SCH 2313 THEORY OF SPECTROSCOPIC METHODS

MASS SPECTROMETRY

 Mass spectrometry is a technique that uses difference in the mass to charge ratio of
ionized atoms or molecules to separate them.
 Mass spectroscopy allows the quantitation of atoms or molecules and provides structural
information by the identification of distinctive fragmentation patterns.
 The mass spectrometer is an instrument that produces ions, separates them according to
their m/z values, detects them, and plots the mass spectrum.
 Mass spectrometry is NOT a spectroscopic technique because absorption of
electromagnetic energy is not involved in any way.
 In the mass spectrometer, analyte molecules are converted to ions by applying energy to
them. The ions formed are separated on the basis of their mass-to-charge ratio (m/z) and
directed to a transducer that converts the number of ions (abundance) into an electrical
signal. The ions of different mass-to-charge ratios are directed to the transducer
sequentially by scanning or made to strike a multichannel transducer simultaneously. The
ion abundance plotted against mass-to-charge ratio is called a mass spectrum.
 The mass-to-charge ratio, m/z, of an ion is the quantity of most interest because the mass
spectrometer separates ions according to this ratio. The mass-to-charge ratio of an ion is
the unitless ratio of its mass number to the number of fundamental charges z on the ion.

Mass spectroscopy allows the quantitation of atoms or molecules and provides structural
information by the identification of distinctive fragmentation patterns of inorganic, organic, and
biological molecules.

Mass spectrometry has a number of applications including:

– Determining molecular mass of a compound

– Finding elemental composition of a compound
– Finding out the molecular formula and structure of an unknown substance
– Verifying the identity and purity of a known substance
– Providing data on isotopic abundance (isotopic ratios of atoms and
samples)

Advantages: superb speed, high selectivity and high sensitivity.

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Mass spectrometers require an elaborate vacuum system to maintain a low pressure in all of the
components except the signal processor and display. Low pressure ensures a relatively low
collision frequency between various species in the mass spectrometer that is vital for the
production and maintenance of free ions and electrons.

The sample is vaporized, and then ionized by being bombarded by a beam of high energy
electrons (usually at 70 eV). The electron beam removes an electron from the molecule of the
injected sample, creating a molecular ion (which is also a radical cation because it has an
unpaired electron and a positive charge).

The radical cation corresponding to the mass of the original molecule is called the molecular ion.

Losing an electron weakens the bond, while the collision gives it extra kinetic energy. The
electron beam transfers so much energy to the molecules that many of the molecular ions break
apart into cations, radicals, neutral molecules and other radical cations.

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 There is a pair of oppositely charged plates in the ionization chamber. The positively
charged one causes the positively charged fragments to accelerate into an analyzer tube.
Neutral fragments are not attracted to the negatively charged plates and therefore are not
accelerated. They are pumped out of the spectrometer.
 The analyzer tube is surrounded by a magnet. Its magnetic field deflects the positively
charged fragment in a curved path in proportion to its mass-to-charge ratio (m/z). The
flight path of the ion depends on its molecular mass, its charge, and the strength of the
magnetic field. The smaller the m/z of fragment, the more the magnet deflects it.
 A collector records the relative number of fragments with a particular m/z passing
through the ion exit slit.

The strength of the magnetic field is varied in increments to produce a mass spectrum, which is a
plot of the relative abundance of each fragment ( y axis) versus its m/z value (x axis). Because the
charge (z) of the fragments that reach the collector is +1, m/z is the molecular weight (m) of the
fragment.

ONLY positively charged fragments reach the detector.

LOOKING AT MASS SPECTRA

The sample is acetone. On the horizontal axis is the value for m/z (as we stated above, the charge
z is almost always +1, so in practice this is the same as mass). On the vertical axis is the relative
abundance of each ion detected. On this scale, the most abundant ion, called the base peak, is set
to 100%, and all other peaks are recorded relative to this value. For acetone, the base peak is at
m/z = 43 - we will discuss the formation of this fragment a bit later. The molecular

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weight of acetone is 58, so we can identify the peak at m/z = 58 as that corresponding to the
molecular ion peak, or parent peak. Notice that there is a small peak at m/z = 59: this is referred
to as the M+1 peak. How can there be an ion that has a greater mass than the molecular ion?
Simple: a small fraction - about 1.1% - of all carbon atoms in nature are actually the 13C rather
than the 12C isotope. The 13C isotope is, of course, heavier than 12C by 1 mass unit. In addition,
about 0.015% of all hydrogen atoms are actually deuterium, the 2H isotope. So the M+1 peak
represents those few acetone molecules in the sample which contained either a 13C or
2
H.

Molecules with lots of oxygen atoms sometimes show a small M+2 peak (2 m/z units greater
than the parent peak) in their mass spectra, due to the presence of a small amount of 18O (the
most abundant isotope of oxygen is 16O). Because there are two abundant isotopes of both
chlorine (about 75% 35Cl and 25% 37Cl) and bromine (about 50% 79Br and 50% 81Br),
chlorinated and brominated compounds have very large and recognizable M+2 peaks.

FORMATION OF IONS:

Electron Impact Ionization

• A sample under high vacuum is bombarded with a beam of high-energy electrons emitted
from a hot filament (like the one in a light bulb) are accelerated through 70 V before
interacting with incoming molecules.

• The impact of the electron beam dislodges a valence electron from the gas-phase
molecules, leaving them with a +1 charge and an unshared electron.

• Some molecules (M) absorb as much as 12–15 electron volts (1 eV = 96.5 kJ/mol), which
is enough for ionization.

• The electron kinetic energy of 70 eV is much greater than the ionization energy of
molecules.

• The resulting molecular ion, can have so much extra energy that it breaks into fragments.

Example: Mass spectrum of the sedative pentobarbital from electron impact ionization

 Does not exhibit a molecular ion peak that would be at m/z 226. Instead,
there are peaks at m/z 197, 156, 141, 112, 98, 69, and 55, arising from
fragmentation of the molecular ion.

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Chemical Ionization

• Chemical ionization produces less fragmentation than electron ionization. Ionization

source is filled with a reagent gas such as methane, isobutane, or ammonia, at a pressure
of 100 Pa.

• Energetic electrons (100–200 eV) convert CH4 into a variety of reactive products:

• CH5+ is a potent proton donor that reacts with analyte to give the protonated molecule,
MH+ which is usually the most abundant ion in the chemical ionization mass spectrum.

Mass spectrum of the sedative pentobarbital from chemical ionization – notice lack of fragments

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 In the chemical ionization spectrum of pentobarbital, MH+ at m/z 227 is the second
strongest peak and there are fewer fragments than in the electron ionization spectrum.

Electrospray Ionization

In ESI, a solution of the analyte is sprayed into the vacuum chamber of the mass spectrometer
from the tip of a high-voltage needle. The extreme electrical potential imparts charge to the
mixture, which on evaporation of the solvent in the mass spectrometer produces charged species
of the analyte.

Electrospray ionization works especially well for mass spectrometry of proteins, carbohydrates,
and nucleic acids.

ESI mass spectrometry used to study protein molecular weights and sequence, enzyme–substrate
complexes, antibody–antigen binding, drug–receptor interactions, and DNA oligonucleotide
sequence, as well as simply for small molecules that cannot be ionized by electron impact.

The Mass Spectrum

• The mass spectrum shows the mass of the molecule and the masses of pieces from it.

• Each m/z value is the nominal molecular mass (mass to the nearest whole number) of the
fragment.

• The mass spectrum of methanol is shown.

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• The peak at m/z= 32 is due to the fragmentation that results when an electron is ejected
from methanol. It is the molecular ion of methanol. The molecular ion peak is the peak
with the largest m/z value (unless all molecular ions break into fragments before they
reach the detector).

• The base peak is the one with the highest intensity (most abundant ion). The peak is
assigned a relative intensity of 100% and the relative intensity of the other peaks is
reported as a % of the base peak.

Mass spectrum of pentane

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• Identify the difference between the m/z value of a given ion and that of the molecular ion.
• m/z =43 in the MS of pentane is 29 units smaller than the molecular ion (M-29=43). This
implies loss of an ethyl group. So, m/z =43 is due to the molecular ion minus an ethyl
radical.
• m/z=57, (M-15=57) can be attributed to the molecular ion minus a methyl radical.
• m/z=15 - methyl cation
• m/z=29 – ethyl cation.
• m/z=43 can further fragment to m/z=42 and m/z=41 by loss of one or two hydrogen atoms.

Isotopes in mass spectrometry

• Although the molecular ion of pentane has m/z = 72, the spectrum shows a small peak at
m/z=73.

• This is called a M+1 peak because the ion responsible for this peak is one unit heavier
than the molecular ion. This is due to presence of one atom that is a heavier isotope.

– Only 13C,15N, and 33S contribute significantly to the M+1 peak

– In the case of carbon, the peak indicates that the molecular ion contains one 13C
instead of a 12C. Can be used to find the number of carbon atoms in a compound.

• M+2 peak has a mass two higher than M.

– The elements that make significant contributions to this peak are 34S, 37Cl, and
81
Br.

– The M:M+2 ratio indicates the presence of S(100:4.4), Cl (100:31.9), or

Br(100:97.2) in the compound.

Isotopes in mass spectrometry-Bromine

 has two isotopes, 79Br (50.69%), 81Br (49.31%) ratio ~1:1

– presence of Br, M+ ~ M+2, 2 mass units apart, of equal relative abundance.

– C-Br bond is weakest bond in resulting radical cation , most easily broken.

– Bond breaks heterolytically.

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Isotopes in mass spectrometry- Chlorine

• Two isotopes, 35Cl (75.77%), 37Cl (24.23%) ratio ~3:1

• M+2 is ~ 1/3 as large as M+

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• Base peak m/z 43 arises from heterolytic cleavage of C-Cl bond to give propyl cation.

• Alternative fragmentation involves homolytic α cleavage of C-C bond & loss of methyl
radical;

• Remaining fragment, still containing Cl in isotopic 3:1 ratio, forms resonance stabilised
cation.

Isotopes in mass spectrometry- Sulphur

• M+2 larger than usual (4% of M+)

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NITROGEN AND HYDROGEN RULES

The Nitrogen Rule states that if m/z for M is odd, then the molecular formula must have an odd
number of nitrogens. If m/z for M is even, then the molecular formula must have an even number
of nitrogens (this includes 0).

The Hydrogen Rule states that the maximum number of hydrogens in the molecular formula is
2C+N+2. In the formula, C: # of carbons, N: # of nitrogens

Nitrogen: Odd number of N = odd MW

Determination of molecular formulas

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Molecular structure from the formula

MOLECULAR FRAGMENTATION PATTERNS

HOMOLYTIC α-CLEAVAGE

• One electron at a time cleavage, 1 electron remains with each fragment.

• Positive charge developed through loss of one electron.
• If electronegative atom (N, O) present, positive charge charge will be on the
electronegative atom.
• Homolytic α-cleavage is promoted by the presence of heteroatoms (N, O, S). Homolytic
α-cleavage is the major fragmentation pattern for molecules containing heteroatoms.
• In molecules containing heteroatoms, it gives to the base peak.
• Loss of the largest possible radical most favored, leads to base peak.
Homolytic α-cleavage of ethanol

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• Many drugs contain heteroatoms, fragmentation is usually homolytic α-cleavage adjacent

to the atoms.
• E.g bupivacaine – nitrogen directs the homolytic α-cleavage in the heterocyclic ring
resulting in an ion at m/z 140 which dominates the spectrum.
• Similar fragmentation occurs for other local anaesthetics like prilocaine and procaine

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HETEROLYTIC α-CLEAVAGE

• Two electrons at a time cleavage. Both electrons stay with one fragment
• Electrons move toward charged site.

• Produces minor ion in the mass spectrum.

CLEAVAGE WITH PROTON TRANSFER

Also common in drug molecules.

Can start with homolytic α-cleavage, followed by loss of neutral hydrocarbon fragment.

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RING REARRANGEMENT
• Fragmentation of molecules that contain a ring in their structure involves the
rearrangement of the molecule structure before fragmentation.
• Cleavage of cyclohexanol with ring rearrangement.

RETRO-DIELS–ALDER FRAGMENTATION

Cycloalkenes can undergo a retro-Diels–Alder fragmentation to an alkene and an alkadienyl

radical cation.

McLAFFERTY REARRANGEMENT

Can occur in carboxylic acids, esters, ketones and amides which have a side chain containing at
least 3 carbon atoms.
Not very common in drug molecules, because where possible homolytic α-cleavage will
dominate.
Example- ketone

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• Requires H in γ position. Involves homolytic β cleavage, H migration from γ C to O, &

extrusion of ethene to produce resonance stabilized cation.

 Multiple McLafferty is possible

Aromatic Compounds - Tropylium Ion

• Readily formed in compounds with a benzyl ring.

• Benzyl CH2 becomes incorporated into the seven-membered ring structure in which the
positive charge on the ion is delocalized around the ring structure giving a very stable
cation.

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Example: The spectrum of levodopa (L-dopa) is dominated by an ion at m/z 123.

• This ion arises from the formation of a tropylium ion.

• In L-dopa, homolytic cleavage next to the N atom also occurs to give a fragment at m/z
73 but this fragment is of lower abundance than the one at m/z 123.

Molecular Formula Determination

1. Figure out the number of carbons = 4

– Use the relative abundance value of your M+1 value. Make sure the relative
abundance of your M value is set to 100%, scale M+1 and M+2 accordingly.

2. Determine the presence of sulphur, chlorine, or bromine.

3. Figure number of N, O and H

– Subtract the mass of the number of carbons and any element present in the M+2
spectrum from the mass of the M isotope.

– Use the remaining mass to determine how many nitrogen, oxygen, and hydrogen
are possible (nitrogen and hydrogen rule). Alternate between possible numbers of
O and N.

4. Eliminate impossible formulas using the hydrogen rule to determine the maximum
number of hydrogens for each proposed structure.

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Molecular Formula Determination - Example 1

1. Find the number of carbons

2. The relative abundance of the M+2 peak indicates the presence (or absence) of S (4.4%),
Cl (33%), or Br (98%) (see Table 9.4). For our unknown, M+2 = 0.3%; thus, we can
assume that S, Cl, and Br are absent.

3. Determine the number of hydrogen atoms and add the appropriate number of oxygen
atoms, if necessary.

– For our unknown the M+ peak at m/z= 72 gives the MW. Also indicates (since it
is even) that N is absent because a compound with four carbons (as established
above) and two N (to get an even MW) would have a molecular weight (76)
greater than that of our compound.

– For a molecule composed of C and H only, H=72 - (4 x12) = 24

– but C4H24 is impossible.

– For a molecule composed of C, H, and one O, H = 72 - (4 x12) – 16= 8

– Our unknown has the molecular formula C4H8O.

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