Biology Reference
In-Depth Information
containing the sample is passed through a beam of highly energetic electrons (≈70 eV),
which are generated from a filament. This causes fragmentation of the molecule in a
pattern that is indicative of the compound. In addition, some of the molecules may
remain intact, producing a molecular ion, from which molecular weight is determined.
The mass spectra produced by EI are fairly reproducible and there are large databases
available with libraries of EI mass spectra.
With some compounds, the use of EI is too energetic to produce a molecular ion,
and in these cases a “softer” method can be used: chemical ionization (CI). Chemical
ionization can either be negative chemical ionization or positive chemical ionization
(PCI), but in both cases, the ionization is achieved through the use of a reagent gas
such as methane. For example, in PCI, the gas-phase reactions with methane form a
strong acid, CH , which can then protonate the sample molecule, forming the molec-
ular ion.
The previous two methods, EI and CI, are primarily used with GC instruments
and thus are restricted to compounds that are thermally stable. However, compounds
that decompose upon heating, or are polar and/or very large are more amenable to
being run on an HPLC apparatus, which requires a different type of system to pro-
duce ions. Much of this is because the amount of solvent exiting the LC column is
far more than exits the GC column. One common method is electrospray ionization
(ESI). In this method, the solvent exits the LC column via a needle that has a high-
voltage charge applied to it. This produces charged droplets that contain solvent mol-
ecules as well as the analyte molecules. As the droplets evaporate, charged molecules are
ejected from the droplet. This technique is particularly useful for large molecules such
as proteins. Other methods include atmospheric pressure chemical ionization (APCI)
and thermospray ionization. Ionization with APCI is similar to ESI, but instead of an
energized needle, the solvent enters a 500°C tube and is nebulized with an inert gas
such as nitrogen. Ions are then formed in the plasma as they exit the tube. This is not a
good choice for compounds that are thermally unstable. Finally, the use of a laser light
can provide a soft ionization method. The most commonly known is that of matrix-
assisted laser desorption (MALDI). This method is not coupled with an inlet such as
GC or HPLC, but rather is used as a more direct method. The sample is embedded in
a matrix that absorbs light at a λ near that of the laser, thus protecting the biomolecule
from being broken apart. However, the energy ionizes the molecules in the sample.
Once the sample has been separated chromatographically and the analytes have
been broken into ionized fragments, those fragments must be separated with a mass
analyzer. The mass analyzer is that part of the instrument that sorts through all of the
molecular ions and fragments that have been generated by the ionization source of the
instrument. Generally speaking, there are two ways that the instrument can do this.
The first is to continuously scan for every possible ion fragment all the time. This is
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