Relative to electron impact
or chemical ionization, “soft”-ionization techniques like electrospray
ionization and atmospheric-pressure chemical ionization require
different skills for interpreting mass spectra. Important points are
Electron impact ionization (EI) is considered a “hard”
ionization process that imparts a large quantity of energy into
molecules. It is very good for producing fragments that generate
information about the structure of the compound. Often, the molecular
ion does not appear in the EI spectrum or is a smaller peak, but if the
molecular ion is observed, it will be the highest mass in the spectrum.
Chemical ionization (CI) also causes fragmentation, but to a lesser
extent than EI. Relatively new “soft” ionization techniques like
electrospray ionization (ESI) and atmospheric-pressure chemical
ionization (APCI) call for different skills for interpreting mass
spectra, which possibly are unfamiliar to those trained to analyze EI
or CI spectra.
With an EI or CI spectrum, the typical approach is to identify the
molecular ion and fragment ions. Fragmentation is studied and general
rules are followed based upon functional groups, bond energetics, and
rearrangements. EI mass spectral patterns are reproducible, and
fragmentation patterns are used as “fingerprints.” EI mass spectra of
many compounds have been published, and these can be used to identify
In interpreting ESI mass spectra of larger molecules, it is important
to consider that the soft ESI process causes less fragmentation. The
ESI spectrum will contain multiply charged molecules, and adduct
formation and noncovalent -mers (dimer, trimer) add to the complexity
of the spectrum.
It is important to understand the ESI process. Droplets are generated
when a high voltage is applied to a liquid stream. In ESI, larger
droplets explode into smaller droplets, and the process is repeated
until the analyte enters the gas phase in ion form. Consequently, ESI
of a single molecular species usually produces a population of multiply
charged molecules that is reflected in the intensity of the peaks in
the spectrum. The number of positive charges that a molecule can carry
is related commonly to the number of basic sites on the molecule. To
promote positive-ion formation from a positive-ion-mode analyte,
dissolve the analyte in a low pH medium. Negative-ion analysis normally
is conducted above the molecule’s isoelectric point, to deprotonate the
Concepts to remember about the general spectrum form, and when
examining ESI spectra, include the following:
The y-axis typically is intensity and is relative to the
tallest peak in the spectrum (also known as the “base peak”) with the
tallest peak set to 100%.
The x-axis is the mass divided by the charge m/z. Here, m is the ion mass, not the
molecular mass, and z is the
number of charges per ion. This is important because often, multiply
charged ions are seen. Initially, this can be unfamiliar, because with
EI, only singly charged ions are formed, and one becomes accustomed to
thinking of the x-axis as representing mass.
Similarly, it is common to use the term “molecular ion”
when referring to an ESI peak, but molecular ions per se are not
observed commonly in ESI. A molecular ion is formed through the loss of
an electron, but in the ESI process, ionization is accomplished through
the loss or gain of a proton (hydrogen).
Steps to interpreting ESI mass spectra and determining the mass of a
Find the tallest peak in the spectrum above a 200–300 m/z value. There will be
considerable solvent noise at the low end of the spectrum.
Look for peaks that are roughly double or half the mass;
this will reveal whether multiply charged species are present.
Calculation of multiply charged peaks is an essential skill (Figure 1).
A single charge is mass + 1 proton (M + 1); m/z is then (M + 1)/1.
A double-charge is mass + 2 protons (M + 2); m/z is then (M + 2)/2.
To convert from m/z
to mass, assume a tripe-charged peak (M + 3) = Q.
To determine mass, reverse the math: (Q x 3) – 3 = mass. (continued)