Unit 13 MASS Spectrometry

UNIT 13

MASS SPECTROMETRY

Mass Spectrometry

Structure 13.1 Introduction Objectives

13.2 Theory of Mass Spectrometry Characteristics of Mass Spectrum Isotopic Peaks

13.3 Instrumentation for Mass Spectrometry Inlet Devices Ionisation Chamber or Ion Sources Analysers Detectors or Ion Collectors Processing and Output Devices

13.4 Applications of Mass Spectrometry Qualitative Applications of Mass Spectrometry Quantitative Applications of Mass Spectrometry

13.5 Summary 13.6 Terminal Questions 13.7 Answers

13.1 INTRODUCTION In this course, you have so far learnt about a number of spectroscopic methods and their analytical applications. You would have understood by now that besides quantitative analysis, one important aspect of the spectroscopic methods is that they help in structure elucidation of the molecules being analysed. In the context of structure determination we take up mass spectrometry in this unit. The mass spectrometry is not a spectroscopic method in true sense of the word because here we do not make use of any electromagnetic radiation; yet it is no less significant. In mass spectrometry we study the consequences of bombardment of the analyte in vapour phase with high energy electrons. We will begin the unit by explaining the principle of mass spectrometry, describe a typical mass spectrum and discuss its characteristics. The discussion on the information available from the mass spectrum will be followed by a detailed account of the instrumental components of mass spectrometers. We will then discuss different applications of mass spectrometry.

Objectives After studying this unit, you should be able to: •

explain the principle behind mass spectrometry,

•

differentiate mass spectrometry from other spectrometries,

•

define and explain the terms like base peak, molecular ion, fragments, etc.,

•

+ + explain the importance of [M+1] . and [M+2] . ion peaks,

•

determine the molecular formula of the compound on the basis of its mass spectrum,

•

identify the common fragmentation patterns in the mass spectra and interpret them to make preliminary leads into the structure of the molecule,

•

outline the essential components of a mass spectrometer,

•

enlist different methods of ionisation and explain the principle behind them, 37

Miscellaneous Methods

•

discuss various fragmentation types and the factors affecting them, and

•

discuss various applications of mass spectrometry.

13.2 THEORY OF MASS SPECTROMETRY Electron volt: It is a unit of energy and is defined as the amount of kinetic energy gained by a single unbound electron when accelerated through a potential difference of 1 volt. 1 eV= 1.602 10 ‒19 J

Have you ever wondered what would happen if we take a molecule and bombard it with a beam of high energy electrons? Taking clue from ionisation of atoms to give ions you may be prompted to say that they would ionise; and you are absolutely right. This is what exactly is done in mass spectrometry. Here the molecule in the vapour phase is bombarded with a beam of high energy (~ 70 eV) electrons. These electrons knock off an electron from the molecule leading to the formation of a positively +

charged molecular ion, M • or more appropriately a radical ion or radical cation. For example, the radical ion formation from ammonia can be represented as

A radical ion is the one which carries a charge and also has an unpaired electron.

+. NH3 + 2 e

NH 3 + e

The radical ions so obtained have a large amount of extra energy which is much more than that required for breaking the covalent bonds. Therefore, the highly energetic molecular ions undergo fragmentation yielding smaller fragments. For example, +. NH3 m/z 17

+

NH2 + H

.

m/z 16 +

NH . + H

.

m/z 15 +

N + H m/z 14

m z denotes a dimensionless quantity obtained by dividing the mass of the ion in unified atomic mass units, u by its charge number.

.

As the molecular ion can fragment in a number of ways depending on the nature of the molecule, the fragments provide clue to the structure of the molecule. The radical ion and the fragments obtained from the molecule are then separated according to their m z value (where m is the mass and z is the charge on the ion) using magnetic and/or electrostatic fields. The record of m z values of these species against their relative amounts or intensities is known as mass spectrum of the sample.

13.2.1 Characteristics of Mass Spectrum The mass spectrum of methanol (molar mass = 32 gmol‒1) is shown as a typical mass spectrum in Fig. 13.1.

Fig. 13.1: Mass spectrum of methanol (CH3OH)

38

The x-axis in the spectrum represents the m z value of the fragment ions and the y-axis gives the relative intensity or abundance of different fragments. For y-axis the intensity of the peak representing the most abundant fragment (CH2OH+ ; m z =31, in this spectrum) is arbitrarily assigned an intensity of 100% and the peak intensities of other ions are measured relative to it. This most intense peak is called as the ‘base peak’ and it need not necessarily be the molecular ion peak which is at m z = 32. Are

Mass Spectrometry

you wondering, why do we observe a small peak at m z = 33? What is the origin of this peak? Let us learn about it.

13.2.2 Isotopic Peaks The peak at m z 33 in the case discussed above is an isotopic peak and is called as +

[M+1] . peak. The origin of this peak can be understood if we take into account the natural abundance of the isotopes of constituent atoms of a molecule. You know that, most of the elements exist in nature predominantly as a single entity i.e., as a collection of identical atoms. However, some elements have isotopes i.e., they exist as a mixture of atoms having same atomic number but different mass numbers. For example, carbon exists in nature as a mixture of 126 C as well as 136 C atoms. The

natural abundance of

13 6C

is 1.1% as compared to 126 C . This means that if we have a

thousand atoms of carbon (having would be of the

13 6C

12 6C

isotope) then there would be 11 atoms that

isotope. In case of the above example it amounts to saying that

for every 1000 molecules of methanol containing a methanol molecules with a

13 6C

12 6C

isotope, there would be 11

in them. These molecules would have a molar mass of

‒1

33 gmol as against the ‘normal’ value of 32 and hence the peak at m z = 33. Further, since oxygen ( 168 O and 188 O ) and hydrogen ( 11 H , and 21 H ) can also exist as isotopes, they would also influence the spectrum and we may expect a very small signal at + + + m z = 34 i.e., at [M + 2] . in addition to the M . and [M + 1] . peaks. The other

peaks in the spectrum appear due to the fragmentation of the molecular ion. The natural abundance of some common elements is compiled in Table 13.1. Table 13.1: Relative isotopic abundance* of some common elements Element

Isotope

Relative abundance

Isotope

Relative abundance

Isotope

Relative abundance

Carbon

12

C

100

13

C

1.11

Hydrogen

1

H

100

2

H

0.016

Nitrogen

14

N

100

15

0.38

Oxygen

16

O

100

17

0.04

18

Sulphur

32

S

100

33

0.78

34

S

4.40

Chlorine

35

Cl

100

37

Cl

32.5

Bromine

79

Br

100

81

Br

98.0

N O S

These contribute to [M+1]

O

+.

ion peak

0.20

These contribute to [M+2]

+.

ion peak

+

In this case M . and [M+2] ion peaks are of comparable intensities.

* Per 100 atoms of the most common isotope.

39

+.

Miscellaneous Methods

The presence and the intensity of peaks at m z values greater than that of the molecular ion can provide important information about the elemental composition of the molecule. As an example let us have a look at the mass spectrum given in Fig.13.2.

Fig. 13.2: A mass spectrum showing the importance of isotopic peaks

What do you observe in this case? Have a look at the Table 13.1 again, what do you infer? Yes it is a typical case of a molecule containing a bromine atom; the intensities +

+

of M . and [M+2] . peaks are comparable. The spectrum is of a simple molecule, bromomethane, CH3Br.

SAQ 1 The mass spectral data shows the following intensity pattern in the region of highest m z values.

m z 78 79 80

Intensity (% of base peak) 24 0.8 8.0

What do you infer about the nature of the compound? You may use Table 13.1 for answering this question. …………………………………………………………………………………………. …………………………………………………………………………………………. …………………………………………………………………………………………. ………………………………………………………………………………………….

13.3 INSTRUMENTATION FOR MASS SPECTROMETRY The essential components of a mass spectrometer are: •

Inlet device

•

Ionisation chamber or Ion Source

•

Analyser

•

Detector

•

Processing and output devices

The arrangement of these components of the mass spectrometer is schematically represented in Fig. 13.3.

40

Mass Spectrometry

Fig.13.3: A schematic diagram showing the components of a mass spectrometer

The inlet device loads the sample into the ionisation chamber where the analyte is ionised by a suitable method and the molecular ion and/or the fragment ions obtained by fragmentation of the molecular ion are directed towards the analyser. In the analyser these ions obtained by the fragmentation of the molecular ion are sorted out on the basis of their m z value by using one of the many available techniques and are sent to the detector (transducer). In the detector the ion flux generates an electrical current proportional to the number of ions reaching it. The processing unit records the magnitude of these electrical signals as a function of m z and gives an output in the form of a mass spectrum. Let us learn about the components of mass spectrometer.

13.3.1 Inlet Devices The purpose of the inlet device is to load the sample into the ionisation chamber. The device used depends on the nature of the sample. The solid samples are placed on the tip of a rod called direct insertion probe which is inserted into the evacuated chamber having a vacuum-tight seal. This is then heated to evaporate or sublime the sample to get the molecules in the gas phase. The gases and heat volatile liquids, on the other hand are generally introduced through specially designed devices with controlled flow. The liquid samples are also suitably evaporated. Once the sample is evaporated the gaseous molecules are then ionised by a suitable technique; this usually is accompanied by fragmentation also. When the analyte is thermally labile i.e., it can decompose upon heating, then we need to use other methods like desorption or desolvation methods (discussed in the next sub section) to bring the analyte into the vapour phase.

13.3.2 Ionisation Chamber or Ion Source As mentioned earlier, the mass spectrometer works by sorting out the charged particles by using magnetic and/or electric fields. Therefore, a compound/molecule must be charged or ionized to be analysed by a mass spectrometer. In the ionisation chamber (also called ion source) the molecule is ionised by using one of the many methods available for the purpose. The ion sources fall into two categories as follows: •

Gas phase sources

•

Desorption sources

Gas phase sources: In these sources the sample is first vaporised and then ionised. These are used for low molecular weight (< 1000 Da) samples which are thermally stable i.e., do not decompose on heating and have low boiling points (< 500 oC). There are three methods that belong to this category. These are:

The choice of ionization method depends on the nature of the sample and the type of information required from the analysis.

Da, dalton is the non-SI unit of mass and is exactly equal to the unified atomic mass, u , which is equal to 1.660 5402 × 10-27 kg

41

Miscellaneous Methods

i)

Electron ionisation (EI)

ii)

Chemical ionisation (CI)

iii)

Field ionisation (FI)

Let us learn about the first two of these as the principle of the third method is beyond the scope of this course. In most of the mass spectrometers the electrons are emitted from a heated tungsten or rhenium filament and are accelerated by applying potential.

Using low energy electrons reduces fragmentation.

Electron ionisation: This is the oldest and probably the best-characterized of all the ionisation methods. In this method, a high energy beam of electrons passes through the sample in the gas-phase. These electrons generally have energy of 10-150 eV. The electrons on colliding with the neutral analyte molecule knock off an electron from it and generate a positively charged ion. This process produces either a molecular ion or one of its fragments. This method is good for volatile compounds but the molecular ion peak is either weak or absent. Chemical ionisation: The chemical ionisation method uses ion/molecule reactions to produce ions from the analyte. For this purpose a reagent gas such as methane, isobutane, or ammonia is passed into the ionisation chamber where it gets ionised by electron ionisation. For example, methane gas gives mainly CH +4 and CH 3+ ions as follows CH4 + e

_

+. CH4 +

2e

_

. + CH3 + H

+. CH4

These reagent gas ions then react with the neutral molecules of the reagent gas as follows +. CH4 +

CH4

+ CH5 + CH3

+ CH3 +

CH4

+ C2H5 +

H2

The products of these ion-molecule reactions react with the analyte molecules (M) to produce analyte ions. The reactions with the above ions can be shown as M +

CH5+

M +

C2H5

+

+

MH + +

MH +

CH4 C2H4

These give an ion at [M+1]. For the analyte M of RH type, we may have reaction that can be represented as RH +

+

CH5

R+ +

CH4 + H2

In this case the ions would be obtained at [M ‒ 1]. Thus, the mass spectrum resulting from chemical ionisation method generally contains well defined [M+1]+ and [M ‒ 1]+ ion peaks. Further since the (M+1)+ ions do not undergo significant fragmentation, the spectra are simpler as compared to the ones obtained in EI method. Desorption sources: In these sources the solid or liquid sample is directly converted into the gaseous ions. These are used for high molecular weight (> 1000 Da) samples which are thermally unstable (i.e., decompose on heating) and are non volatile. A number of sources belonging to this category are available but we shall take up just one of them, namely the fast atom bombardment (FAB) method.

42

Fast atom bombardment (FAB): In this method the analyte is dissolved in a liquid matrix like glycerol and a small amount of this is placed on a target. The target is then bombarded with a beam of fast atoms (e.g., xenon or argon atoms at several keV). These desorb positive and negative analyte ions from the sample. +

A B

Fast

+

A +

B

Atoms

Mass Spectrometry

FAB is a good method for polar high molecular weight species.

As the sample heating is achieved very quickly the fragmentation of the analyte ions is greatly reduced. Thus, the ionisation chamber generates a stream of ions (primarily positive ions) which is accelerated by applying suitable potential and is sent to the analyser. Let us understand how the analyser separates these ions.

13.3.3 Analysers The analysers in the mass spectrometers are like the grating in the spectroscopic instruments. In a way, these disperse the fragment ions according to their mass to charge ( m z ) ratios. Several types of analysers are available; we shall take up only two such analysers that are commonly employed in the mass spectrometers. These are:

•

Magnetic sector analyser

•

Double focussing analysers

Magnetic sector analyser: As the name suggests, these analysers make use of a permanent magnet (or an electromagnet) to separate the fragment ions. It consists of an evacuated curved metallic tube through which the fragment ions pass on their way from the ion source to the detector. The electromagnets are mounted perpendicular to the tube and provide a stable and uniform magnetic field (Fig 13.4).

Other types of analysers are: • Time-of-Flight Mass Analyser • Quadrupole Mass Analyser • Ion Trap Mass Analyzer • Fourier Transform Mass Analyser

Fig.13.4: Schematic diagram of a magnetic sector mass analyser

As the ions entering the analyser have approximately the same kinetic energy, they have different velocities depending on their masses; heavier ions would be slower. The field of the magnet makes these ions to travel in a circular path generally of 60, 90 or 180 degrees. The mass to charge ratio of the fragment ions is related to the parameters of the instrument as per the equation.

43

Miscellaneous Methods

m B2 r 2e = z 2V where, B = magnetic field strength, r = radius of curvature of the magnetic sector (metallic tube) , V= the accelerating voltage applied on the ion in the ionisation chamber and e = the electronic charge. According to this equation the mass spectrum can be obtained by varying any of the three parameters viz., B,V or r. In most of the modern spectrometers, the separation is achieved by varying the field strength of the electromagnet keeping V and r constant. At a given magnetic field strength, only the fragments of a certain m z value would be able to travel through the circular path in the analyser tube; the ions of larger or smaller m z values would strike the tube and get destroyed. Thus, with the gradual increasing strength of the electromagnet, the fragment ions of increasing m z values successfully pass through the analyser and get detected by an ion collector. Double focussing analysers: In describing the magnetic sector analyser we mentioned that the ions entering the analyser have the same kinetic energy. In fact these fragments have a small range of kinetic energies and when passed through the magnetic field these get focussed at the same position. Thus, the spread of kinetic energies cause a broadening of the beam reaching the detector leading to a loss in resolution.

For a better resolution and more accurate mass determinations double focussing instruments are used which employ an electrostatic field prior to the magnetic sector. This combination of electrostatic and magnetic fields causes the energy focusing as well as the directional focusing respectively. A schematic diagram of the double focussing mass analyser is given in Fig. 13.5.

Fig. 13.5: Schematic diagram of a double focussing mass analyser

The electrostatic analyser consists of two curved metallic plates as shown in Fig. 13.5 and a dc potential is applied across them. This allows the fragment ions with only a limited range of kinetic energy to go over to the magnetic sector. As in case of the magnetic sector, here also the ions with greater and smaller kinetic energies are destroyed at the plates.

44

13.3.4 Detector or Ion Collector

Mass Spectrometry

In the mass spectrometers the ions after passing through the analyser are generally detected by a suitable electron multiplier. The electron multipliers are capable of providing quick response times and high current gains. The electrical signal so obtained can be processed, stored or suitably displayed.

13.3.5 Processing and Output Devices A typical mass spectrum contains a large amount of structural data in terms of the m z values and the relative intensities of all the fragments obtained from the molecule. Further, for the data to be dependable and useful a number of instrumental parameters need to be monitored and controlled. This means a large amount of data and its manipulation. It is achieved with the help of microprocessors and microcomputers that are an integral part of all mass spectrometers. The mass spectrometer data systems also include softwares for quantification, interpretation, and identification of the molecules using on-line spectral libraries. In the processing units the ion-current signals obtained from the detector is digitalised and extensively processed before being displayed in terms of a mass spectrum. The spectrum displays the m z values of all the fragments and their intensities relative to that of the most intense peak called base peak. Sometimes, the data is also displayed in the form of a table wherein the m z values are listed in an increasing order and the corresponding relative peak intensities are given in numbers.

SAQ 2 Arrange the following fragment ions in the order of their detection in the mass spectrometer. +

OCH3, +OCCH3, +CH2CH3

…………………………………………………………………………………………. …………………………………………………………………………………………. …………………………………………………………………………………………. …………………………………………………………………………………………. ………………………………………………………………………………………….

SAQ 3 List three ion sources used in mass spectrometers. Give one characteristic feature of each one of them. …………………………………………………………………………………………. …………………………………………………………………………………………. …………………………………………………………………………………………. ………………………………………………………………………………………….

13.4 APPLICATIONS OF MASS SPECTROMETRY Mass spectrometry finds extensive applications in diverse areas like chemical analysis, biochemistry, clinical chemistry, environmental pollution monitoring, food adulteration, doping in sports, and archaeology, etc. We will not get into the

45

Miscellaneous Methods

specialised areas but be contented with the most common qualitative and quantitative applications.

13.4.1 Qualitative Applications of Mass Spectrometry The most common qualitative applications of the mass spectrometry are: • •

Determination of molar mass of the analyte Determination of molecular formula of the analyte

• Determination of the structure of the analyte • Identification of the analyte ( alone or in a mixture) As an organic chemist our aim is primarily to determine the structure of the compound (III above) under analysis. In fact the listing above forms a logical sequence for the same. An analytical chemist on the other hand would be interested in the identification of the analyte and its quantification. Let us learn about these in the following paragraphs.

Determination of Molar Mass of the Analyte An accurate molar mass determination from mass spectrometry essentially means the correct identification of the molecular ion peak. You would recall (subsection 13.3.2) that depending on the method of ionisation we get the molecular ion (M+.) peak or (M+1)+ and (M ‒1)+ ion peaks. You would also recall that the molecular ion is quite unstable and quickly undergo fragmentation. In fact, in about 20% of the cases the molecular ion peak is not observed at all. However, in a large number of cases the cluster of peaks towards the highest m z values includes the molecular ion peak along with the corresponding isotopic peaks and can be easily identified. In cases when the molecular ion peak is absent or too weak we may resort to running spectrum with a higher concentration of the analyte or at higher sensitivity. If the nature of the molecule is known then sometimes the molecular ion peak can be reconstructed from the fragments. Many a times, an important rule called Nitrogen rule comes handy in deciding about the molecular ion. According to this rule a molecule having an even number molar mass must have none or an even number of nitrogen atoms in it; similarly a molecule with an odd number molar mass must have one or an odd number of nitrogen atoms in it. Here again the fragmentation pattern as well as the nature of the molecule helps in sorting out the issue. In cases of doubt, additional spectra obtained by alternative ionisation methods are useful.

Determination of Molecular Formula of the Analyte The determination of molecular formula can be taken up once the molecular ion peak is identified and the molar mass determined accurately. In case the isotopic peaks are reasonably intense and can be accurately determined then the molecular formula determination is quite straight forward. For this we can refer to the exhaustive tables that collate the molecular formulae and the corresponding isotopic peak intensities for a given molar mass. In addition, knowledge about the nature of the molecule is also useful. Let us take an example.

Example 1 The mass spectrum of a compound containing only carbon, hydrogen and oxygen gave the following intensity pattern in the region of molecular ion.

46

Mass Spectrometry

Intensity (% of molecular ion peak) 100 (M) 100 101 (M+1) 6.67 102 (M+2) 0.41

m z

Deduce its molecular formula.

Solution Since the molecular ion peak is at m z of 100 we need to check for the possible molecular formulae from the tables referred above. The following table gives the possible molecular formulae containing C, H and O and having a molar mass of 100. M =100

M+1 M+2 Formula mass

C4H4O3

4.50

0.68

100.0160

C5H8O2

5.61

0.53

100.0524

C6H13O

6.71

0.39

100.0888

It is quite obvious that the given mass spectrum is for the compound with a molecular formula as, C6H13O; the compound is cyclohexanol; as the (M+1) is 6.71 and (M+2) = 0.39 which matches with 6.67 and 0.41 given in the data. If you look at the table given in the solution to the example 1 you can appreciate that these tables can be used in a yet another way. Suppose we take a high resolution mass spectrum of the compound and find that the molar mass obtained from molecular ion peak is 100.0890 we are quite sure that the compound is C6H13O. Thus, the molecular formula of an analyte can be obtained either from the intensity pattern of the isotopic peaks or by accurate molar mass determination from high resolution mass spectrum. Let us learn how to determine the structure of the analyte molecule once the molecular formula is known?

Determination of the Structure of the Analyte You know that the molecular ion obtained in the course of ionisation in the ion source is highly energetic and undergoes fragmentation. In the process of fragmentation the molecular ion loses free radicals or small neutral molecules to give smaller fragments. The fragmentation does not occur randomly instead take place in a way so as to give the stable fragments. The characteristic fragments obtained in the mass spectrum of a compound can provide important leads into its structure. Detailed studies on the mechanism of fragmentation and characteristic fragments of different classes of organic compounds have led to certain general rules which can be used to decipher the structure of an organic compound from its mass spectrum. A detailed account of the fragmentation of different types of functional groups is beyond the scope of this course. For these you may refer to the books given at the end of the unit. However, let us take up some common fragmentation patterns. Fragmentation by Cleavage at a Single Bond

A simple cleavage of the single bond in the molecule is quite common and important fragmentation mechanism. In such a cleavage of a radical cation we get a radical and a

Remember that it is the positively charged ion which is detected by mass spectrometry.

47

Miscellaneous Methods

cation. For example, the simple cleavage of C-C single bond in molecular ion radical of propane can be represented as follows. +

+

CH3CH2

. CH3

m/z = 29

+.

CH3CH2CH3

. CH3CH2

+

+

CH3

m/z = 15

In this cleavage we get two cations with the m z values of 29 and 15 respectively and both of them are observed in the mass spectrum of propane. However, the intensity of the peak at m z 29 is much more than that of the peak at m z 15. In fact the m z 29 peak is the base peak which is the most intense peak. The intensities of the peaks is determined by the stability of the fragment ions; the secondary ethyl carbocation (CH3CH2 +) is much more stable than the primary methyl carbocation (CH3 +). For the same reason the cleavage is favoured at more alkyl substituted carbons in branched alkanes. Fragmentation by α -cleavage in Molecules with Heteroatoms

The bond on the carbon atom next to a herteroatom (N, O, halogen) in a molecule is easily cleaved. This is called α-cleavage and in such a cleavage the charge goes with the fragment containing the heteroatom. For example, two such cleavages are shown below in case of an alcohol (i) R

+. CH

OH

(i)

+ CH

(ii) H

. .. .. + R OH ..

H (ii) CH

R

+ CH

. .. .... + H OH

+ OH ....

H

m/z= M-1

R

CH

+ . .OH ..

The resulting cations are resonance stabilised due to the participation of the nonbonding pair of electrons on the heteroatom as shown above. Fragmentation due to Intramolecular Rearrangement: McLafferty Rearrangement

Sometimes the origin of the fragments observed in a mass spectrum can not be attributed to a formal cleavage; instead it involves some kind of an intramolecular rearrangement. Such rearrangements often explain the existence of prominent characteristic peaks in the mass spectrum and are therefore very useful in structure elucidation. One of the most important rearrangements, called McLafferty rearrangement is observed in the molecules containing a carbonyl group having abstractable hydrogen at a position γ to the carbonyl group.

48

Mass Spectrometry . .O . +

H CHR

C Y

. .O . +

CH2 C H2

H +

RCH=CH2

C Y

CH2

Y= H, R, OR, OH, NH2 .....

As an example, let us take the mass spectrum of butanal (CH3CH2CH2CHO) given in Fig. 13.6.

Fig. 13.6: Mass spectrum of butanal The prominent peaks in the spectrum are at m z 72, 57, 44 and 29. Of these, the peak

at m z 72 is the molecular ion peak and the ones at 57 (M ‒15) and 29 (M ‒43) can be explained in terms of methyl and propyl radicals respectively. The peak observed at m z 44 (M ‒28) can be explained in terms of McLafferty rearrangement as shown below.

. .O . +

H CH2

C H

.. . O +

CH2 C H2

H +

CH2=CH2

C H

CH2 m/z = 44

m/z = 28

In addition to the common fragmentation patterns discussed above, the origin of a number of peaks in the mass spectrum can be explained in terms of loss of small stable neutral molecules like H2, NH3, CO, H2S, alcohols, alkenes etc. These however, are not being taken up here. You may look into any standard text on Organic Spectroscopy for the same. Some commonly lost fragments and the stable fragments observed in the mass spectrum are compiled in Table 13.2. These would be useful when you attempt to use the mass spectral data for the structure elucidation of the molecules. This exercise would be taken up in Unit 14, the last Unit of this course.

49

Miscellaneous Methods

Table 13.2: Some commonly lost fragments and the stable fragments in the mass spectrum Commonly lost fragments Fragment lost

Peak obtained

.

CH3

M.

.

OH

M.

CN

M.

.

H 2C

+

15

+

17

+

CH 2

. CH2CH3

+

M.

+

M.

Fragment lost

Peak obtained

. OCH

+ M.

.

3

- 31

+ M . - 35

Cl

. O

M +. - 43

OCH2CH3

+ M . - 45

26

CH 3C

28

.

+

29

. CH2

M.

- 91

C

O

Common stable ions m/z values

m/z = 43

Ion CH 3

+ C

O . +

.

m/z = 91

+

+

CH2

O.

m/z = M . - 1

+

R

CH

R

SAQ 4 The mass spectrum of butanoic acid gives a characteristic peak at m z = 60. Justify the same. …………………………………………………………………………………………. …………………………………………………………………………………………. …………………………………………………………………………………………. …………………………………………………………………………………………. ………………………………………………………………………………………….

Identification of the Analyte, Single or in a Mixture Structure determination of an analyte from the mass spectrum is an elaborate job. As you have learnt above that it involves the identification of the molecular ion peak to determine the molar mass followed by determination of the molecular formula and thereafter interpretation of the characteristic fragments. After all these efforts an experienced mass spectrometrist can come close to a number of probable structures. The exact identification is then achieved by comparing the observed mass spectrum with the authentic spectra of the likely compounds. Many a times the analytical chemist is aware of the likelihood of the analyte; in such cases the identification is obtained simply by comparison spectra. This may sound quite simple however, it may not be so straight forward because the nature of spectrum depends a great deal on the

50

instrumental parameters like, ionisation method, temperature, pressure, etc. It may not always be possible to have reference spectra under the conditions of the sample being analysed.

Mass Spectrometry

Now-a-days most of the modern mass spectrometers are equipped with computerised library search systems. The identification of the analyte can be achieved by performing the library search on the expected group of compounds. Once a reasonable match is obtained the spectra of the analyte and the reference compound can be plotted simultaneously and compared. Very often we deal with mixtures of substances. The complexity of the spectra for mixtures necessitates that the components be separated prior to their mass spectral analysis. Such a separation is usually achieved by gas chromatography and the gas chromatograph is coupled with the mass spectrometer. In this hyphenated technique, the components from the GC are allowed to directly enter the spectrometer in the vapour phase one by one and get analysed. Recent advances have allowed even the liquid chromatographs, supercritical fluid (SCF) chromatographs, and capillary electrophoresis (CE) devices to be coupled directly to mass spectrometers.

13.4.2 Quantitative Applications of Mass Spectrometry The quantification of the analyte, besides its identification, is one of the main aims of an analytical chemist. Mass spectrometry has an important role to play in achieving this objective of the analytical chemist. It can be effectively used in the quantitative determination of the analyte alone or in a complex system with a large number of components, especially in the areas of petroleum, pharmaceuticals and environment The hyphenated techniques wherein a mass spectrometer is coupled with a liquid or a gas chromatograph or an electrophoretic column are employed for this purpose. The analyte from the chromatographic or electrophoretic column is passed continuously through the spectrometer which analyses the aliquots reaching it as a function of time. The spectrometer may be instructed to monitor any one or a number of characteristic peaks of the analytes. The area under the peaks is a measure of their concentration. In a yet another strategy the spectrum of the mixture is run and the different analytes in it are monitored in terms of their unique fragment peaks. The heights of the unique peaks of different analytes in the mixture are indicative of their concentrations. A calibration curve of the peak height as a function of the analyte concentration is quite useful in these circumstances. Having learnt about the principle of mass spectrometry, the mass spectrum and its characteristics, instrumentation of mass spectrometers and the applications of mass spectrometry let us sum up what all have we learnt in this unit. However, why don’t you assess your understanding of mass spectrometry by solving the following SAQ before that?

SAQ 5 How can you analyse a mixture of substances using mass spectrometry? …………………………………………………………………………………………. …………………………………………………………………………………………. …………………………………………………………………………………………. …………………………………………………………………………………………. ………………………………………………………………………………………….

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Miscellaneous Methods

13.5 SUMMARY In this unit, you have learnt about mass spectrometry which is an analytical tool with wide ranging applications. In mass spectrometry a molecule in the vapour phase is bombarded with a beam of high energy electrons that knock off an electron from the +

molecule to give a positively charged molecular ion, M • . The energetic molecular ions undergo fragmentation yielding smaller fragments. The molecular ion and the fragments obtained from it are then separated according to their m z ratio using magnetic and/or electrostatic fields. The record of m z values of these species against their relative amounts or intensities is known as mass spectrum of the sample. The most intense peak in the spectrum is called the base peak and the peaks at m z values greater than that of the molecular ion are called isotopic peaks. These arise due to the natural abundance of the isotopes of different elements present in the molecule and are useful in the determination of molecular formula of the compound. In a mass spectrometer the sample is loaded into the ionisation chamber using a direct insertion probe for solid samples or by controlled flow devices for the gases and heat volatile liquids. In gas phase ion sources like electron impact ionisation and chemical Ionisation the sample is first vaporised and then ionised. In the former a high energy beam of electrons passes through the sample and generates a positively charged ion by knocking off an electron from it whereas in the later ion-molecule reactions are used to produce (M+1)+ and (M-1)+ ions. In fast atom bombardment method – a desorption ion source, the analyte is dissolved in a liquid matrix and bombarded with a beam of fast atoms to generate ions. The molecular ion and the fragment ions produced from it are directed towards the analyser where these are sorted out on the basis of their m z ratio. In magnetic sector analyser which consists of an evacuated curved metallic tube, an electromagnet mounted perpendicular to the tube is used to separate the fragment ions. The separation is achieved by continuously varying the field strength of the electromagnet. In double focussing instruments, an electrostatic field is applied prior to the magnetic sector to achieve better resolution. The ions after being separated according to their m z ratios are sent to the detector where these generate electrical signal which are digitalised, processed and recorded by the processing and output device. These devices use a number of microprocessors and microcomputers as a large amount of data needs to be handled to generate a mass spectrum. These data systems also include softwares for quantification, interpretation, and identification of the molecules using on-line spectral libraries. Mass spectrometry finds extensive applications in diverse areas like chemical analysis, biochemistry, clinical chemistry, environmental pollution monitoring, food adulteration, doping in sports, archaeology, etc. The determination of molar mass, molecular formula and structure of the analyte and the identification of the analyte, alone or in a mixture are the most common qualitative applications of the mass spectrometry.

13.6 TERMINAL QUESTIONS

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1.

Explain in brief the principle of mass spectrometry.

2.

Mass spectrometry is different from other spectroscopic methods. Comment.

3.

What are the characteristics of a mass spectrum?

4.

What are isotopic peaks and in what way are these useful?

Mass Spectrometry

5.

Describe the working of a double focussing mass analyser.

6.

What is McLafferty rearrangement? Explain with the help of an example.

7.

The mass spectrum of hexane is given below. Explain the origin of important fragment ion peaks observed in the spectrum.

Fig.: The mass spectrum of hexane

13.7 ANSWERS Self Assessment Questions +

+

1.

Since the intensity ratio of the M • and [M + 2] • ion peaks is 24:8 :: 3:1, we can infer (using Table 13.1) that the molecule contains a chlorine atom.

2.

In mass spectrometer the fragment ions are detected in the increasing order of their m z values. Since there is unit charge in all the given species, these will be detected in the increasing order of their masses. The order of their detection would therefore be: +

CH2CH3, +OCH3, +OCCH3

3.

The three ion sources used in mass spectrometers and their characteristic features are as follows: Electron ionisation: The method is good for volatile compounds but the molecular ion peak is either weak or absent. Chemical ionisation: This method uses ion-molecule reactions to produce ions from the analyte. Fast atom bombardment: This method is good for polar high molecular weight species and uses a beam of fast atoms that desorb positive and negative analyte ions from the sample.

4.

The butanoic acid has a carbonyl group with abstractable hydrogen at γ position; therefore it can easily undergo McLafferty rearrangement. Accordingly the peak at m z =60, [M-28] can be explained as follows.

. .O . +

H CH2

C HO

. .O . +

CH2 C H2

H +

CH2=CH2

C HO

CH2 m/z = 60

m/z = 28

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Miscellaneous Methods

5.

A mixture of substances can be analysed by coupling the mass spectrometer with a suitable chromatographic separation technique like GC or HPLC etc.

Terminal questions 1.

In mass spectrometry, the analyte in the vapour phase is bombarded with a beam of high energy which knock off an electron from it to form a positively charged + radical or molecular ion, M • . The radical ions so obtained undergo fragmentation yielding smaller fragments. The radical ion and the fragments obtained from the molecule are then separated according to their m z values using magnetic field analysers or double focussing mass analysers. These are then detected by a suitable transducer or analyser and an output is generated after suitable processing.

2.

Most of the spectral techniques generally involve absorption, emission or scattering of radiation from different regions of electromagnetic spectrum; NMR had an additional requirement of keeping the sample in a homogenous magnetic field. On the other hand in mass spectrometry the molecule is ionised using a suitable method and fragmented; the fragments being then analysed.

3.

The mass spectrum gives a plot of the intensities (proportional to the amounts) of different fragments as a function of their m z values. The most intense peak in the spectrum is given an intensity of 100 and is called the base peak; the intensity of other peaks is given with respect to the base peak in fragmentation pattern. Another characteristic of the spectrum is the presence of peaks called isotopic peaks, observed due to the natural abundance of the isotopes of constituent atoms of a molecule.

4.

The isotopic peaks in a mass spectrum refer to the peaks observed at m z values greater than the molecular ion peak. These arise due to the natural abundance of the isotopes of constituent atoms of a molecule. These are useful in the determination of the molecular formula of the compound. These may also be used for the determination of the accurate masses of the isotopes.

5.

In double focussing mass spectrometer, the molecular and the fragment ions coming out of the ionisation chamber are passed through an electrostatic analyser. This allows the fragment ions with only a limited range of kinetic energy to go over to the magnetic sector. In other words this analyser causes the energy focusing of the ions. In the magnetic sector analyser these ions are then dispersed according to their m z ratios and sent to the detector.

6.

McLafferty rearrangement is a mechanism of fragmentation that accounts for prominent characteristic peaks in the mass spectrum of a number of molecules belonging to different classes. The essential requirement for the rearrangement is that the molecules containing a carbonyl group must have abstractable hydrogen at a position γ to the carbonyl group. For example, the mass spectrum of phenylpropylketone gives at a m z of 120 and its existence can be accounted for by McLafferty rearrangement as follows:

. .O . +

H CH2

C

CH2 C H2

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.. . O +

H +

C CH2 m/z = 120

CH2=CH2

7.

The molecular ion peak is observed at m/z = 86 and the prominent fragment ions are at m/z = 71, 57, 43 and 29. A little observation reveals that these fragment ions differ by a m/z of 14 units. This indicates that the different fragments differ by –CH2 – units. Accordingly, the possible fragmentation pattern leading to the observed peaks can be depicted as:

Mass Spectrometry

+ CH3CH2CH2CH2CH2 + .CH3 m/z= 71

+ CH3CH2CH2CH2 + .CH2CH3 m/z= 57

CH3CH2CH2CH2CH2CH3

+.

+ . CH3CH2CH2 + CH2CH2CH3 m/z= 43

+ CH3CH2 + .CH2CH2CH2CH3 m/z= 29

+ CH3 + .CH2CH2CH2CH2CH3 m/z= 15

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