systems – Single channel – 4 channel sequential

– Quad3 UV – 12 channel parallel with 12 channel UV detection

– Simple air driven systems – 1Liter basic system •

Fully manual, good for sample mass up 5g

– Flash 75 systems •

Manual system but can be automated with Isolera system, sample loads up 50g

– Flash 150 systems •

Manual systems, good for sample loads up to 500g

– Flash 400 skid mounted system

Simple FLASH Purification Jeff Horsman

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

Optimize Purification with TLC 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

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 =

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

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

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

Combining Natural Product purification with V-10 and Isolera and Quad3 UV

Isolera Purification System •

State-of-the-art automated flash purification system – –

• • •

Touchscreen graphic user interface with new, intuitive software Two piston pump Two detector options – –







Biotage fixed 254 nm UV Biotage variable UV detector

Large fraction collection volume – –



Single cartridge 4-cartridge sequential

Standard (4.8-L maximum) Extended bed (9.6-L maximum) Molded plastic racks

Uses SNAP, Flash+, and Flash 75 cartridges Elevated solvent reservoir

Two wavelength fractionation 3

• Collect compounds otherwise missed using a single wavelength • Increase compound recovery

1

2

– Compound 1 – At 254 nm – At 330 nm

100% 0%

– Compound 2 – At 254 nm – At 330 nm

100% 0%

– Compound 3 – At 254 nm – At 330 nm

93.8% 94.3%

Conditions System: Isolera One Cartridge: SNAP 50g KP-SIL Solvent A: Heptane Solvent B: Ethyl Acetate Gradient: 10% B for 1 CV 10%-70% B in 5 CV 70% B for 2 CV Flow rate: 40 mL/min Load: 1.5g via Samplet 1 (red): 254 nm 2 (black): 330 nm Fractionation: Low slope

Quatro-binary gradient •

Switching between solvents A/B to B/C during the gradient can… – Elute strongly retained compounds – Clean the cartridge – Convert normal phase systems to reversed-phase without additional priming/flushing

TLC Conditions Solvent system:

1:1 Hexane/EtOAc

Rf N-Phenylbenzylamine Nifedipine 0.25 4-(Dimethylamino)antipyrine Promethazine hydrochloride 0.00

0.76 0.08

A to B Rf 0.76

B to C Rf 0.08

Rf 0.25 Rf 0.00

Conditions System: Isolera One Cartridge: SNAP 50g KP-SIL Solvent A: Heptane Solvent B: Ethyl Acetate Solvent C: Methanol Gradient: 20% B for 1 CV 20%-100% B in 7 CV 100% B for 0.5 CV 10%-60% C in 6 CV 60%C for 2CV Flow rate: 40 mL/min Load: 1 g via Samplet 1 (red): 254 nm 2 (black): 280 nm Fractionation: Low slope

Availability •

Eight system configurations: – One or four flow paths – Fixed (254 nm) detector or variable dualwavelength (200-400 nm) detector – Single or expanded fraction collection bed Single bed



Systems shipped with: – Five SNAP 10g, five SNAP 50g cartridges, and their Samplets, and KP-SIL TLC plates – Five 25g cartridges will be added after their launch

– Four 16 x 150 mm racks (eight with expanded bed)

Expanded bed

u-Wave with V-10 Simple Sample Preparation

Reactions:

2. 60 x 5mL reactions overnight 3. De-capped filtered through Isolute SPE cartridges, transferred to 20mL scintillation a) Filter cartridges b) Phase separator c) Supported liquid extraction cartridge 4. Add 1.25g silica 5. Dry reaction mix onto silica using V-10

Simple Sample addition to cartridge

Sample load: 2. Remove vial from V-10 3. Tap vial to loosen silica 4. Pour sample loaded silica into Snap Cartridge 5. Replace Snap top, ready to run

Running Quad3 UV with 12 samples • Load all 12 samples the same way • Sample preparation and work-up in simple steps – – – – –

Evaporation time 5 minutes/sample Loading time 1 minute/sample Total time for all 12 samples 6 x 12 = 72 minutes Run time for 12 samples on Quad3 UV 20minutes Total time from reaction mix to 12 purified samples 92 minutes!

Quad3 UV system

Full system with new Snap Rack Screen shot with all 12 channels

Data analysis selected channels

Quad3 UV with V-10 Evaporator • Quad3 UV removes purification bottleneck – 12 samples purified in 20 minutes with UV traces

• Next bottleneck is fraction combination and evaporation • Biotage V-10 fully automated system – Will combine fraction and concentrate samples from multiple purification runs – Saves chemist time and avoids mis-matches of samples – Open access software allows simple operation

V-10 with Liquid Handler

V-10 system with liquid handler 2. load Quad3 test tube rack onto liquid handler bed 3. Program selected tubes to combine from for each track 4. System will automatically combined and evaporate samples into the vial of choice

Simple, Efficient Sample Work-up, Organic synthesis or Natural Product extracts Jeff Horsman

Suzuki Coupling Reactions • Two aqueous Suzuki reactions • 1a, 4-Bromo-acetophenone – Simple work-up

• 1b, 4-Iodoaniline – Weigh Iodoaniline directly into u-wave vial – Simple work-up

Reaction 1a

Reaction 1b

Suzuki Reactions simple work-up • De-cap reaction vial • Add 3mL DCM – Shake to extract

• Set up Isolute Phase separator on gravity rack – – – –

Ensure drain valve is closed Pour mixture into Isolute Phase separator Place 20mL scintillation vial under drain valve When DCM separates open drain valve – DCM drains through while retaining the aqueous

Suzuki Reactions Purification • Weigh out 1.25g of silica onto weigh paper • Add this to the DCM solution in the vial • Evaporate DCM using the Biotage V-10 solvent evaporator • Prepare Snap 10g cartridge by removing Snap insert – Pour dried silica/sample mix into the recess – Add Snap top frit and screw top cap back on

• Purify on Isolera using the relevant Suzuki method, 1a or 1b

Suzuki Reactions Purification Suzuki 1a separation

Suzuki 1b separation

Aqueous Suzuki Coupling Workup and Purification

Expected yield of peak 3 = 70mgs Peak 3

Actual mass of recovered peak 3 = 71.6mgs Actual yield = 102%

Suzuki Coupling work-up Options • Traditional work-up 30 to 45 minutes – Manually extract into organic (MeCl2) from DME/EtOH/H2O – Add NaSO4 to MeCl2 to remove trace water – RotaVap to remove organic solvent – Manually Re-dissolve for FLASH purification

• Liquid-liquid extraction typically results in loss of yield.

• Direct dry with V-10, 12 minutes – Pour reaction mixture into 20mL scintillation vial – Dry the aqueous reaction mixture directly with V-10 using Aqueous mode, 12 mins to reduce to ¼ volume – Ready to load onto 25+M C18 cartridge

• 100% yield with no losses and saves 75% of time as well as 4manual steps

V-10 Automated Back Extractions • Small Library 12 reactions – Reaction solvent acetonitrile – Solvent exchange into ethylacetate – Extract impurities with acidified water

• Entire process automated on V10 • V-10 used to: – Evaporate acetonitrile, then re-dissolve in ethylacetate – Add acidified water then use re-dissolve function to extract impurities into aqueous

• Process automated – Better utilization of Chemists time – This type of library work-up very time consuming

Simple, Efficient Evaporation the V-10 Jeff Horsman

Sample Evaporation • A novel approach – New evaporating technology allows rapid low temperature evaporation of solvents from vials under precisely controlled conditions without bumping

V10 Vortex and Vacuum evaporation system

Performance Comparisons Time to evaporate 8-mL of solvent @ the appropriate preset method

Typical Applications • Evaporate liquids with atmospheric boiling points up to 205°C, at temperatures less than 50°C • Evaporation of solvents in a general bench chemistry environment • Open access drying of chromatography fractions, Prep LC or Flash • Preparation of reaction mixture for Flash purification • Concentration of extractions and / or dilute HPLC fractions for analysis V-10 with GX-271 Liquid Handler

The Compound Factory

Simple work-up Synthetic or natural product extracts

V-10 evaporator

Automated Fraction combination and evaporation Parallel UV directed Flash

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