Interpretation of a Mass Spectrum

Certainly the most important applications of mass spectrometry are the identification of complex molecules and the elucidation of their structures. It might be expected that a given molecule would give a unique fragmentation pattern that would distinguish it from all other substances. This expectation is realized often, but not 

Mass spectrometry is an extremely information-rich technique, producing many signals or peaks for a single substance. Its chief advantage over other information-rich tools (such as NMR, IR, and x-ray spectroscopy) is its sensitivity— useful spectra can be obtained for samples as small as one nanogram. However, a complete understanding of all the fragmentation mechanisms has not yet been achieved. 

In the spectrum of methane (CH4), a small peak located at mje =17 has an intensity 1.1% that of the M+ peak at mje = 16. The signal at mje = 17 arises because carbon consists of two naturally occurring stable isotopes and Assigning 

The analyst can make use of the natural abundance of to assign the number of carbon atoms in M+. For example, if M+ is 100% and (M + 1)+ is 7.7%, contains 7 carbons. Often is not the largest peak in a mass spectrum and therefore is not assigned an intensity of 100% (the largest peak in a spectrum is usually arbitrarily assigned an intensity of 100% and all other peaks are measured relative to this). In that case, a useful formula is 

Other elements have isotopic contributions helpful in determining how many atoms of that element are contained in M . Table 16.2 gives some examples. The halogens are noteworthy an that contains one Cl must have an M -I- 2 peak with at least 1/3 the abundance of M and a bromine-containing molecule will have nearly a 1 1 ratio of M (M + 2). 

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A mass spectrum is a graphic representation of the ions observed by the mass spectrometer over a specified range of m/z values. The output is in the form of an x,y plot in which the x-axis is the mass-to-charge scale and the y-axis is the intensity scale. If an ion is observed at an m/z value, a line is drawn representing the response of the detector to that ionic species. The mass spectrum will contain peaks that represent fragment ions as well as the molecular ion (see Figure 1.3). Interpretation of a mass spectrum identifies, confirms, or determines the quantity of a specific compound. 

There are certain rules determining fragmentation of organic compounds in a mass spectrometer. That is why on the basis of the fragmentation pattern it is possible to define the molecular mass, elemental composition, presence of certain functional groups, and often the structure of an analyte. There are a lot of similarities in the mass spectrometric behavior of related compounds. This fact facilitates manual interpretation of a mass spectrum, although it requires some experience. It is also worth mentioning that mass... 

Manual interpretation of a mass spectrum from basic principles is always important for identification. Although several specialised works have been published on the topic, this type of analysis still requires a lot of experience. Organic chemists are usually familiar with these methods of interpretation since more reaction mechanisms lead to decomposition than those observed in the condensed state. However, because of the very short period of time between ion formation and ion detection (a few microseconds), species that are unstable under normal conditions can be observed. 

Due to the distinctive mass spectral patterns caused by the presence of chlorine and bromine in a molecule, interpretation of a mass spectrum can be much easier if the results of the relative isotopic concentrations are known. The following table provides peak intensities (relative to the molecular ion (M+) at an intensity normalized to 100%) for various combinations of chlorine and bromine atoms, assuming the absence of all other elements except carbon and hydrogen.1 The mass abundance calculations were based on the most recent atomic mass data.1... 

In addition to a library computer search for matching spectra, the usual procedure for interpretation of a mass spectrum involves (1) identification of the molecular ion peak and, if possible, determination of the elemental composition of the molecule based on the observed mass number (m/z) for the molecular ion, (2) establishment of the fragmentation patterns of the molecular ion, and finally (3) reduction of the number of possible structures for the compound, based on the interrelationships of the observed fragmentation patterns. The absence of meta-stable ions mass spectra obtained by GC-MS analysis makes (2) very difficult to achieve. 

A further important aspect of the abundances of ions is their relationship to the initial structure of the sample molecule. It is often possible to deduce this structure, or elements of it, by comparison of the MS fragmentation reactions of related compounds. Examples of mass spectra are shown in Figures 1.6, 1.12 and 1.13. The reasoning used in the interpretation of a mass spectrum is based on the accumulated knowledge from the rationalisation of fragmentation mechanisms of known compounds and supported by labelling studies and the accurate mass measurement of ions. Detailed information of such mechanisms can be found in specific textbooks [14] and throughout the literature. 

In developing the example of the mass spectrum of V-TFA—Val—Gly—Ala— OMe and in the interpretation of other mass spectra later in this chapter, it will be seen that the interpretation of a mass spectrum of a peptide relies on this consistent manner of bond cleavage. The electron-impact mass spectrum (EIMS) of V-TFA— Val—Gly—Ala—OMe shown in Figure 4.5 contains a molecular ion M+ at m/z 355 and a peak at m/z 324, proving the occurrence of the primary cleavage process A—> B—>C illustrated in Scheme 4.1, since a loss of 31 atomic mass units from the intact... 

A second fruitful approach to the interpretation of a mass spectrum is to examine the low-mass fragment peaks (below, say, mIz 150) for characteristic ions. 

Since the 1960s, mass spectrometry has played a pivotal role in the field of structure elucidation and identification of organic compounds. Over the years, a wealth of knowledge has been gained on reactions of gas-phase ions from the use of a variety of mass spectrometric techniques. Mass spectrometry can be used to identify unknown compounds or to perform de nova stmcture determination. The former is relatively easy if one knows accurate mass and a reference spectrum. The latter is much more difficult and requires detailed knowledge of the rules for interpretation of a mass spectrum. Providing this knowledge is the focus of this chapter. 

Chemists often use mass spectra primarily for the determination of molecular mass and molecular formula. Very rarely do they attempt a full interpretation of a mass spectrum, which can be very time consuming, difficult, and dependent on the experimental details of the ionization. The mass spectrum of dopamine (Figure 14.2), for example, contains at least 45 peaks with intensity equal to or greater than 0.5% of the intensity of the base peak. We have neither the need nor the time to attempt to interpret this level of complexity. Rather, we concentrate in this section on the fragmentation mechanisms giving rise to major peaks. 

The LC/MS combination is another valuable aid in the structure elucidation of eluted solutes but the system is not as comprehensive as the LC/NMR combination. The successful interpretation of a mass spectrum for structure identification can occasionally require other spectroscopic information such as the IR spectrum of the substance to indicate the presence of specific functional groups. Nevertheless the LC/MS combination can be a very useful analytical technique. 

Therefore, a detailed interpretation of a mass spectrum both for understanding the reactivity of organic radical cations and for a structure analysis of the... 

The universality of El (aU organic molecules are ionizable by the process) justifies the success of this technique, which is by far the most used in GC-MS coupling. It nevertheless happens that this techniqne is unsuitable for certain analyses. In structural elucidation, the fact that the molecular ion is not always present poses a crucial problem. Indeed, the interpretation of a mass spectrum implies establishing the observed fragmentations from a molecular ion. Confronted with the electron ionization spectrum of an unknown product, it is difficult if not impossible to start the interpretation since one cannot know whether the highest m/z ratio present in the spectrum corresponds to the molecular ion or not. Chemical ionization (see next section) is very valuable in... 

The interpretation of a mass spectrum cannot be improvised experience shows that chemists who are not properly trained in mass spectrometry are often anbarrassed when they start tackling the interpretation of mass spectra. This is because the chan-istry of ions in the gaseous phase is often different from the chemistry of the same ions in solution. Most chemists learned to deal with solutions using techniques such as pH metrics, titration, and synthesis. Furthermore, except for photochanistry, classical training treats species with even numbers of electrons molecules and MH+ or [M-H]- ions, respectively, emitted from protonated or deprotonated molecules. 

In the case of chemical elements presenting several isotopes in relative proportions that are sufficiently abundant for each one to be detectable, as is the case of chlorine and bromine (see below), the shape of an isotope pattern is essential for the interpretation of a mass spectrum because it reveals the number of chlorine or bromine atoms included in the raw formula of the ion. To illustrate isotope patterns, we will study carbon, chlorine, and bromine elements. 

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