Biology Reference
In-Depth Information
when current is applied, these cations migrate toward the cathode, which causes a fluid
migration through the capillary. This flow can be adjusted by changing the dielectric
strength of the buffer, altering the pH, adjusting the voltage, or changing the viscosity.
Under these conditions both anions and cations are separated in a single separa-
tion, with cations eluting first. Neutral molecules (e.g., pesticides) can be separated by
adding a detergent (e.g., sodium dodecyl sulfate) to the buffer, forming micelles into
which neutral molecules will partition based on their hydrophobicity. Because the
micelles are attracted to the anode, they move toward the cathode at a slower rate than
does the remainder of the fluid in the capillary, thus allowing separation. This process is
called micellar electrokinetic capillary chromatography. Many of these analyses can be
carried out in 5 to 10 min with sensitivities in the low parts per billion range.
Mass Spectroscopy
Although there are many types of detectors available, especially for GC, the primary
type of detector used for studies such as pesticide biotransformation is the mass spec-
trometer. The mass spectrometer is an outstanding instrument for the identification of
a wide range of compounds. It is widely used as a highly sensitive detection method
for GC and is increasingly used with HPLC and CE because technological hurdles
have been solved, allowing these instruments to be interfaced with a mass spectrom-
eter. Chromatographic techniques (e.g., GC, CE, HPLC) are used to separate indi-
vidual components as previously described. A portion of the column effluent passes
into the mass spectrometer, where it is bombarded by an electron beam. Electrons or
negative groups are removed by this process, and the ions produced are accelerated.
After acceleration they pass through a magnetic field, in which the ion species are sep-
arated by the different curvatures of their paths under gravity. The resulting pattern is
characteristic of the molecule under study. Although there are many varieties of MS
instruments, they share four common components: a system to introduce the sample,
a method to produce ions, a method to separate and resolve the charged particles, and
a detector. For example, a sample extract is injected into a gas chromatograph and the
compounds are eluted into the mass spectrometer, the individual compounds are bom-
barded by an ionization source, and the resulting fragments are separated within the
detector based on their mass-to-charge ratio (
m/z
). The resultant output, or the mass
spectrum of a given compound, is unique, much like a fingerprint. Of the four com-
ponents listed above, we have already discussed the first one. With the exception of the
use of a direct injection probe to analyze the sample, MS is generally paired with an
analytical instrument, such as a gas chromatograph or an HPLC apparatus to achieve
separation of multiple analytes. So let us now turn our attention to the various meth-
ods of ionization. The most well established method, especially with regard to volatile
samples, is electron-impact (EI) ionization. In this type of instrument, the mobile phase
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