Agriculture Reference
In-Depth Information
9.3.2.
Mobile Phases
In HPLC the mobile phase is a liquid or a mixture of liquids. The common
elutants are water, aqueous solutions, acetonitrile, and methanol. Almost any
other common solvent, compatible with the column packing and the detector,
may be used. In some cases the HPLC instrument will be capable of making
a mixture of elutants or changing the mixture of elutants during chromatog-
raphy. If this is done, care must be taken to make sure that the elutant mixture
is compatible with the detector.
9.3.3.
Stationary Phases
In HPLC columns (Figure 9.4,
C
) the most common packing is a solid with an
organic group attached to it. For instance, the solid may have a hydrocarbon
containing 18 carbons attached to it, making it hydrophobic. This type of
column would be called a C18 column or reverse-phase column. Columns can
be made with varying polarity and functionality and thus be used to carry out
a wide variety of separations.
9.3.4.
Detection
In HPLC four different types of detectors are common: ultraviolet, refractive
index, conductivity, and mass spectrometry. The ultraviolet detector is an ultra-
violet source that passes a specific wavelength of UV light through the sample
as it exits the chromatographic column. The absorbence of the compound is
then recoded. The source of ultraviolet light may be a deuterium lamp with a
filter to remove all except the desired wavelength of light. Alternatively, it may
be designed like a spectrometer such that the analytical wavelength of light
being used can be changed. In the most sophisticated cases the whole spec-
trum of the compound may be taken as it exits the column. In this case the
ultraviolet spectrum may be used to identify the compound.
Refractive index and conductivity detectors are much simpler but cannot
identify compounds eluting from the chromatographic column. The refractive
index of the elutant will change as its composition changes. Thus, as compounds
elute from the column, the refractive index (RI) changes, and this is recorded
to obtain a chromatogram.The conductivity detector is used when water is used
as the elutant and the materials being separated are ionic. When no ions are
present, the conductivity of water is very low; when ions emerge from the
column, the conductivity of the water increases and is recorded, producing the
chromatogram. Chromatographic procedures that involve changing the elutant
during the chromatography will not generally be suitable for these detectors.
The quadrupole mass spectrum detector is used in the same way as it is with
gas chromatography with the same sensitivity and ability to identify com-
pounds. In use most or all the solvent is removed before the sample enters
the MS.
