Sunday, April 20, 2008

lecture 2- The mass spectrum and its interpretation

2. The Nature of the Mass Spectrum:
Mass spectra are routinely obtained at an electron beam energy of 70 electron volts.
The mass spectrum produced will usually be presented as a vertical bar graph, in which each bar represents an ion having a specific mass-to-charge ratio (m/z) and the length of the bar indicates the relative abundance of the ion. The most intense ion is assigned an abundance of 100, and it is referred to as the base peak. The abundance of other ions formed is presented as pecentage of the most abundant ion (base peak). Most of the ions formed in a mass spectrometer have a single charge, so the m/z value is equivalent to mass itself. Modern mass spectrometers easily distinguish (resolve) ions differing by only a single atomic mass unit (amu), and thus provide completely accurate values for the molecular mass of a compound. The highest-mass ion in a spectrum is normally considered to be the molecular ion (produced due to the simplest event that occurs in the ion source which is the removal of a single electron from the molecule in the gas phase by an electron in the electron beam). On the other hand the lower-mass ions are fragments from the molecular ion, assuming the sample is a single pure compound.


  • Most stable organic compounds have an even number of total electrons, reflecting the fact that electrons occupy atomic and molecular orbitals in pairs. When a single electron is removed from a molecule to give an ion (the molecular ion)M+
  • The molecular ion in a mass spectrum is always a radical cation, but the fragment ions may either be even-electron cations or odd-electron radical cations, depending on the neutral fragment lost. The simplest and most common fragmentations are bond cleavages producing a neutral radical (odd number of electrons) and a cation having an even number of electrons. A less common fragmentation, in which an even-electron neutral fragment is lost, produces an odd-electron radical cation fragment ion. Fragment ions themselves may fragment further. As a rule, odd-electron ions may fragment either to odd or even-electron ions, but even-electron ions fragment only to other even-electron ions.

Generally the tendency has been to represent the molecular ion with a delocalized charge. There is a different approach which is to localize the positive charge on either a л bond (except in conjugated ystems), or on a heteroatom.

The probability of cleavage of a particular bond is related to the bond strength, to the possibility of low- energy transmitions, and the stability of the fragments both charged and uncharged formed in the fragmentaion process. Our knowledge of pyrolytic cleavages , can be used to some extent, to pridict likely modes of cleavage of the molecular ion. Because of the extremely low vapur pressure in the mass spectrometer, there are very few fragment collisions; we are are dealing largely with unimolecular decompositions.

Whereas conventional organic chemistry deals with reactions initiated by chemical reagents, by thermal energy, or by light, mass spectrometry is concerned with the consequences suffered by an organic molecule struck by an ionizing electronic beam at a vapour pressure of about 10-5 mmHg.

A number of general rules for predicting prominent peaks in electron impact spectra can be written and reaction analized by using standard concepts of physical oreganic chemistry:

  1. Relative hight of the molecular ion peak is greatest for the straight-chain compound and decreases as the degree of branching increases.
  2. The relative hight of the molecular ion peak usually decreasing with increasing molecular weight in a homologous series. Fatty esters appear to be an exception.
  3. Cleavage is favoured at alkyl substituted carbon;the more substituted, the more likely is cleavage. This is a consequence of the increased stability of a tertiary carbocation over over a secondary, which in turn is more stable than primary. Generally, the largest substituent at a branch is eliminated most readily as a radical, persumably because a long-chain radical can acheive some stability by delocalization of the lone electron.
  4. Double bonds, cyclic structures, and especially aromatic (or heteroaromatic) rings stabilize the molecular ion,and thus increase the probability of its appearance.
  5. Double bonds favor allylic cleavage and give the resonance stabilized allylic carbonium ion.
  6. Saturated rings tend to lose side chains at the α-bond. This is merely a special case of branching (Rule 3). The positive charge tends to stay with the ring fragment.
  7. In lkyl-substituted aromatic compounds, cleavage is very probable at the bond beta to the ring, giving the resonance-stabilized benzyl ion or, more likely, tropylium ion.
  8. C-C bonds next to heteroatom are frequently cleaved, leaving the charge on the fragment containing the heteroatom whose nonbonding electrons provide resonence stabilization.
  9. Cleavage is always associated with elemination of small stable neutral moleculs, such as carbon monoxide, olefins, water, ammonia, hydrogen sulfide, hydrogen cyanide, mercaptans, ketenes, or alcohol.

The fragmentaion rules enumerated above apply to EI mass spectrometry. Other ionizing techniques often produce molecular ions with much lower energy which are governed by different fragmentaion rules of the molecular ion. The table below lists some of the common fragments lost during fragmentation, as well as, some common stable ions which give common peaks found in mass spectra of different organic compounds.

Common small ions

Common Neutral Fragments

19H3O, F18H2O
26C2H2, CN19F
28C2H4, CO, H2CN27C2H3, HCN
29C2H5, CHO28C2H4, CO
33SH, CH2F32CH4O,S
34H2S33CH3+H2O, HS
41C3H5, C2H3N42C3H6, C2H2O, C2H4N
42C3H6, C2H2O, C2H4N43C3H7, CH3CO
43C3H7,CH3CO44CO2, CONH2

Recognition of the molecular ion peak:

If the molecular ion appears, it will be the highest mass in an EI spectrum (except for isotope peaks discussed below). This peak will represent the molecular weight of the compound. Its appearance depends on the stability of the compound. Double bonds, cyclic structures and aromatic rings stabilize the molecular ion and increase the probability of its appearance and thus increase its abundance.There are two situations in which the identification of the molecular ion peak may be difficult.

1-The molecular ion does not appear or is very weak. The obvious remedy in most cases is to run the spectrum at maximum sensitivity (and accept the resulting loss in resolution) and to use a larger sample. (Sometimes a large sample exaggerates the M+1 peak) Still the molecular ion may not be evident and other sources of information may be useful. The type of compound may be known, and the molecular mass may be deduced from the breakdown pattern. For example alcohol usually give a very weak parent molecular ion peak but often show a pronounced peak resulting from loss of water (M-18).

2- The molecular ion is present but is one of several peaks which may be as prominent. In this situation the question is that of purity. If the compound can be assumed to be pure, the usual problem is to distinguish the molecular ion peak from a more prominent M-1 peak. One good test isto reduce the energy of the bombarding electron beam to near the appearance potential. This will reduce the intensities of all peaks, but will increase the intensity of the molecular ion realtive to other peaks, including fragmentaion peaks(but not molecular ion peaks) of impurities. Another test frequently used is to increase the size of the sample, or increase the time the sample spends in the ionization chamber by decreasing the ion repeller voltage. In either case the net effect is to increase the oppertunity for bimolecular collisions to occur in the ion chamber. The most commen result of the bimolecular collision of a molecular ion containing a heteroatom (O,N or S)is a contribution to the M+1 peak (i.e.,the net effect is the transfer of a hydrogen atom from a neutral molecule to the molecular ion).
Thus an increase in peak size relative to other peaks, as sample size is increased or the repeller voltage is decreased,designates that peak as the M+1 peak and affords an indirect identification of the molecular ion. . Of course the dependance of the M+1 on a sample size must be kept in mind when the peak is used to establish a molecular formula of a compound containing a heteroatom.Many peaks can be ruled out as possible molecular ions simply on grounds of reasonable structure requirements.

1- The Nitrogen rule is often helpful identifying the molecular ion peak. It states that a molecule of even-numbered molecular weight must contain no nitrogen or an even number of nitrogen atoms; an odd-numbered molecular weight requires an odd number of nitrogen atoms. This rule holds for all compounds containing carbon, hydrogen, oxygen, nitrogen, sulfur, and the halogens, as well as many of the less usual atoms such as phosphorous, boron, silicon, arsenic, and the alkaline earths.

This distinction is illustrated nicely by the follwing two examples. The unsaturated ketone, 4-methyl-3-pentene-2-one, below has no nitrogen so the mass of the molecular ion (m/z = 98) is an even number. Most of the fragment ions have odd-numbered masses. Diethylmethylamine, on the other hand, has one nitrogen and its molecular mass (m/z = 87) is an odd number. A majority of the fragment ions have even-numbered masses (ions at m/z = 30, 42, 56 & 58 are not labeled). The weak even -electron ions at m/z=15 and 29 are due to methyl and ethyl cations (no nitrogen atoms).

2-A useful corollary states that fragmentation at a single bond gives an odd-numbered ion fragment from an even-numbered molecular ion, and an even-numbered ion fragment from an odd numbered molecular ion.For this corollary to hold, the fragment must contain all nitorgrn( if any) of the molecular ion.

3-Consideration of the breakdown pattern coupled with other information will also assist in identifying molecular ions. It should be kept im mind that; The intensity of the molecular ion peak depends on the stability of the molecular ion. The most stable molecular ions are those of purely aromatic systems. If subsitiuents that have a favourable mode of cleavage are present, the molecular ion peak will be less intense, and the fragment peaks relatively more intense.In general the following group of compounds will, in order of decreasing ability, give prominent molecular ion peaks: aromatic compounds>conjugated alkenes>cyclic compounds>organic sulphides>short, normal alkanes.
Recognizable molecular ions are usually produced for these compounds in order of decreasing abaility: Ketones>amines>esters>carboxylic acids-aldehides-amides-halides.The molecular ion is ferquently not detectable in aliphatic alcohols, nitrites, nitrates, nitro compounds, nitriles and in high.

The presence of M-15 peak (loss of CH3) or an M-18 peak (loss of H2O) or an M-31 Peak (loss of OCH3 from methyl esters),etc., is taken as confiramtion of the molecular ion peak. An M-1 peak is common and occasionally an M-2 peak (loss of H2 by either fragmentation or thermlysis or even a rare M-3 peak(from alcohol) is resonable.Peaks in the range of M-3 to M-14, however, indicate that contaminants may be present or that the persumed moecular ion peak is actually a fragment ion peak.Loss of fragments of masses 19 to 25 are also unlikely (except for the loss of F=19 or HF=20 from flourinated compounds). Loss of 16 (o), 17(OH), or 18 (H2O)are likely only if an oxygen atom is in the molecule.

3-Another way of confirming a molecular ion is that all fragments in the spectrum (other than impurities) should come from the assigned molecular ion with logical loss of neutral fragments. If a fragment or two do not comply this will mean either the assigned molecular ion is not really the molecular ion but a fragment ion or the fragment does not belong to the compound but to a background contaminant.

3. Isotopes
Since a mass spectrometer separates and detects ions of slightly different masses, it easily distinguishes different isotopes of a given element. This is manifested most dramatically for compounds containing bromine and chlorine, as illustrated by the following examples. Since molecules of bromine have only two atoms, the spectrum on the left will come as a surprise if a single atomic mass of 80 amu is assumed for Br. The five peaks in this spectrum demonstrate clearly that natural bromine consists of a nearly 50:50 mixture of isotopes having atomic masses of 79 and 81 amu respectively. Thus, the bromine molecule may be composed of two 79Br atoms (mass 158 amu), two 81Br atoms (mass 162 amu) or the more probable combination of 79Br-81Br (mass 160 amu). Fragmentation of Br2 to a bromine cation then gives rise to equal sized ion peaks at 79 and 81 amu.

The center and right hand spectra show that chlorine is also composed of two isotopes, the more abundant having a mass of 35 amu, and the minor isotope a mass 37 amu. The precise isotopic composition of chlorine and bromine is: Chlorine: 75.77% 35Cl and 24.23% 37Cl Bromine: 50.50% 79Br and 49.50% 81Br
The presence of chlorine or bromine in a molecule or ion is easily detected by noticing the intensity ratios of ions differing by 2 amu (isotopic pattern). In the case of methylene chloride, the molecular ion consists of three peaks at m/z=84, 86 & 88 amu, and their diminishing intensities may be calculated from the natural abundances given below. Loss of a chlorine atom gives two isotopic fragment ions at m/z=49 & 51amu, clearly incorporating a single chlorine atom. Fluorine and iodine, by contrast, are monoisotopic, having masses of 19 amu and 127 amu respectively. It should be noted that the presence of halogen atoms in a molecule or fragment ion does not change the odd-even mass rules given above.

Two other common elements having useful isotope signatures are carbon, 13C is 1.1% natural abundance, and sulfur, 33S and 34S are 0.76% and 4.22% natural abundance respectively. For example, the small m/z=99 amu peak in the spectrum of 4-methyl-3-pentene-2-one (above) is due to the presence of a single 13C atom in the molecular ion. Although less important in this respect, 15N and 18O also make small contributions to higher mass satellites of molecular ions incorporating these elements.



Relative abundance


Relative abundance


Relative abundance






















Now uppose that a compound contains one carbon atom. Then for every 100 moleculaes containing a 12C atom, about 108 "molecules" contain a 13C atom, and these molecules will produce an M+1 peak of about 1.08% the intensity of the molecular ion peak; the 2H will make an additional very small contribution to M+1 peak. If the compound contains one sulfur atom, the M+2 peak will be about 4.4% of the parent peak.

In practice measured isotope peaks will be slightly higher than th calculated contributions because of incomplete resolution, bimolecular collsions or contribution coincident peak of an impurity. The presence of Sulfur, Chlorine, Bromine is usually readily apparent because of a large isotope contribution to M+2. We shall see thal the number of sulfur, chlorine and bromine atoms can be determined from the isotopic ratio pattern produced. As mentioned before Iodine, Fluorine, and phoeporous are monoisotopic. Their presence can usually be deduced from a suspeciously small M+1 peak relative to the molecular weight, from the fragmentation pattern, from other spectra with which we are concerned, or form the history of the compound.

If only C, H, N, O, F, P, I are present, the appropriate expected % (M+1) and % (M+2) data can be calculated by using the following fprmulas.

=1.1xnumber of C atoms + 0.36 x number of N atoms

These equations are useful for cases in which one has a precoceived notion about the molecular formula for the compound of interest.
It is difficult to over imphasize the importance of locating the molecular ion peak. It will be stressed again that this gives an exact numerical molecular wight. Even in cases in which the molecular ion peak is very small ( and therefore an accurate determination of M+1 and M+2 is impossble) only a little extra information can often lead to identification. This information can be available from the source and history of the sample, from the fragmentation pattern, and from other spectra. Let us work through the selection of a molecular ion formula from the isotope abundance data obtained on an organic compound we are given the following information.

m/e .........................................%

The molecular ion peak mass 150; thus we have the molecular weight. The M+2 peak obviously does not allow the presence of sulfur or halogen atoms. The M+1 peak abundance is 10.2% of the paren peak. Teh formulas whose calculated isotope contribution to the M+1 peak falls (albitrarily) between 9.0 and 11.0) are listed below along with the calculated M+2.


























On the basis of the Nitrogen rule we immediately eliminate three of these fromulas because they contain an odd number of nitrogen atoms. Our M+2 peak is 0.88% of the parent. This best fits C9H10O2. However, C8H10N2O cannot be ruled out without additional evidence. Not that mass 150 is the sum of the masses of the common isotopes of these molecular formulas; the isotope masses used are whole numbers (12 for carbon, 14 for nitrogen etc.).


Rearrangement ions are fragments whose origin cannot be described by simple cleavage of bonds in the molecular ion, but are aresult of intramolecular atomic rearrangement during fragmentation. Rearrangements involving migration of hydrogen atoms in molecules that contain a heteroatom are especially common. One important example is Mclafferty rearrangement:

To undergo Mclafferty rearrangement, a molecule mus possess; an appropriately located heteroatom (e.g. O), a π-system (usually a double bond) and an abstractable hydrogen γ to the C=O system. Such rearrangements often account for prominent characteristic peaks and are consquently very useful for our purpose. They can frequently be rationalized on the basis of low-energy transitions and increased stability of the products. Rearrangements resulting in elemination of stable neutral molecule are common (e.g. the olefin product in the Mclafferty rearrangement). Rearramgement peaks can be recognized by considering the mass (m/e) number for fragment ions and for their corresponding molecular ions. A simple (no rearrangement) cleavage of an even-numbered molecular ion gives an odd numbered fragment ion and simple cleavage of an odd-numbered molecular ion gives an even-numbered fragment. Observation of a fragment ion mass different by 1 unit from that expected for a fragment resulting from simple cleavage (e.g., an even-numbered fragment mass from an even-numbered molecular ion mass) indicates rearrangement of hydrogen has accompanied fragmentation.Rearrangemetn peaks may be recognized by considering the corollary to the "nitrogen rule". Thus even-numbered peak derived derived from an even-numbered molecular ion is a result of two cleavages, which may involve a rearramgement.

Now let's examin the following diagram which displays the mass spectra of three simple gaseous compounds, carbon dioxide, propane and cyclopropane.

The molecules of these compounds are similar in size, CO2 and C3H8 both have a nominal mass of 44 amu, and C3H6 has a mass of 42 amu. The molecular ion is the strongest ion in the spectra of CO2 and C3H6, and it is moderately strong in propane. The unit mass resolution is readily apparent in these spectra (note the separation of ions having m/z=39, 40, 41 and 42 in the cyclopropane spectrum). Even though these compounds are very similar in size, it is a simple matter to identify them from their individual mass spectra. Even with simple compounds like these, it should be noted that it is rarely possible to explain the origin of all the fragment ions in a spectrum. Also, the structure of most fragment ions is seldom known with certainty. Shown below are the individual mass spectra and diagram of thier fragmentaion pattern

Since a molecule of carbon dioxide is composed of only three atoms, its mass spectrum is very simple. The molecular ion is also the base peak, and the only fragment ions are CO (m/z=28) and O (m/z=16). The molecular ion of propane also has m/z=44, but it is not the most abundant ion in the spectrum. Cleavage of a carbon-carbon bond gives methyl and ethyl fragments, one of which is a carbocation and the other a radical. Both distributions are observed, but the larger ethyl cation (m/z=29) is the most abundant, possibly because its size affords greater charge dispersal. A similar bond cleavage in cyclopropane does not give two fragments, so the molecular ion is stronger than in propane, and is in fact responsible for the the base peak. Loss of a hydrogen atom, either before or after ring opening, produces the stable allyl cation (m/z=41). The third strongest ion in the spectrum has m/z=39 (C3H3). Its structure is uncertain, but two possibilities are shown in the diagram. The small m/z=39 ion in propane and the absence of a m/z=29 ion in cyclopropane are particularly significant in distinguishing these hydrocarbons.

How can mass spectrometric data be used for structure analysis?

We have already seen the mass spectrum of a simple molecule, carbon dioxide. A more complex example is the mass spectrum of acetone, C3H6O, in figure 9. This mass spectrum shows many fragment ions in addition to the molecular ion at m/z 58. The most intense ions have been labeled with their mass-to-charge ratio. The fragment ions are used by mass spectrometrists to deduce molecular structures. (Sometimes the symbols [or +·, "plus dot"] and [or -·, "minus dot"] are used to indicate radical [odd-electron] ions. This can be useful in understanding ion fragmentation patterns.) For example, the loss of 15 Da from the molecular ion of acetone to give an ion at m/z 43 indicates the presence of a methyl group(CH3) in the original molecule. A subsequent loss of 28 Da to give an ion at m/z 15 suggests the presence of CO. By rationalizing such losses and drawing reasonable structures for the resulting ions, the structures of the original compounds can often be deduced. Some commonly observed lossed are 18 Da for water, H2O; 17 DA for ammonia, NH3; and 77 Da for the phenyl group, C6H5.

Another aid in determining molecular composition is exact mass measurement. Every isotope of every element (except carbon which is assigned exactly 12.00000 Da) has a unique, non-integer mass. Exact mass measurement thus allows determination of chemical composition. As illustrated in the mass spectrum to the right with high resolution it is possible to distinguish between carbon monoxide (CO, m/z 27.995) and nitrogen (N2, m/z 28.006) by exact mass measurement. This spectrum s was recorded using an ultra-high resolution FT-ICR instrument. Notice that, unlike the simple histogram depictions of other spectra , this spectrum is shown as a plot of the acquired data.

For those molecules that can be vaporized without decomposition , EI is often used to generate ions for mass analysis . As previously discussed, however, ionization by electrons accelerated through a potential of 70 volts is a highly energetic or "hard" process and may lead to entensive fragmentation that leaves very little or no trace of a molecular ion. Because molecular mass and structure are not easily determined in the absence of a molecular ion, lower energy or "soft" ionization techniques have been developed based on chemical and desorption ionization.

EI ionization introduces a great deal of energy into molecules. It is known as a "hard" ionization method. This is very good for producing fragments which generate information about the structure of the compound, but quite often the molecular ion does not appear or is a smaller peak in the spectrum. Spectra interpretation can become complicated as initial fragments undergo further fragmentation, and as rearrangements occur. However, a wealth of information is contained in a mass spectrum and much can be determined using basic organic chemistry "common sense".

In contrast to electron ionization, most applications of chemical ionization (CI) produce ions by the relatively gentle process of proton transfer. The sample molecules are exposed to a large excess of ionized reagent gas. Transfer of a proton to a sample molecule M, from an ionized reagent gas such as methane in the form of CH5+, yields the [M+H]+ positive ion. For example, the mass spectrum of ephedrine shows no molecular ion at m/z 165 under electron ionization conditions. However, under positive CI conditions the protonated molecule at m/z 166 and the important fragement ion corresponding to the loss of water (18 Daltons) have significant intensity. In both spectra an intense ion is seen at m/z 58 but note that the fragmentation patterns of protonated molecules, [M+H]+, are not necessarily the same as the fragmentation patterns of molecular ions, M+ .Negative ions can also be produced under chemical ionization conditions. Transfer of a proton from M to other types of reagent gas or ions can leave [M-H]-, a negatively charged sample ion. Addition of an electron to M, a process facilitated by collisionally decreasing the energy of electrons generated in the source, can yield an intense M- ion. Such ions, often the only ion generated, can be used to detect species by mass spectrometry with great sensitivity.Desorption ionization is a term used by mass spectrometrists to decribe the process by which a molecule is both evaporated from a surface and ionized although the exact mechanism may not be understood. For the first four desorption ionization methods listed below, samples are desorbed and ionized by an impact process that involves bombardment of the sample with high velocity atoms, ions, fission fragments, or photons of relatively high energy. The impact deposits energy into the sample, either directly or via the matrix, and leads to both sample molecule transfer into the gas phase and ionization.In field desorption, the sample is coated as a thin film onto a special filament placed within a very high intensity electric field. In this environment, ions created by field-induced removal of an electron from the molecule are extracted into the mass spectrometer.

Mass Spec Calibration Compounds and Spectra :
Calibration Compounds are used by the mass spectrometer user to adjust the mass spec calibration scale as well as the relative intensities of mass spec peaks from low to high mass. This is particularily necessary for the setup of quadrapole mass spectrometers. The most popular calibration compound is FC-43, also known as Perfluorotributylamine. Other calibration compounds are used to adjust the high resolution mass spectrometer calibration scale. The following pages list the data available on the most common calibration compounds in various mass spec techniques including low resolution, high resolution, Electron Impact, Chemical Ionization and Negative Ion Mass Spectrometry.