CH437 ORGANIC STRUCTURE ANALYSIS
CLASS 5: MASS SPECTROMETRY 5
Synopsis. Determination of ion formulas (elemental composition) from accurate mass measurement and from isotope peak intensities. Fragmentation: molecular ions, general fragmentation modes, metastable ions.
Interpretation of Mass Spectra: Identification of Ions by Formula Mass
The most important information that can be obtained from a mass spectrum is the formula mass of each ion (this includes relative molecular mass from the molecular ion). This information helps the chemist to devise fragmentation pathways that may ultimately give clues about the structure of an unknown compound.
There are always several molecular formulas possible for a particular formula mass. For example, if the mass spectrum of an unknown compound reveals a molecular ion at m/z = 110, the most likely molecular formulas are C8H14, C7H10O, C6H6O2 and C6H10N2. These all have nominal masses of 110. There are various ways of distinguishing amongst these: sometimes information from other areas or other kinds of spectra can eliminate one or more of these possibilities, but mass spectrometry can be used alone, as described below.
Determination of Accurate Mass
High-resolution mass spectrometers (such as double focusing BE or FTICR instruments) can provide mass measurements that are accurate to better than 0.0001 mass unit, making it possible to distinguish between formulas with the same nominal mass. For example, both C5H12 and C4H8O have formula mass 72, but the accurate masses differ beyond the decimal point: C5H12 has mass 72.0939 amu and C4H8O has mass 72.0575 amu. A high-resolution instrument can easily distinguish between these.
Isotope Peak Intensities
Most elements have less common heavier isotopes (e.g. hydrogen - 2H, carbon – 13C, nitrogen – 15N, etc). The peak representing the molecular ion results from ionization of the molecule containing all the common isotopes (1H, 12C, 15N, etc). Peaks at higher m/z values than the molecular ion (usually of much lower intensity), at M + 1 and M + 2, etc, arise from molecules containing the heavier, less abundant isotopes.
These isotopic peaks make it possible to determine molecular formulas, without the need to determine accurate mass. Knowing the relative natural abundance of isotopes, it is possible to calculate the relative intensity of for each isotopic peak (M + 1, M + 2, etc) for a particular molecular formula. The actual calculations are not dealt with in this course, but the data is tabulated (see, for example, and can be used to match the relative intensities of the M + 1 and M + 2 peaks (etc) of an unknown ion. An example is shown below.
M+1 and M+2 isotopic abundances and exact masses, for CHON ions up to a nominal mass of 100, are given in a separate hand-out.
Elements with high abundance heavier isotopes are easily spotted in mass spectra, by the appearance of high intensity peaks at m/z values two units apart and with intensities matching the relative abundance of the isotopes:
More examples of isotope cluster patterns that are commonly found in the mass spectra of organic compounds are given below.
The lines are in the unenclosed pictures are 2 m/z units apart. In organic molecules, carbon (and nearly always hydrogen and often oxygen) will also be present and their isotopes must be taken into account, as in the enclosed picture, but usually the isotopic patterns arising from combinations of Br, Cl and S can be clearly seen in the presence of many carbon and other atoms, as in the two examples below.
Fragmentation
Fragmentation is a characteristic feature of many mass spectra and is pronounced in those spectra that have been achieved by “hard” ionization (EI) of the compound, but may also be present to a more limited extent in CI, APCI and other “soft” ionization spectra.
Molecular Ions
Generally, organic molecules ionize by losing an electron from:
(i) the highest energy occupied molecular orbital (HOMO) of multiple-bonded or aromatic systems.
(ii) non-bonded orbitals of heteroatoms, such as N, O or S.
Stable (high abundance) molecular ions include those of aromatic derivatives, whereas unstable (low abundance) molecular ions include those of highly branched or long chain molecules and most alcohols.
Fundamental Fragmentation Processes
The way in which a molecular ion breaks up (called the fragmentation pattern or scheme) is a function of its structure and hence can be very useful in the elucidation of structure.
There are three basic fragmentation modes:
(i) Ejection of a radical from a radical ion
(ii) Ejection of a small neutral molecule from a radical ion
These frequently occur via rearrangement.
(iii) Ejection of a small molecule from an ion
A generalization of the above is known as the “even electron rule”, to which there are very few exceptions (e.g. fragmentations of the type, mp+ mf+. + m., are extremely rare).
Representation of Charged Species
Charged species (radical ions and ions) can be represented by generalized structures thus:
More "precise" structures can be used to aid rationalization of fragmentation:
Direction of Cleavage in fragmentation Steps
At least one bond cleavage occurs in a fragmentation step, producing a charged species (ion or radical ion) and a neutral species (radical or molecule). Generally, the fragment that takes the charge is the one whose structure can best stabilize that charge: that is, the fragment that has the lowest ionization energy (IE). The ionization energies of some compounds and radicals are given in the table below.
Compound / IE (eV) / Compound / IE (eV) / Compound / IE (eV) / Compound / IE (eV)CHCH / 11.4 / RCOOR’ / ~10.2 / RCONH2 / ~9.8 / I. / 10.5
CH2=CH2 / 10.5 / n-ROH / ~10.1 / RCH=NH / ~9.6 / SH. / 10.4
n-Alkanes / ~10.4 / RCHO / ~9.8 / Pyridine / 9.3 / CH3. / 9.8
R2CHCHR’2 / ~10.2 / CH3COCH3 / 9.7 / RCH=NR’ / ~9.1 / CH2=CH. / 8.8
C6H12 / 9.9 / ArCOOH / 9.7 / RCONR’2 / ~8.8 / ROCO. / ~8.6
Benzyne / 9.7 / CH2C=O / 9.6 / n-RNH2 / ~8.7 / CH3O. / 8.6
n-Alkenes / ~9.6 / R2O / ~9.5 / Pyrrole / 8.2 / Ar. / ~8.1
Benzene / 9.2 / ArCOR / ~9.4 / R2NH / ~8.0 / CH2=CHCH2. / 8.1
RCH=CHR’ / ~9.1 / n-RSH / ~9.1 / ArNH2 / ~7.7 / HCO. / 8.1
1,3-Butadiene / 9.1 / Thiophene / 8.9 / n-RF / ~12.5 / n-R. / ~8.0
ArCH3 / 8.9 / Furan / 8.9 / n-RCl / ~10.7 / HOCH2. / 7.6
Cyclohexene / 8.8 / ArOH / ~8.5 / n-RBr / ~10.1 / Branched alkyl. / ~6.7
– 7.5
ArCH=CH2 / 8.4 / R2S / ~8.4 / n-RI / ~9.2 / CH3CO. / 7.0
Naphthalene / 8.1 / ArOR / ~8.2 / ArCl / ~9.1 / ROCH2. / ~6.9
CO / 14.0 / CH3SSCH3 / 7.4 / ArBr / ~9.0 / Cyclic C7H7. / 6.2
CO2 / 13.6 / N2 / 15.6 / F. / 17.4 / R2NCR’2.
(R=H or Alkyl) / ~5.4
– 6.1
H2O / 12.5 / HCN / 13.6 / Cl. / 13.0
H2C=O / 10.9 / NH3 / 12.5 / Br. / 11.8
For example, examination of the molecule cocaine indicates that the
tertiary nitrogen group has the lowest IE and hence ionization is most likely to occur this position. This in turn means that fragmentation will probably be based upon this radical ion:
In practice, where there is a reasonable choice of direction of cleavage, both will occur, but the dominant pathway will be that which gives the more stable charged species.
Some Empirical Rules Regarding Fragmentation
The Nitrogen Rule
This “rule” helps in the prediction of whether charged species in the fragmentation pattern are radical ions (odd electron ions, OE+.) or ions (even electron ions EE+), according to whether there are no nitrogen atoms (or an even number of N) or an odd number of nitrogen atoms in the species.
If the precursor species has an even m/z value, the situation is straightforward, as shown below:
If the charged species has an odd m/z value, the situation is not quite as certain as above, but a useful scheme can be drawn, as above.
In practice, an ion of odd m/z value is likely to have an odd number of nitrogen atoms in its structure.
Other Empirical Rules
- It is impossible to lose CH2 (mass 14) from an ion, because the IE of CH2 (methylene carbene) is too high.
- It is almost impossible to lose fragments of m/z values between 2 and 15 from ions containing C, H, (O), (N). If a mass loss of 3 occurs in the mass spectrum of a compound, it must be accompanied by mass losses of 2 and 1. N (mass 14) is not lost from nitrogen-containing ions.
- Mass losses between 20 and 26 and also between 36 and 42 are hardly ever observed.
Common Neutral Losses
Finally, some common neutral losses are given in the table below, along with possible identities.
Loss / Possibilities / Loss / PossibilitiesM – 1 / H. / M – 32 / CH3OH S*
M – 15 / CH3. / M – 33 / HS.*
M – 16 / O (rare, N-oxides); NH2. / M – 35 / Cl.*
M – 17 / OH.; NH3 (rare) / M – 36 / HCl*
M – 18 / H2O / M – 42 / CH2C=C=O CH2=CHCH3
M – 19 / F. / M – 43 / CH3CO. C3H7.
M – 20 / HF / M – 44 / CO2
M – 26 / HCCH CN. / M – 45 / C2H5O. COOH.
M - 27 / HCN H2C=CH2 / M – 46 / NO2 (nitro compounds
M – 28 / CO CH2=CH2 / M – 57 / C4H9. C2H5C.=O
M – 29 / C2H5. HCO. / M – 77 / C6H5. (phenyl)
M – 30 / NO (nitro compounds) CH2O / M – 79 / Br.*
M – 31 / CH3O. / M – 91 / C6H5CH2. (benzyl)
M – 127 / I.
* Isotope peak intensity pattern should be checked
Appearance of a Mass Spectrum
The most common representation of EIMS is as a bar diagram, with % relative abundance (or relative intensity) plotted against m/z. Two different representations of the EI mass spectrum of diethyl 2-acetoglutarate are shown below.
The bar diagram is preferred nowadays, but the profile of ion abundances plot enables the study of small, but significant peaks, such as those of metastable ions (marked *).
Metastable Ions
Metastable ions are characteristic of electron ionization: they appear as broad peaks (usually of low abundance) at non-integral values of m/z, as seen in the BE mass spectrum above.
Origins
Most ions that reach the detector are produced by very rapid decompositions in the ion source: within 10-5 s of the ionization event. However, a few fragmentations require a somewhat longer time and actually occur “in flight”, somewhere in the field-free region between the ion source and the analyzer. These are called metastable ions. The ability to detect these ions depends on the analyzer: the transmission quadrupole (Q) cannot detect them, because the motions of ions in the analyzer are not dependent on conditions that the ions experience before their arrival: a product ion will be detected as such, irrespective of whether it was formed in, or just outside the ion source. Magnetic sector (B) instruments, on the other hand, are able to detect metastable ions, because when a precursor ion fragments in the field-free region before reaching the magnetic field of the analyzer, the fragment ion will lose some kinetic energy to the neutral fragment and so it reaches the detector via a different trajectory to the “normal” fragment ion. See below.
It does not appear at mf on the m/z scale but at a lower value, m*, given by
m* = mf2/mp. (m* is typically non-integral)
The metastable peak is broad because of a narrow range of kinetic energies the fragment ions actually possess. It is of low abundance because only ions that decompose in a narrow time “window” (~10-4 – 10-6 s) are detected in this way. Fragment ions that are produced more rapidly (<~10-6 s) are recorded as “normal” fragment ions, whereas ions that are produced by fragmentations in the analyzer are not focused at a point and hence contribute only to the “background” of the mass spectrum.
The observation of metastable ions is indicative of a process mp mf occurring in one step (i.e. mf is formed directly from mp) and hence is a valuable aid to structure determination.
On the other hand, the absence of metastable ions does not necessarily mean that mf is not formed from mp.