Developing a Purification Strategy

for Natural Product Extracts

Jeff Horsman
Commercial Product Manager
Phone: 434 825 0058
E-mail: Jhorsman@Biotage.com
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 Solvent Solvent
Methanol II
Ethanol II
? C
2-Propanol II A
B B
Tetrahydrofuran III C ?
?
Acetone VIa
Ethyl Acetate VIa ?
A
Acetonitrile VIb
Dichloromethane V Origi Origi

Toluene VII
Hexane/EtOAc 100%
Chloroform VIII CH2Cl2

(From L.R. Snyder, J. Chromatogr., 92, 223

 Solvent Strength

Solvent Solvent Strength

Solvent Solvent
Methanol .95
Ethanol .88
2-Propanol .82
Acetonitrile .65
Ethyl Acetate .58
Tetrahydrofuran .57
Acetone .56
Dichloromethane.42
Chloroform .40
Origi
Diethyl Ether .38 Origi

Toluene .29 Hexane/EtOAc

Hexane .01 Hexane/CH2Cl2

Calculated Solvent 0.30 0.28

Strength
 CV vs. Rf

A A A

Origin
Origin

Origin
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
B B B

A
A A

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
9 10 9 10 Column 9 10

• No Rf change with lowering of Rf

• Increasing CV with decreasing Rf
• Predict maximum sample loading better with
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 CV • Lower Rf values mean larger CV
0.10 Rf 10.0 CV
and CV values
0.15 0.05 6.7 • Equal changes in Rf (Rf) do
Optimal 1.7
0.20 0.05 5.0 not translate to equal changes
Range 1.0
0.25 0.05 4.0 in CV (CV)
0.30 0.05 3.3 • Optimal Rf range(0.15 – 0.35)
0.35 2.8 – For compound of interest
0.40 2.5 with isocratic elution
0.45 2.2 – Maximum resolution
0.50 2.0 – Maximum loading capacity
0.60 1.6 – Minimal solvent consumption
0.70 1.4
0.80 1.25
 Optimal Performance
Rf 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Rf
1
20 0
.05 .00
0 10.0 0
0 13.3 3 0
0 15.0 5 1 0
.20 0 .00 .67 .00
0 16.0 6 2 1 0
0 16.6 6 3 1 0 0 Delta CV
0 17.1 7 3 2 1 0 0
.35 4 .14 .81 .14 .14 .48 .00
0 17.5 7 4 2 1 0 0 0
0 17.7 7 4 2 1 1 0 0 0
0 18.0 8 4 3 2 1 0 0 0 0
.50 0 .00 .67 .00 .00 .33 .86 .50 .22 .00
0 18.1 8 4 3 2 1 1 0 0 0 0
0 18.3 8 5 3 2 1 1 0 0 0 0 0
0 18.4 8 5 3 2 1 1 0 0 0 0 0 0
.65 6 .46 .13 .46 .46 .79 .32 .96 .68 .46 .28 .13 .00
0 18.5 8 5 3 2 1 1 1 0 0 0 0 0 0
0 18.6 8 5 3 2 2 1 1 0 0 0 0 0 0 0
0 18.7 8 5 3 2 2 1 1 0 0 0 0 0 0 0 0
.80 5 .75 .42 .75 .75 .08 .61 .25 .97 .75 .57 .42 .29 .18 .08 .00
0 18.8 8 5 3 2 2 1 1 1 0 0 0 0 0 0 0 0
0 18.8 8 5 3 2 2 1 1 1 0 0 0 0 0 0 0 0 0
0 18.9 8 5 3 2 2 1 1 1 0 0 0 0 0 0 0 0 0 0
.95 5 .95 .61 .95 .95 .28 .80 .45 .17 .95 .77 .61 .49 .38 .28 .20 .12 .06 .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
– Single cartridge
– 4-cartridge sequential
• 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
– 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 100%
– At 330 nm 0%
– Compound 2 Conditions
System: Isolera One
– At 254 nm 100% Cartridge: SNAP 50g KP-SIL
Solvent A: Heptane
– At 330 nm 0% Solvent B: Ethyl Acetate
Gradient: 10% B for 1 CV
– Compound 3 10%-70% B in 5 CV
– At 254 nm 93.8% 70% B for 2 CV
Flow rate: 40 mL/min
– At 330 nm 94.3% Load: 1.5g via Samplet
1 (red): 254 nm
2 (black): 330 nm
Fractionation: Low slope
Quatro-binary gradient

A to B B to C
• Switching between
solvents A/B to B/C during Rf 0.76 Rf 0.08
the gradient can…
– Elute strongly retained
compounds
– Clean the cartridge Rf 0.25
– Convert normal phase Rf 0.00
systems to reversed-phase
without additional
priming/flushing

TLC Conditions Conditions

Solvent system: 1:1 Hexane/EtOAc System: Isolera One
Cartridge: SNAP 50g KP-SIL
Solvent A: Heptane
Rf Solvent B: Ethyl Acetate
N-Phenylbenzylamine 0.76 Solvent C: Methanol
Nifedipine 0.25 Gradient: 20% B for 1 CV
4-(Dimethylamino)antipyrine 0.08 20%-100% B in 7 CV
Promethazine hydrochloride 0.00 100% 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 dual-
wavelength (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 • Direct dry with V-10, 12

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 • 100% yield with no
• Liquid-liquid
extraction typically
results in loss of
yield.
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
losses and saves 75%
of time as well as 4-
manual 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

V-10
evaporator
Simple
work-up

Synthetic or natural Automated Fraction

product extracts combination and
evaporation

Parallel UV
directed Flash