The first step in the identification of any compound is to obtain that material in pure form. The most common way to achieve this goal today is to use chromatography.
CHROMATOGRAPHIC PURIFICATION
The
first step in the identification of any compound is to obtain that material in
pure form. The most common way to achieve this goal today is to use
chromatography. While a discussion of the many separation and purification
techniques which utilize some form of chromatography are outside the focus of
this book, all rely in one way or another on the interaction of molecules with
a surface. Such interactions depend much more on the chemical properties of a
molecule (functional groups, polarity, unsaturation, etc.) than on physical
properties of the bulk substance (boiling point, vapor pressure, etc.). Furthermore
the interactions of a compound with a surface allow it to be resolved
(separated) from other molecules by placing it in a flowing system (mobile
phase). When the molecule is not adsorbed to the surface, it moves over the
surface at the same velocity as the mobile phase (Figure 11.1).
When
it is adsorbed to the surface, it does not advance with the mobile phase. Since
adsorption is an equilibrium process, those compounds which are only weakly
adsorbed (M in Figure 11.1) to the surface spend a greater portion of
Because different compounds
are adsorbed differently on the surface, each can travel at a different rate
over the surface. By collecting the effluent from the surface as a series of fractions,
individual com-pounds can be separated cleanly from other components in the
original mixture because each component exits the surface at a different time.
Many
different mobile phases have been utilized to provide the forward velocity for
nonadsorbed molecules. If the mobile phase is a gas, then the technique used is
gas chromatography (GC). In GC, the surface to which the molecules adsorb can
be a wide variety of materials which are often prepared by coating an inert
surface with a polymer whose properties are related to its structure. In this
way the surface properties and hence adsorption of the solid surface can be
varied to give the best chromatographic resolution.
If
a liquid is used as the mobile phase, the technique used is liquid chromatography
(LC). The solid adsorbent is constrained in a tube or column through which the
liquid mobile phase flows. Any number of solvents, buffer solutions, or
supercritical fluids can be used as liquid mobile phases. High-pressure liquid
chromatography (HPLC) is used if pressure is needed to force the liquid phase
through the tube. If the liquid phase moves over a thin adsorbent surface
propelled by capillary action, the technique used is thin-layer chromatography
(TLC). In general, two types of surfaces are used as the solid phase.
In
“normal”-phase LC systems, the solid
phase is a polar solid such as silica gel (most common) or alumina and the
liquid is generally an organic solvent of low polarity. In such a case, polar
compounds bind more strongly to the polar silica gel surface and thus travel
more slowly along the surface, whereas nonpolar components have a lower
affinity for the polar surface and a greater affinity for the nonpolar eluting
solvent. They consequently elute from the column more rapidly. In reversed-phase systems, the surface of
silica gel is modified to produce a nonpolar hydrocarbon-derivatized surface,
and the mobile phase often is a polar, aqueous solvent mixture. In this case
polar compounds have a low affinity for the nonpolar surface; they remain
dissolved in the polar mobile phase and elute more rapidly. Nonpolar components
have a higher affinity for the nonpolar surface than the polar mobile phase and
elute more slowly. Using various chromatographic techniques, it is possible to
separate most mixtures into the individual components efficiently and very
rapidly.
In
addition to chromatographic techniques, traditional purification methods such
as recrystallization, distillation, or sublimation are also employed. Such
methods often require much more material than chromatographic techniques.
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