Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

12 FLASH CHROMATOGRAPHY AND LOW PRESSURE CHROMATOGRAPHIC TECHNIQUES FOR SEPARATION OF PHYTOMOLECULES
12.4 Adsorbent Selection and Mode of Separation
Typical sorbents for normal phase fl ash chromatography are
polar (e.g. silica, NH 2 ) and elution solvents are non-polar (e.g. hexane, heptane,
dichloromethane, sometimes modifi ed with small amounts of more
polar solvents such as isopropanol). The sample is usually applied in a weak
(very non-polar) solvent, and separation occurs when the elution solvent is
applied. In normal phase fl ash chromatography, the most non-polar sample
component elutes fi rst, followed by successively more polar compounds.
12.4.1 Isolute Flash Columns
Isolute fl ash columns (Biotage) are polypropylene columns prepacked
with Isolute fl ash sorbents. These columns are appropriate for use
in both off-line and on-line fl ash chromatography. In off-line fl ash chromatography,
chemists apply the sample and elution solvent volumes to the
top of an Isolute column fi xed in a FlashVac Sample Processing Station. In
off-line fl ash chromatography, chemists perform isocratic or step gradient
separations. In on-line fl ash chromatography, an Isolute column is mounted
on a system that connects the column to an external liquid pump system to
produce a continuous fl ow of solvent through the column. Depending on the
capability of the pump, the solvent composition can be isocratic (a single
solvent or solvent mixture) or a gradient with an increasing proportion of
stronger solvent (either in a step gradient or a linear gradient).
12.4.2 Method Development Using Isolute Flash Columns
12.4.2.1 Column Equilibration
Prior to sample loading, the column should be prepared for the
separation by equilibration (prewetting) with a suitable solvent:
• Off-line. Apply the equilibration solvent to the top of the column
and allow it to fl ow through the column under gravity.
• On-line. Equilibrate the flash column for on-line mode separation
in the off-line mode using a vacuum manifold such as the
FlashVac system or by mounting the column on the on-line
apparatus and pumping a suitable volume of equilibration solvent
through the column.
12.4.2.2 Typical Equilibration Solvents
Suitable solvents are non-polar, e.g. hexane or pentane. For
best results, prewet the silica and NH 2 columns prior to use. A suitable volume
for column equilibration is approximately two bed volumes (Table 3).
200