Types of Chromatography
Flash chromatography
Flash chromatography is a type of
preparative liquid chromatography used for the separation of organic
compounds. This is adsorption chromatography for the routine
purification of organic compounds. By using the flash technique
chromatographers can scale up normal phase chemistries from thin layer
chromatography (TLC) helping to satisfy the demands of the
pharmaceutical and biotech industries in the transition to large scale
purification of organic compounds and peptides. The technique utilizes
an air pressure driven hybrid of medium pressure and short column
chromatography optimized for particularly rapid separations.1
Flash is very similar to traditional column chromatography except that
solvent is driven through the column by applying positive pressure.
Resolution is measured in terms of the ratio of retention time (r) to
peak width (w, w/2). The technique simply uses a set of chromatography
columns and flow controller valves. Modern flash chromatography systems
are very convenient, being sold as prepackaged plastic cartridges with
solvent being pumped through the cartridge.
Column
Chromatography
Column chromatography (which is the
basis for flash chromatography) follows the same principles as thin
layer chromatography (TLC). The main difference is that TLC separates
miniscule amounts of material whereas column chromatography can be used
to separate large amounts of material. If the solvent flows down the
column by gravity or percolation the technique is called gravity column
chromatography. If the solvent is forced down the column by positive air
pressure it is called flash chromatography. The term flash
chromatography was first used by Dr. W. Clark at Columbia University
because the technique allows organic compounds to be purified “in a
flash”.
Column chromatography involves
stationary and mobile phases. In column chromatography the stationary
phase (a solid absorbent) is placed in a vertical column and the mobile
phase (liquid) is added to the top and flows down through the column by
either gravity or external pressure. In column chromatography the
stationary phase is most commonly either silica (Si02) or alumina
(Al2O3). The columns packed with silica usually have a defined particle
size of 40-60 microns. The mobile phase is normally a mixture of hexane
and ethyl acetate. Mobile phases with low viscosity require smaller
particle sizes. The stationary phase is normally more polar than the
mobile phase.
By increasing the polarity of the solvent system all components of the
mixture move faster. By lowering the polarity all components move more
slowly.
The eluting power of
organic solvents
The highest polarity being the most
powerful eluters (at the top of the list)
- Acetic acid
- Alcohol
- Acetone
- Ethyl acetate
- Diethyl ether
- Halogenated hydrocarbons (methylene
chloride)
- Toluene
- Alkanes (hexanes, petroleum ether)
Dry Column Chromatography
Dry
column chromatography (DCC)
is a versatile Prep LC method
Basically, any sample that can be
separated on silica gel or neutral alumina TLC plate can also be
separated by the corresponding DCC-setup. The dry-column procedure
has been successfully applied for the preparation of dye-stuffs,
alkaloids, and other heterocyclic substances which are known to be
separated on other types of columns, but, with
considerable difficulties. Lipids have also been successfully
separated.
Dry column chromatography bridges the gap between analytical
TLC and preparative classical column chromatography. The cost is
much less
than the cost incurred in instrumental pressure associated
with preparative liquid chromatography.
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The load sample versus adsorbent is maintained at
approximately less than 1:500 in TLC while the ratio is
1:300 or even higher for dry column chromatography.
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The
Dry Column Technique
Bridges the gap between preparation
column chromatography and analytical thin-layer chromatography.
Dry Column
Chromatography
This is a unique
and simple method for purifying material. If inexpensive and fast. It
is single column elution technique. Below is a schematic form of the
method.
Dry Column
Chromatography DCC Compared to TLC
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Chromatographic
Parameters
|
TLC |
DCC |
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Solvent
Reservoir
Solvent Force
“Charge” Addition of Sample
Support
Adsorbent
Adsorbent Activity
Equilibrium with solvent vapor
Dimensions of width: thickness: length
Adsorbent bed
Detection
Techniques for Recovery
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tank
capillary
pipette
glass, plastic
silica, alumina, polyamide
low
partial
(sometimes
controlled)
width: thickness: length
200 :1: 200
visible, UV
spray techniques
scrape off
elute |
overhead
gravity
pipette
nylon tubes
silica. alumina
low
none
1 :1: 20
visible,
UV
cut into sections
elute |
