Download - Flash chromatography
FLASH CHROMATOGRAPHY.P R E S E N T E D B Y : -
M I S S . P R A C H I T E E P R A K A S H A Y A R E .
M . P H A R M F I R S T Y E A R .
( Q U A L I T Y A S S U R A N C E )
U N D E R T H E G U I D A N C E O F
M R S . V I N E E T A K H A N V I L K A R .
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INTRODUCTION
It is also called as “Medium Pressure chromatography”.
An air pressure driven hybrid of medium and short column
chromatography.
Invented by Clark & Still of Columbia University in 1978.
An alternative to slow and often inefficient gravity-fed
chromatography.
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Differs from the conventional technique in 2 ways:
Slightly smaller silica gel particles (40 –
63 µm) are used.
Due to restricted flow of solvent caused by the
small gel particles, pressurized gas (10-15 psi)
used to drive the solvent through the column of
stationary phase
The net result is a rapid “over in a flash”and high
resolution chromatography.
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TRADITIONAL COLUMN CHROMATOGRAPHY
Column Chromatography
In the traditional column
chromatography system, the user fills
the glass columns with silica gel.
The sample is placed on the top of the
column. column runs by simply gravity
flow
The separation is very slow and is
restricted to an isocratic solvent
mixture.
At the end of the run, the silica gel must
be removed, cleaned, dried and re-
packed.
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In the modern Flash Chromatography
system the glass columns are replaced
with pre-packed plastic cartridges.
Faster flow rates can be achieved by
using a pump or by using compressed
gas (e.g. air, nitrogen, or argon) to push
the solvent through the column.
Systems may also be linked with
detectors and fraction collectors.
The introduction of gradient pumps
means quicker separations, less
solvent usage and greater flexibility.
EVOLUTION TO THE FLASH
CHROMATOGRAPHY
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PRINCIPLE OF FLASH CHROMATOGRAPHY.
The eluent is, under gas pressure (normally nitrogen or compressed air)
rapidly pushed through a short glass column with large inner diameter.
The glass column is packed with an adsorbent of defined particle size. The
most used stationary phase is silica gel 40 – 63 μm.
STATIONARY PHASE
MOBILE PHASE
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INSTRUMENTATION
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Pump Systems
Pump Controller
Vacuum Pump/peristaltic Pump
Sample Injection Systems
Columns, Filling Sets & Column Valves
Precolumns
Fraction Collector
Detectors and Chart Recorders
Computerize LCD Display
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PROCEDURE FOR MICROSCALE FLASH COLUMN
CHROMATOGRAPHY
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then tamp it down before
scooping more out
Another way to fill the column is to
pour the gel into the column using
a 10 mL beaker
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PRE-ELUTION OF COLUMN
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LOADING OF THE SAMPLE.
1.Wet loading method.
2.Dry loading method.
1.The sample to be purified is dissolved insmall amount of solvent. This solution isloaded onto the column.
2.Dissolve the sample in small amount ofsolvent & add silica gel. Swirl the mixture untilsolvent evaporates & only dry powderremains. This powder is loaded on the top ofthe column.
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ELUTION THROUGH THE COLUMN
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ANALYSIS OF THE FRACTION
If the fractions are colored, combine like-colored fractions, although TLC
before combination is usually advisable.
If the fractions are not colored, they are analysed by TLC .
Once the composition of each fraction is known, the fractions containing the
desired compound(s) are combined.
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Biotag Releveris
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DETECTORS USED IN FLASH CHROMATOGRAPHY
UV-DETECTOR
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ADVANCED DETECTION TECHNIQUES FOR
FLASH CHROMATOGRAPHY
Compounds are often difficult to detect due to a lack of chromophores or, in
the case of natural products, the compound absorbance is unknown.
Evaporative Light Scattering Detection (ELSD) has long been used for High
Performance Liquid Chromatography, but has only recently been employed
for Flash chromatography.
All-Wavelength Collection allows the collection of compound with unknown
absorbance or collection in the presence of interfering solvent absorbance.
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WORKING OF ELSD
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CURRENT TRENDS IN FLASH CHROMATOGRAPHY…
GREEN FLASH CHROMATOGRAPHY.
The ultimate flash chromatographic technology that achieves
most efficient sample purification.
The sample run is always carried out with minimum eluting
volume.
Eco-friendly.
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FEATURES OF GREEN FLASH CHROMATOGRAPHY
Optimal parameters for flow rate, run time, fraction volume, etc. will be
calculated and set automatically upon selecting a column on “Green
Flash” software. The default parameters will be shown in System
Setting window.
Software provides the maximum sample load information for the
selected column.
State-Of-The-Art Software Based On True Theory of Chromatography.
Sample Eluting Position and Resolution Can Be Fully Controlled for
Systems.
Automatic Method Setup for Reverse Phase Chromatography.
Parallel Detection of UV Detector and RI Detector or ELSD.
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ADVANTAGES
Large quantities of the sample can be separated (0.5-2g)
Fast ( 1o to 15 minutes)
Cost efficient
If high resolution is required, flash chromatography is carried out before HPLC to avoid contamination of the expensive columns.
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APPLICATIONS
Flash Chromatography has various applications in following fields
SYNTHETIC CHEMISTRY
It is used as a tool to monitor the reaction progress and
to isolate and identify a mixture’s compounds.
It is used for separation of Closely related organic compounds (isomer).
It is used to purify ,collect and identify the various aromatic
compounds in a semi-synthetic extracts.
Mestranol Purification During Chemical Synthesis.
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Amino modified silica is used with normal phase solvents and is better
suited for nitrogen heterocyclic purification.
It has received increased attention as a lead investigation and
optimization tool in drug discovery.
Bile Acid Purification During Lead Generation in Drug Discovery.
In Anti-malarial Drug Purification in Drug Discovery.
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BIOLOGICAL STUDY
It is used for Purification of Protected Peptides.
Normal phase Flash chromatography purified the protected Peptides
segments and reversed phase Flash chromatography purified final free
peptides.
This two step purification eliminated the traditional costly & difficult
HPLC purification step
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PHYTOCHEMISTRY
Flash chromatography is a very valuable technique in the field of natural
compounds research because it provides a fast and economical way to
separate important phytochemical constituents of complex plant
extracts.
1.Separation and Isolation of α-Santalol and β-Santalol from Sandalwood
Extraction
2 . Isolation and Purification of Chromophoric and Nonchromophoric
Compounds in Giant Knotweed Rhizome.
3. Isolation and Purification of Flavonoids from Ginkgo Biloba Leaves
Extract.
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4 . Isolation and Purification of Ginsenosides from Red
Panax Ginseng Extract.
5. Isolation and Purification of Catechins from Green Tea
Extract .
6 .In Purification of Galla Chinensis.
7. Purification of Ferulic Acid in Rhizoma Chuanxiong
Extract
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REFERENCES
1. Still WC. Kahn M., Mitra A, Flash chromatography, J.Org.Chem. 43(14)1978; 2923-2925.
2. A. B. Roge*, S. N. Firke, R. M. Kawade, S. K. Sarje, and S. M. Vadvalkar, BRIEF REVIEW ON: FLASH CHROMATOGRAPHY, IJPSR (2011), Vol. 2, Issue 8,1930-1937.
3. William CSand Hill DC. General methods for flash chromatography using disposable column. Mol. Divers, 13(2), 2009, 247-252.
4. Hetal Chaudhari*, Falguni Chaudhari, Madhavi Patel, P.K. Pradhan, U.M. Upadhyay, A REVIEW ON A FLASH CHROMATOGRAPHY, International Journal of Pharmaceutical Development & Technology, 2 (2), 2012, 80-84.
5. www.chem.rochester.edu/how to flash.html
6. www.saiadsorbants.com
7. www.sorbeadindia.com
8. www.orgchem.colorado.edu.
9. www. Buchi Preparative Chromatography.
10. Dane Ganesh D*, Raka KC, Honde BS, Bhawal GS, Tajane PJ, REVIEW ON FLASH CHROMATOGRAPHY, Vol 3,Issue 1, 2013 ,45-49.
12. Dewick, P.; John Wiley & Sons, Inc., Hoboken, Medicinal natural products; a biosynthetic approach, 3rd edition ,New Jersey, 2009.
13. www.discoverysciences.com
14. www.pretech.nu/products
15. http://www.sigmaaldrich.com
16. http://yamazenusa.com 31
