Extraction and Separation of Natural Products - Natural Products Chemistry: Sources, Separations, and Structures - page 13

2.4 REFINED ISOLATION TECHNIQUES

AND CHROMATOGRAPHY

2.4.1 S eparationS of n onpolar C ompoundS
There are three common separation techniques. Thin layer chromatography (TLC)
is by far the simplest, cheapest, and most rapid. Mixtures are applied to precoated
silica gel plates, which are then immersed in a chamber containing a sufficient
amount of the eluting solvent, to allow for solvent migration. As the solvent migrates
up the plate the mixture is separated on the coated plate due to each component
having a different retention time. TLC is used mostly for small quantities of mate-
rial under analysis and is a very versatile technique for nonpolar compounds. For
separation of larger quantities of nonpolar materials, silica gel column chromatog-
raphy (often referred to as normal phase) is commonly used. In this case, an eluent
is passed through a column packed with silica gel. The mixtures of compounds are
separated and are eluted off the silica gel column in the eluting solvent. Two methods
are generally used to prepare a column: the dry method and the wet method. For the
former, the column is first filled with dry stationary phase powder, followed by the
addition of the mobile phase, which is flushed through the column. In the latter case,
a slurry comprised of eluent and the stationary phase powder is mixed and poured
into the column. The stationary phase is often silica gel, but alumina, cellulose, and
polyamide are quite commonly used as well.
The mobile phase (eluent) is either a single solvent or more commonly a mixture
of miscible solvents. The eluent can be optimized in small-scale pretests, often using
TLC with the same stationary phase. One popular form of normal phase chromatog-
raphy is called “flash chromatography” based on the same principle as described ear-
lier, where the particle size of silica gel is much smaller, typically 250-400 mu, and
the mobile phase is passed rapidly over the stationary phase using medium pressure.
For more volatile and nonpolar compounds, gas chromatography (GC) is used.
In this case, a carrier gas (e.g., nitrogen/helium) is passed over a coated and heated
column. This method has been successfully used for the separation of oils, volatiles,
terpenes, and esters of fatty acids.
An example is the separation of long chain fatty acids in fish oils. In order to achieve
a well-resolved GC analysis, it is often necessary to convert these fatty acids to their
respective methyl esters. A polar stationary phase may be used for the chromatographic
separation and a typical analysis of the methyl ester derivatives of cod liver oil is shown
in Figure 2.2 using a fused silica column coated with Carbowax 20M™, a packing
material that contains polyethylene glycol (PEG) polymers bound to silica.

2.4.2 S eparationS of p olar C ompoundS
There are several different chromatographic systems amenable to the separation of
polar compounds. Polar compounds are usually soluble in aqueous systems and if
the compound of interest possesses a charge (due to the presence of either positive or
negative functional groups) then ion exchange chromatography is very useful. The