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12 FLASH CHROMATOGRAPHY AND LOW PRESSURE CHROMATOGRAPHIC TECHNIQUES FOR SEPARATION OF PHYTOMOLECULES
12.2 Flash Chromatography
Flash chromatography, also known as medium pressure chromatography,
is a rapid form of preparative column chromatography that uses
optimized, prepacked columns through which a solvent is pumped at a high
fl ow rate. Initially developed in 1978 by W. C. Stills of Columbia University,
New York, USA, fl ash chromatography is now a method of purifi cation and
separation using normal phases. Use of reverse phase packing materials is
opening up the technique to a wider range of preparative separations. Currently,
it is considered to be a simple and economical approach to preparative
liquid chromatography (LC).
Flash chromatography differs from conventional techniques
in two ways. First, slightly smaller silica gel particles (250-400 mesh) are
used. Second, due to the limited fl ow of solvents caused by the small gel
particles, pressurized gas (10-15 psi) is used to drive the solvent through
the column of stationary phase. The net result is rapid (“over in a fl ash”) and
high resolution chromatography.
12.2.1 Theory of Flash Chromatography
Chromatography is a separation method that exploits the differences
in partitioning behavior between a mobile phase and a stationary
phase to separate the components in a mixture. Compounds of a mixture
may interact with the stationary phase based on charge, relative solubility
or adsorption. Retention is a measure of the speed at which a substance
moves in a chromatographic system. In continuous development systems
like high performance LC (HPLC) and gas chromatography (GC), where the
compounds are eluted with the eluents, retention is usually measured as
the retention time (Rt or t R ), i.e. the time between injection and detection.
In uninterrupted development systems like thin layer chromatography (TLC),
retention is measured as the retention factor (R f ), i.e. the run length of the
compound divided by the run length of the eluent front:
R f =
Distance travelled by the analyte
Distance travelled by the solvent front
12.2.2 Converting TLC to Flash Chromatography
TLC separations can be used to help determine effective solvent
compositions for fl ash chromatography. R f is a common TLC unit and
ΔR f is the distance between the compounds:
ΔR f = R f1 – R f2
The ideal solvent system for TLC is one that moves the compound
of interest in the mixture to an R f of 0.15-0.35 and that separates
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