Express, an International Journal of Multi Disciplinary Research ISSN: , Vol. 2, Issue 7,July 2015 Available at:

Transcription

1 2052, Vol. 2, Issue 7,July 2015 Available at: Principles and Fragments of Mass Spectra Dr. Nagham Mahmood Aljamali Organic Chemistry, Chemistry Department.,College of Education. (To corresponding : dr.nagham_mj@yahoo.com),(lectures of Dr. Nagham) Abstract : A mass spectrum 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. Most of the ions formed in a mass spectrometer have a single charge, so the m/z value is equivalent to mass itself. Keywords : mass,spectra, spectrum Introduction The formation of molecular ions When the vaporised organic sample passes into the ionisation chamber of a mass spectrometer, it is bombarded by a stream of electrons. These electrons have a high enough energy to knock an electron off an organic molecule to form a positive ion. This ion is called the molecular ion - or sometimes the parent ion. The molecular ion is often given the symbol M + or - the dot in this second version represents the fact that somewhere in the ion there will be a single unpaired electron. That's one half of what was originally a pair of electrons - the other half is the electron which was removed in the ionisation process. Fragmentation The molecular ions are energetically unstable, and some of them will break up into smaller pieces. The simplest case is that a molecular ion breaks into two parts - one of which is another positive ion, and the other is an uncharged free radical. The uncharged free radical won't produce a line on the mass spectrum. Only charged particles will be accelerated, deflected and detected by the mass spectrometer. These uncharged particles will simply get lost in the machine - eventually, they get removed by the vacuum pump.

2 2052, Vol. 2, Issue 7,July 2015 Available at: The ion, X +, will travel through h the mass spectrometer just like any other positive ion - and will produce a line on the stick diagram. mass spectra Below is typical output for an electron-ionization MS experiment (MS data in the section is derived from the Spectral Database for Organic Compounds, a free, web-based service provided by AIST in Japan. 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 thebase 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 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 13 C rather than the 12 C isotope. The 13 C isotope is, of course, heavier than 12 C by 1 mass unit. In addition, about 0.015% of all hydrogen atoms are actually deuterium, the 2 H isotope. So the M+1 peak represents those few acetone molecules in the sample which contained either a 13 C 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 18 O (the most abundant isotope of oxygen is 16 O). Because there are two abundant isotopes of both chlorine (about 75% 35 Cl and 25% 37 Cl) and bromine (about 50% 79 Br and 50% 81 Br), chlorinated and brominated compounds have very large and recognizable M+2 peaks. Fragments containing both isotopes of Br can be seen in the mass spectrum of ethyl bromide:

3 2052, Vol. 2, Issue 7,July 2015 Available at: Much of the utility in electron-ionization MS comes from the fact thatt the radical cations generated in the electron-bombardment process tend to fragment in predictable ways. Detailed analysis of the typical fragmentation patterns of different functional groups is beyond the scope of this text, but it is worthwhile to see a few representative examples, even if we don t attempt to understand the exact process by which the fragmentation occurs. We saw, for example, that the base peak in the mass spectrum of acetone is m/z = 43. This is the result of cleavage at the alpha position - in other words, at the carbon-carbon bond adjacent to the carbonyl. Alpha cleavage results in the formation of an acylium ion (which accounts for the base peak at m/z = 43) and a methyl radical, whichh is neutral and therefore not detected. After the parent peak and the base peak, the next largest peak, at a relative abundance of 23%, is at m/z = 15. This, as you might expect, is the result of formation of a methyl cation, in addition to an acyl radical (which is neutral and not detected). A common fragmentation pattern for larger carbonyl compounds is called the McLafferty

4 2052, Vol. 2, Issue 7,July 2015 Available at: rearrangement: The mass spectrum of 2-hexanone shows a 'McLafferty fragment' at m/z = 58, while the propene fragment is not observed because it is a neutral species (remember, only cationic fragments are observed in MS). The base peak in this spectrum is again an acylium ion. When alcohols are subjected to electron ionization MS, the molecular ion is highly unstable and thus a parent peak is often not detected. Often the base peak is from an oxonium ion.

5 2052, Vol. 2, Issue 7,July 2015 Available at: Other functional groups have predictable fragmentation patterns as well. By carefully analyzing the fragmentation information that a mass spectrum provides, a knowledgeable spectrometrist can often put the puzzle together and make some very confident predictions about the structure of the starting sample. Gas Chromatography - Mass Spectrometry Quite often, mass spectrometry is used in conjunction with a separation technique called gas chromatography (GC). The combined GC-MS procedure is very useful when dealing with a sample that is a mixture of two or more different compounds, because the various compounds are separated from one another before being subjected individually to MS analysis. We will not go into the details of gas chromatography here, although if you are taking an organic laboratory course you might well get a chance to try your hand at GC, and you willl almost certainly be exposed to the conceptually analogous techniques of thin layer and column chromatography. Suffice it to say that in GC, a very small amount of a liquid sample is vaporized, injected into a long, coiled metal column, and pushed though the column by helium gas. Along the way, different compounds in the sample stick to the walls of the column to different extents, and thus travel at different speeds and emerge separately from the end of the column. In GC-MS, each purified compound is sent directly from the end of GC column into the MS instrument, so in the end we get a separate mass spectrum for each of the compounds in the original mixed sample. Because a compound's MS spectrum is a very reliable and reproducible 'fingerprint', we can instruct the instrument to search an MS database and identify each compound in the sample. The extremely high sensitivity of modern GC-MS instrumentation makes it possible to detect and identify very small trace amounts of organic compounds. GC-MS is being used increasingly by environmental chemists to detect the presence of harmful organic contaminants in food and water samples. Airport security screeners also use high-speed GC-MS instruments to look for residue from bomb-making chemicals on checked luggage. Mass spectrometry of proteins - applications in proteomics Mass spectrometry has become in recent years an increasingly important tool in the field

6 2052, Vol. 2, Issue 7,July 2015 Available at: of proteomics. Traditionally, protein biochemists tend to study the structure and function of individual proteins. Proteomics researchers, in contrast, want to learn more about how large numbers of proteins in a living system interact with each other, and how they respond to changes in the state of the organism. One very important subfield of proteomics is the search for protein biomarkers for human disease. These can be proteins which are present in greater quantities in a sick person than in a healthy person, and their detection and identification can provide medical researchers with valuable information about possible causes or treatments. Detection in a healthy person of a known biomarker for a disease such as diabetes or cancer could also provide doctors with an early warning that the patient may be especially susceptible, so that preventive measures could be taken to prevent or delay onset of the disease. New developments in MS technology have made it easier to detect and identify proteins that are present in very small quantities in biological samples. Mass spectrometristss who study proteins often use instrumentation thatt is somewhat different from the electron-ionization, When proteins are being analyzed, the object is often to magnetic deflection system described earlier. ionize the proteins without causing fragmentation, so 'softer' ionization methods are required. In one such method, called electrospray ionization, the protein sample, in solution, is sprayed into a tube and the molecules are induced by an electric field to pick up extra protons from the solvent. Another common 'soft ionization' method is 'matrix-assisted laser desorption ionization' (MALDI). Here, the protein sample is adsorbed onto a solid matrix, and protonation is achieved with a laser. Typically, both electrospray ionization and MALDI are used in conjunction with a time-of-flight (TOF) mass analyzer component. The ionized proteins are accelerated by an electrode through a column, and separation is achieved because lighter ions travel at greater velocity than heavier ions with the same overall charge. In this way, the many proteins in a complex biological sample (such as blood plasma, urine, etc.) can be separated and their individual masses determined very accurately. Basic principles of Mass Spectra : Mass spectrometry is based on slightly different principles to the other spectroscopic methods. The physics behind mass spectrometry is that a charged particle passing through a magnetic field is deflected along a circular path on a radius that is proportional to the mass to charge ratio, m/e.

7 2052, Vol. 2, Issue 7,July 2015 Available at: In an electron impact mass spectrometer, a high energy beam of electrons is used to displace an electron from the organic molecule to form a radical cation known as the molecular ion. If the molecular ion is too unstable then it can fragment to give other smaller ions. The collection of ions is then focused into a beam and accelerated into the magnetic field and deflected along circular paths according to the masses of the ions. By adjusting the magnetic field, the ions can be focused on the detector and recorded. Probably the most useful information you should be able to obtain from a MS spectrum is the molecular weight of the sample. This will often be the heaviest ion observed from the sample provided this ion is stable enough to be observed.

8 2052, Vol. 2, Issue 7,July 2015 Available at: Terminology Spectra Molecular ion The ion obtained by the loss of an electron from the molecule Base peak M + The most intense peak in the MS, assigned 100% intensity Symbol often given to the molecular ion Radical cation + ve charged species with an odd number of electrons Fragment ions Lighter cations formed by the decomposition of the molecular ion. These often correspond to stable carbcations. The MS of a typical hydrocarbon, n-decane is shown below. The molecular ion is seen as a small peak at m/z = 142. Notice the series ions detected that correspond to fragments that differ by 14 mass units, formed by the cleaving of bonds at successive -CH 2 - units The MS of benzyl alcohol is shown below. The molecular ion is seen at m/z = 108. Fragmentation via loss of 17 (-OH) gives a common fragment seen for alkyl benzenes at m/z = 91. Loss of 31 (-CH 2 OH) from the molecular ion gives 77 corresponding to the phenyl cation. Note the small peaks at 109 and 110 which correspond to the presence of small amounts of 13C in the sample (which has about 1% natural abundance).

9 2052, Vol. 2, Issue 7,July 2015 Available at: Isotope patterns Mass spectrometers are capable of separating and detecting individual ions even those that only differ by a single atomic mass unit. As a result molecules containing different isotopes can be distinguished. This is most apparent when atoms such as bromine or chlorine are present ( 79 Br : 81 Br, intensity 1:1 and 35 Cl : 37 Cl, intensity 3:1) where peaks at "M" and "M+2" are obtained. The intensity ratios in the isotope patterns are due to the natural abundance of the isotopes. "M+1" peaks are seen due the the presence of 13 C in the sample. The following two mass spectra show examples of haloalkanes with characteristic isotope patterns. The first MS is of 2-chloropropane. Note the isotope pattern at 78 and 80 that represent the M amd M+2 in a 3:1 ratio. Loss of 35 Cl from 78 or 37 Cl from 80 gives the base peak a m/z = 43, corresponding to the secondary propyl cation. Note that the peaks at m/z = 63 and 65 still contain Cl and therefore also show the 3:1 isotope pattern.

10 2052, Vol. 2, Issue 7,July 2015 Available at: The second MS is of 1-bromopropane. Note the isotope pattern at 122 and 124 that represent the M amd M+2 in a 1:1 ratio. Loss of 79 Br from 122 or 81 Br from 124 gives the base peak a m/z = 43, corresponding to the propyl cation. Note that other peaks, such as those at m/z = 107 and 109 still contain Br and therefore also show the 1:1 isotope pattern.

11 2052, Vol. 2, Issue 7,July 2015 Available at: All sorts of fragmentations of the original molecular ion are possible - and that means that you will get a whole host of lines in the mass spectrum. For example, the mass spectrum of pentane looks like this: It's important to realise that the pattern of lines in the mass spectrum of an organic compound tells you something quite different from the pattern of lines in the mass spectrum of an element. With an element, each line represents a differentisotope of that element. With a compound, each line represents a different fragment produced when the molecular ion breaks up.

12 2052, Vol. 2, Issue 7,July 2015 Available at: The molecular ion peak and the base peak In the stick diagram showing the mass spectrum of pentane, the line produced by the heaviest ion passing through the machine (at m/z = 72) is due to the molecular ion. The tallest line in the stick diagram (in this case at m/z = 43) is called the base peak. This is usually given an arbitrary height of 100, and the height of everything else is measured relative to this. The base peak is the tallestt peak because it represents the commonest fragment ion to be formed - either because there are several ways in which it could be producedd during fragmentation of the parent ion, or because it is a particularly stable ion. Using fragmentation patterns This section will ignore the information you can get from the molecular ion (or ions). That is covered in three other pages which you can get at via the mass spectrometry menu. You will find a link at the bottom of the page. Working out which ion produces which line This is generally the simplest thing you can be asked to do. The mass spectrum of pentanee Let's have another look at the mass spectrum for pentane: What causes the line at m/z = 57? How many carbon atoms are there in this ion? There can't be 5 because 5 x 12 = 60. What about 4? 4 x 12 = 48. That leaves 9 to make up a total of 57. How about C 4 H + 9 then? C 4 H + 9 would be [CH 3 CH 2 CH 2 CH 2 ] +, and this would be produced by the following fragmentation:

13 2052, Vol. 2, Issue 7,July 2015 Available at: The methyl radical produced will simply get lost in the machine. The line at m/z = 43 can be worked out similarly. If you play around with the numbers, you will find that this corresponds to a break producing a 3-carbon ion: The line at m/z = 29 is typical of an ethyl ion, [CH 3 CH 2 ] + : The other lines in the mass spectrum are more difficult to explain. For example, lines with m/z values 1 or 2 less than one of the easy lines are often due to loss of one or more hydrogen atoms during the fragmentation process. You are very unlikely to have to explain any but the most obvious cases in an A'level exam. The mass spectrum of pentan-3-one This time the base peak (the tallest lest peak - and so the commonest fragment ion) is at m/z = 57. But this isn't produced by the same ion as the same m/z value peak in pentane. If you remember, the m/z = 57 peak in pentane was produced by [CH 3 CH 2 CH 2 CH 2 ] +. If you look at the structure of pentan-3-one, it's impossible to get that particular fragment from it. Work along the molecule mentally chopping bits off until you come up with something that adds up to 57. With a small amount of patience, you'll eventually find [CH 3 CH 2 CO] + - which is produced by this fragmentation: You would get exactly the same products whichever side of the CO group you split the

14 2052, Vol. 2, Issue 7,July 2015 Available at: molecular ion. The m/z = 29 peak is produced by the ethyl ion - which once again could be formed by splitting the molecular ion either side of the CO group. Peak heights and the stability of ions The more stable an ion is, the more likely it is to form. The more of a particular sort of ion that's formed, the higher its peak height will be. We'll look at two common examples of this. Examples involving carbocations (carbonium ions) Applying the logic of this to fragmentation patterns, it means that a split which produces a secondary carbocation is going to be more successful than one producing a primary one. A split producing a tertiary carbocation will be more successful still. Let's look at the mass spectrum of 2-methylbutane. 2-methylbutane is an isomer of pentane - isomers are molecules with the same molecular formula, but a different spatial arrangement of the atoms. Look first at the very strong peak at m/z = 43. This is caused by a different ion than the corresponding peak in the pentane mass spectrum. This peak in 2-methylbutane is caused by: The ion formed is a secondary carbocation - it has two alkyl groups attached to the carbon with the positive charge. As such, it is relatively stable.

15 2052, Vol. 2, Issue 7,July 2015 Available at: The peak at m/z = 57 is much taller than the corresponding line in pentane. Again a secondary carbocation is formed - this time, by: You would get the same ion, of course, if the left-hand CH 3 group broke off instead of the bottom one as we've drawn it. In these two spectra, this is probably the most dramatic example of the extra stability of a secondary carbocation. Examples involving acylium ions, [RCO] + Ions with the positive charge on the carbon of a carbonyl group, C=O, are also relatively stable. This is fairly clearly seen in the mass spectra of ketones like pentan-3-one. The base peak, at m/z=57, is due to the [CH 3 CH 2 CO] + fragmentation that produces this. ion. We've already discussed the Using mass spectra to distinguish between compounds Suppose you had to suggest a way of distinguishing between pentan-2-one and pentan-3-one using their mass spectra. pentan-2-one CH 3 COCH 2 CH 2 CH 3 pentan-3-one CH 3 CH 2 COCH 2 CH 3 Each of these is likely to split to produce ions with a positive charge on the CO group.

16 2052, Vol. 2, Issue 7,July 2015 Available at: In the pentan-2-one case, there are two different ions like this: [CH 3 CO] + [COCH 2 CH 2 CH 3 ] + That would give you strong lines at m/z = 43 and 71. With pentan-3-one, you would only get one ion of this kind: [CH 3 CH 2 CO] + In that case, you would get a strong line at 57. You don't need to worry about the other lines in the spectra - the 43, 57 and 71 lines give you plenty of difference between the two. The 43 and 71 lines are missing from the pentan-3-one spectrum, and the 57 line is missing from the pentan-2-one one. The two spectra look like this:

17 2052, Vol. 2, Issue 7,July 2015 Available at: Fragmentation Mechanisms Cleavage of M + (also written M +. ) ions from electron impact ionization or [M +1 ] + ions from chemical ionization at bonds near the site of ionization gives smaller fragment ions. The appearance of fragment ions in the mass spectrum and an understanding of fragmentation mechanisms can provide information about the structure of unknown compounds. Electron impact ionization yields molecular radical ions, also called odd electron ions. Their fragmentation is very common and is often predicted through an understanding of free radical chemistry. Chemical ionization yields even electron ions that are less prone to fragmentation. The fragmentation that does occur is often predicted through an understanding of cation chemistry. A general rule is that the most favored fragmentation pathway cleaves the weakest bond or bonds near the site of ionization to give the most stable cation and/or radical products. Mechanisms for odd electron fragmentation are written with fish hook arrows to show the migration of single electrons, and mechanisms for fragmentation of even electron ions, with double headed arrows to show the migration of electron pairs. A few simple rules for fragmentation of ions are as follows: 1. If one bond of an odd electron ion cleaves, the products are an even electron cation and an odd electron neutral radical. Only the cation is detected. 2. If two bonds of an odd electron ion cleave, either simultaneously of sequentially, the products are an odd electron cation and an even electron neutral fragment.

18 2052, Vol. 2, Issue 7,July 2015 Available at: 3. If one (or even two bonds) of an even electron ion cleaves, the products are an even electron cation and an even electron neutral fragment. 4. Odd electron ions can also undergo structural (also called skeletal) rearrangement of their atoms prior to fragmentation. Rearrangements are sometimes predictable but often make mass spectral fragment ion interpretation difficult. 5. Fragment ions can undergo additional fragmentation to give smaller fragment ions. In terms of structure determination, the most important fragment ions are those of higher m/z that result from fragmentation of the molecular ion. Identification of the parent ion, the ion undergoing fragmentation, as the molecular ion or a fragment ion undergoing subsequent fragmentation is not always simple. More advanced mass spectral techniques facilitate this identification.

19 2052, Vol. 2, Issue 7,July 2015 Available at:

20 2052, Vol. 2, Issue 7,July 2015 Available at:

21 2052, Vol. 2, Issue 7,July 2015 Available at: Application of fragmentationn mechanisms for the assignment of some of the higher m/z fragment peaks in appearing in EI mass spectra

22 2052, Vol. 2, Issue 7,July 2015 Available at:

23 2052, Vol. 2, Issue 7,July 2015 Available at: References : 1. R. M. Silverstein, G. C. Bassler and T. C. Morrill, Spectrometric Identification of Organic Compounds, 5th Ed., Wiley, The Theory of NMR - Chemical Shift 3. Donald C. Hofer; Vincent N. Kahwaty; Carl R. Kahwaty, "NMR field frequency lock system", issued Balci, M., in Basci 1H- and 13C-NMR spectroscopy (1st Edition, Elsevier), 5. Gottlieb HE; Kotlyar V; Nudelman A (October 1997). "NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities". J. Org. Chem. 62 (21): F. A.Carey and R. J. Sundberg "Advanced Organic Chemistry" part A:strures and Mechanisms, 2 nd ed., Plenum Press. New York, p. 243, (1983). 7. Nagham M Aljamali., As. J. Rech., 7,9, , C.O.Wilson and O. Givold, "Text book of Organic Medicinal and pharmaceutical Chemistry", 5 th Ed., Pitman Medical Publishing Co. LTD, London coppy right. Cby. J. B. LippinCott Company (1966). 9. Nagham M Aljamali., As. J. Rech., 2014, 7, Nagham M Aljamali., Int. J. Curr.Res.Chem.Pharma.Sci. 1(9): , (2014):. 11. Nagham M Aljamali., Int. J. Curr.Res.Chem.Pharma.Sci. 1(9):):88-120, (2014). 12. Y. Ju, D. Kumar, R. S. Varma, J. Org. Chem, 71, , N. Iranpoor, H. Firouzabadi, B. Akhlaghinia, R. Azadi, Synthesiss, , Y. Liu, Y. Xu, S. H. Jung, J. Chae, Synlett, ,2012.

24 2052, Vol. 2, Issue 7,July 2015 Available at: