Flash Chromatography
Presented by
Yusuf Bhimani
Contents
Flash Chromatography
definition & history
Instrumentation of Flash Chromatography
Advantages
Apllications
Introduction
Chromatography is a Greek word chroma "colour" and graphein "to write".
chromatography is a family of analytical chemistry techniques for the
separation of mixtures.
It was the Russian botanist "Mikhail Tsvet" who invented the first
chromatography technique in 1901.
Flash Chromatography
Flash chromatography, also known as medium pressure chromatography.
"An air pressure driven hybrid of medium and short column chromatography
optimized for rapid separation“.
It was popularized several years ago by Clark Still of Columbia University.
An alternative to slow and often inefficient gravity-fed chromatography.
Differs from the conventional technique in 2 ways:
Slightly smaller silica gel particles (250-400 mesh) 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.
Theory
Chromatography exploits the differences in partitioning behavior between a
mobile phase and a stationary phase to separate the components in a
mixture.
Compounds of the mixture interact with the stationary phase based on
charge, relative solubility or adsorption.
The retention is a measure of the speed at which a substance moves in a
chromatographic system.
Principle
The principle is that the eluent is, under gas pressure (normally nitrogen or compressed
air) rapidly pushed through a short glass column.
The glass column is packed with an adsorbent of defined particle size with large inner
diameter.
The most used stationary phase is silica gel 40 -63 um, but obviously packing with other
particle sizes can be used as well.
Particles smaller than 25 µm should only be used with very low viscosity mobile phases,
because otherwise the flow rate would be very low.
Normally gel beds are about 15 cm high with working pressures of 1.5-2.0 bars.
In the mean time, however, and parallel to HPLC, reversed phase materials are used
more frequently in flash chromatography.
The computerized system control the Working of Flash chromatography.
Selection of Stationary Phase
The most important stationary chromatography is silica.
phase in column Silica gel (SiO2) and alumina (A1203) are two adsorbents
commonly used by the organic chemist for column chromatography.
Adsorbent particle size affects how the solvent flows through the column.
Smaller particles (higher mesh values (70-230}) are used for flash
chromatography; larger particles (lower used for gravity mesh values (230-
400}) are used chromatography.
The amount of silica gel depends on the Rf difference of the compounds to be
separated, and on the amount of sample.
Adsorbents which are mainly used in
flash chromatography
Silica: Slightly acidic medium. Best for ordinary compounds, good separation
is achieved.
Alumina: Basic or neutral medium. Can be effective for easy separations, and
purification of amines.
Reverse phase silica: The most polar compounds elute fastest, the most
nonpolars lowest.
Solvent Systems
Flash column chromatography is usually carried out with a mixture of two
solvents, with a polar and a nonpolar component.
[Link]: pentane, petroleum ether,hexanes.
2. Ether and dichloromethane (very similar polarity)
3. Ethyl acetate
[Link]/Petroleum
[Link]/Hexane
[Link]/Pentane
[Link] Acetate/Hexane
[Link]/Dichloromethane
Parts of flash chromatography
[Link] Systems.
Pump Controller.
Sample Injection Systems.
Glass Columns, Filling Sets & ColumnValves.
Pre columns.
Fraction Collector.
Detectors and Chart Recorders.
Computerize LCD Display.
Pump Systems
Pump Controller :
A pressure range up to either 10 bar or 50 bar gives optimum separation
results fora broad range of applications.
The pump modules can be controlled by three different units.
Pump Controller C610
Pump Manager C615
Control Unit C620
1. Pump Controller C-610
The Pump Controller C-610 for one Pump Module C-601 is designed for
isocratic separations.
The flow rate can be easily adjusted by turning a knob and is indicated
by a large illuminated LCD-display.
Delivered with a overpressure sensor for maximum safety.
Pump Manager C-615
Pump Manager C-615 is designed for both isocratic and gradient
separation.
Fast operation, easy programming and a large graphical display allows a
quick and easy set up.
The unit has Input/Outputs for 2 solvent valves and level sensors and
includes a pressure sensor and mixing chamber.
Control Unit C-620
The Control Unit C-620 in combination with Sepacore Control provides
precise control of the chromatography system. The following components
can be connected to the Control Unit C-620.
2 to 4 Pump Modules C-601 or C-605
Up to 2 Fraction Collectors
Up to 8 Detectors e. g. UV, RIS sequential Modules C-623 or C-625 for
autori sequential chromatography on up to 5 columns
Type of pump
Pump Module C-601, 10 bar :-
The Pump Controller C-610 with a Pump Module C-601 is used for fast isocratic Flash
separations.
No programming is needed.
The pump module provides a constant, pulse-free flow from 2.5 to 250 ml/min and
ensures reproducible, fast separation at a maximum working pressure of 10 bar/145 psi. ›
Pump Module C-605, 50 bar :>
The Pump Manager C-615 with a Pump Module C-601/C-605 isused for isocratic Flash
separations.
Pump Manager C-615 :-
Pump Manager C-615 with two Pump Modules C-601/C605 for binary solvent gradients.
The efficient solvent mixing under pressure and the pulsation free solvent flow
eliminate vapour bubbles and result inmaximum separation performance.
Sample injection
Column
Glass Columns :.
A wide range of columns offer maximum flexibility for every situation.
Depending on the nature and the quantity of the sample offers a series
of column types which vary in form, size and performance.
Plastic +Glass Column :
Plastic+Glass-coated Glass Columns are available for larger amounts of
samples and higher pressure applications on a high safety level.
Precolumns :
Pre column are minimizing dead volumes and enhance the life time of
the maincolumn by trapping contaminants.
The small Pre column, fits to Glass Columns of inner diameter of ID 15,
26, 36and 49 mm.
The large Pre column, fits to Glass Columns of ID 70 and 100 mm inner
diameter.
Filling Sets for Glass Columns
Dry Filling Set : The Dry Filling Set is employed for filling glass columns
with silica gel using compressed [Link] gel in the size range of 25-
200micro meters can be packed with this method.
Load the sample onto the silica gel column:
Two different methods are used to load the column.
[Link] Method
[Link] Method
Wet Loading Method
In the wet method, the sample to be purified(or separated into
components) is dissolved in a small amount of solvent, such as hexanes,
acetone, or other solvent.
This solution is loaded onto the column.
Sometimes the solvent choice to load the sample onto the column is
more polar than the eluting solvents. In this case, if we use the wet
method of column loading, it is critical that we only use a few drops of
solvent to load the sample.
If we use too much solvent, the loading solvent will interfere with the
elution and hence the purification or separation of the mixture.
In such cases, the dry method of column loading is recommended.
Dry Loading Method
In dry Loading Method First dissolve the sample to be analyzed
in the minimum amount of solvent and add about 100 mg of
silica gel.
The mixture until the solvent evaporates and only a dry powder
remains.
Place the dry powder on a folded piece of weighing paper and
transfer it to the top of the prepared column.
Add fresh eluting solvent to the top now we are ready to begin
the elution process.
Elute the column
Add a good part of elution solvent to the column.
Apply pressure to force the solvent through the column by pressing with
apipette bulb.
The pressure should be the minimum necessary to keep a steady stream
coming out of the column.
The collection beaker is changed as soon as the colored compound begins to
elute.
The process is complicated if the compound is not colored.
In such experiments, equal sized fractions are collected sequentially and
carefully labelled for later analysis.
Advantages
Fast and economic methods for the synthesis laboratory.
Ideal for the separation of compounds up to gram quantities.
No expensive equipment required.
ideal way transfers results from TLC to CLC.
Automated changes between normal phase and reversed phase
chromatography.
APPLICATIONS OF FLASH CHROMATOGRAPHY
Natural Products/Nutraceuticals Application
Separation and Isolation of a-Santalol and ẞ-Santalol from Sandalwood Extraction.
Isolation and Purification of Flavonoids from Ginkgo Biloba Leaves Extract.
Isolation and Purification of Catechins from Green Tea Extract.
In Purification of Galla Chinensis.
Isolation and Purification of Ginsenosides from Red Panax Ginseng Extract.
Carbohydrate Application
Purification of Conjugated Quercetin and Rutinose.
Impurity Isolation of Valproic Acid from Cyclodextrine During Encapsulation.
Isolation of Aminosugar and Acarbose.
Isolation of Aminoglycoside Antibiotics.
Lipids Application:
[Link] of Fatty Acid Methyl Esters (FAMES).
2. Purification of a Mixture of Glycerides, Mono-, Di-, and Tristearin.
3. Purification of Sterols.
Conclusion
Purification of drug is an important step in any branch of research.
Preparative chromatography is used to separate the components of a mixture
for more advanced use and isthus a form of purification.
Flash Chromatography can be alternative to preparative HPLC as it saves time
and solvent.
