Flash Chromatography Method
Development
Michael Ye, Boris Polanuyer, Lew Kurtzman, Daniel Vitkuske,
and Tom Henderson
Supelco, 595 North Harrison Road, Bellefonte, PA 16823
Shaoyin Wang, Brenda Nye, Becky Caproni, and Michael Singer
Sigma RBI, 1 Strathmore Road, Natick, MS 01760
T405018H
HST
Introduction
What is Flash Chromatography?
• A type of preparative purification for rapid isolation of compounds
including reaction mixtures, where the target (synthesized)
molecule must be separated from excess reagents, by-products,
and side-products; natural products – compound of interest have
be separated from matrix and impurities
• Sample range varies from several mg to over 150 g
• Linear flow rates up to 15 cm/min.
• Pressure 10-100 psi
Early Flash Chromatography:
Glass Flash Columns
• 1903 - Michael Tswett reported
separation of plant pigments using
glass columns packed with calcium
carbonate.
• 1978 - Clark Still reported fast flow
glass column chromatography – flash
chromatography
• Do-by-yourselves style
• Significant labor involved in
packing, unpacking and
washing columns
• Safety risks in shattering glass
columns
• Limited flow rate and pressure
• Reproducibility concerns
Modern Flash Chromatography
(1994-Present)
1994 –disposable cartridges for flash
chromatography were introduced
1. Disposable plastic cartridges –
time and reproducibility
2. Cartridges of different size –
easy scale-up
3. Solid sample module and
injection valve – easy sample
loading
4. Pressure up to 100 psi – fast
separation
5. Narrow particle size distribution Low backpressure and higher
efficiency
Modern Flash Chromatography Saves
Time and Money
Glass
Glass
Cost
Time
Flash
Purification Steps
Flash
Areas
Experimental and Results
Flash Chromatography System
The Supelco VersaFlash System
was used in the experiments. It
fits a wide range of cartridge
sizes and cartridge change-out
is very convenient.
Materials in contact with sample
are stainless steel, Teflon®,
PEEK, and polypropylene,
which are inert to most of
samples.
Method Development Examples
• Purification of a synthetic compound
• Purification of plant pigments
• Purification of a synthetic peptide
Purification of a Synthetic Product
1. Product Properties
This a derivative of 3-Indolylacetonitrile, which is a naturally
occurring plant growth hormone. Total of 4 g of reaction
mixture needs to be purified. The mixture was a semi-solid and
soluble in dichloromethane and methanol.
O
CN
H2N
N
H
Purification of a Synthetic Product
2. TLC Method Development
O
CN
H2N
N
H
P
Origin
Conditions:
TLC plate: Silica
Solvent: DCM:MeOH 5:95
Rf values:
Product: 0.38
Impurity: 0.75
Impurity: 0.63
Impurity: 0.58
Impurity: 0.21
Impurity: 0.18
Impurity: 0.17
Purification of a Synthetic Product
3.
Transfer the Method from TLC to
Flash Chromatography
a. Under ideal conditions
Vs =
1
Rf
Vc
Vs is the sample elute volume
Rf is the TLC retention factor
Vc is the cartridge bed volume
b. In practice, we expect some deviations of the equation.
Factors, such as sample quantity, particle size differences,
and moisture content will affect the direct transfer.
Purification of a Synthetic Product
4. Flash Chromatography Conditions and Results
Conditions:
sample size: 4 grams
cartridge size: 150 mm x 40
mm I.D. (96g
silica)
sample loading
method: direct load with
a syringe
mobile phase: DCM:MeOH 5:95
flow rate: 50 mL/min.
Results:
• Total solvent volume: 5 L
• Volume of fractions: 30 mL
• Yield: 56%
• Purity: 100% by TLC
• Total time (sample prep to
purity confirmation): 3
hours
Flash Chromatography of Plant Pigments
1. Product Properties
Chlorophylline (1), astaxanthene (2), and carotene (3) represents
typical groups of plant pigments. There is 0.5 mL, 10 mg/mL of each
compound in dichloromethane. All compounds are colored.
2
1
3
Flash Chromatography of Plant Pigments
2. TLC Method Development
A
B
C
Development Solutions:
A = dichloromethane:methanol, 4:1, v/v
B = dichloromethane:methanol, 10:1, v/v
C = dichloromethane
Carotene (yellow)
Astaxanthene (red)
Chlorophylline (green)
No single mobile phase can provide
isocratic separation in a reasonable
time
Flash Chromatography of Plant Pigments
3. Separation Strategy
2
1
Carotene (yellow)
Astaxanthene (red)
Chlorophylline (green)
1)
Carotene - dichloromethane
(100 mL)
2)
Astaxanthene dichloromethane:methanol
4:1, v/v, (100 mL)
3)
Chlorophyll - invert column to
change direction of flow,
methanol (60 mL)
Flash Chromatography of Plant Pigments
4. The Results
Experimental Conditions:
cartridge: 75 x 40 mm I.D. silica
sample: 0.5 mL, 10 mg/mL of
each compound in
dichloromethane
detection: by the compound
color
Samples from left to right
Chlorophylline (green)
Astaxanthene (red)
Carotene (Orange)
Purification of a Synthetic Peptide
1. Product Properties
Approximately 20 mg of synthetic peptide
Val-Gln-Ala-Ala-Asp-Tyr-Ile-Asn-Gly
In accordance with Expasy ProtParam analysis (www.expasy.ch)
this peptide has the following properties
• Molecular weight: 1063.1
• Theoretical pI: 3.80
Acidic
• Total number of negatively charged residues (Asp + Glu): 1
• Total number of positively charged residues (Arg + Lys): 0
• Aliphatic index: 127.00
Hydrophobic, retention on C18
Purification of a Synthetic Peptide (contd.)
2. Selection of HPLC Conditions
column:
mobile phase:
temp.:
det.:
Discovery C18, 5 cm x 2.1 mm I.D., 5µm particles
linear gradient of acetonitrile:water from 0 to 100%
acetonitrile in 15 min.
room temperature
UV at 254 nm
Product
11.324
600
400
9.251
mAU
200
1.438
6.249
0
0
2
4
6
8
Time (min)
10
12
14
Purification of a Synthetic Peptide (contd.)
3. Selection of Flash Chromatography Conditions
Flash cartridge 75 x 40 mm I.D., packed with spherical silica (C18
bonded; carbon content 26%). Peptide samples (10 mg/mL in 0.02M
KOH, 2mL) were injected using a stop-flow injector.
Based on the HPLC results – the following step gradient conditions
were used:
1.
20% acetonitrile – 200 mL
2.
60% acetonitrile – 200 mL
3.
80% acetonitrile – 200 mL
4.
100% acetonitrile – 200 mL
Purification of a Synthetic Peptide (contd.)
4. Results
The compound of interest was found in the 80% acetonitrile fraction with
95%+ purity and 75% yield. Most of the impurities were concentrated in
60% acetonitrile, the 100% acetonitrile fractions contained no detectable
components.
600
11.305
400
mAU
200
0
0
2
4
6
8
Time (min)
10
12
14
Summary of Method Development
• Understand the physical and chemical properties of the sample,
such as solubility, hydrophobicity, sensitivity, and so on.
• Method development on TLC or HPLC. For isocratic conditions,
TLC is more preferable. For gradient conditions, HPLC is more
preferable.
• Select the appropriate flash chromatography cartridges, size and
type of material.
• Transfer the method to the flash cartridge.
• Recover sample and characterize.
Conclusion
• Modern flash chromatography with disposable cartridges is
applicable to a wide range of compound types.
• Saves time and solvents.
• HPLC linear gradients can be transformed into step gradients
in flash chromatography.
• Flash chromatography can be a reliable and cost-effective
alternative to preparative HPLC.