1. Presented By :- Ms. ARTI R RAJPUT M.Pharm, (SUCOP,Pune) Mass Spectrometry 1

2. Contents  Introduction of Mass spectrum.  Types of Ions Molecular ion, Metastable ions, Fragment ions.  Fragmentation procedure  Fragmentation patterns  Fragment characteristics  Relative abundances of isotopes. 2

3. Introduction of MS  The impact of a stream of high energy electrons causes the molecule to lose an electron forming a radical cation.  A species with a positive charge and one unpaired electron Molecular ion (M + ) m/z = 16 3

4. Introduction of MS  Only cations are detected. - Radicals are “invisible” in MS  The amount of deflection observed depends on the mass to charge ratio (m/z). -Most cations formed have a charge of +1 so the amount of deflection observed is usually dependent on the mass of the ion.. 4

5. Molecular ion The ion obtained by the loss of an electron from the molecule also called parent ion Base peak The most intense peak in the MS, assigned 100% intensity M+ Symbol often given to the molecular ion. Mol. With an unpaired e- Radical cation +ve charged species with an odd number of electrons Fragment ions Lighter cations formed by the decomposition of the molecular ion. also called daughter ion

6. Mass Spectrum  The resulting mass spectrum is a graph of the mass of each cation vs. its relative abundance.  Relative abundance of an ion means the % of total ion current.  Mass spectrum is an analytical techniques which can provide information concerning the molecular structure of organic comp.  Base peak is the highest peak or the most intense peak in the spectrum. 6

7. Types of Ion  Types of ion produced in MS 1.Molecular ions (parent ion) 2.Metastable ions 3.Fragment ions (Dissociation process) 4.Rearrangement ions 5.Multiple charged ions 6.Isotopes ions 7.Negative ions 8.Base peak 7

8. Molecular ion  Molecular ion (parent ion): -The radical cation corresponding to the mass of the original molecule  The molecular ion is usually the highest mass in the spectrum 8

9. Molecular ion  When a sample sub.is bombarded with electrons of energies of 9 to 15eV, the molecular ion is produced by loss of a single electron.  This will give rise to a very simple mass spectrum with essentially all of the ion appearing in one peak called parent peak.  M + e = M + + 2e -  Most important ion. 9

10. Molecular ion  In organic compound there is generally a small peak appearing one mass unit higher than the parent peak (M+1) due to small but observable,natural abundance of C 13 and H 2 in these compound.  The relative height of parent peak decreases in the following order, aromatic>conjugated olefins>sulphides> unbranched>hydrocarbon>ketones>amine>ester> ethers >carboxylic acid>branched hydrocarbons. 10

11. Molecular ion  If a molecule yields the parent peak due to molecular ion,the exact molecular weight can be calculated.  Molecular ion are formed in the ground state and in the electronically excited state. 11

12. Mass Spectrum Mass spectrum of ethanol (MW = 46) M+M+  Mass spectrum of ethanol (MW = 46) M+M+ 12

13. Introduction of MS. The mass spectrum of ethanol base peak M+M+ 13

14. Fragment ion  The molecular ion produced in MS is generally left with considerable excess energy.  This energy is rapidly lost by the molecular ion resulting in one or more cleavages in it with or without some rearrangement.  One of the fragment retains the charge where as the remaining fragment may be stable molecule or radicals. 14

15. Fragment ion  If the electron beam energy is further increased to apparent potential of a molecule,then the excited molecule ions undergoes decomposition to give rise to variety of fragment ions which leaves smaller masses than the molecular ion.  Formed by both heterolytic and homolytic cleavage of bond.  They are formed by simple cleavage and rearrangement process. 15

16. Fragment ion  Bond dissociation energy stability of neutral fragment are steric factors are some of the major factor which determine formation of fragment ions.  E.g. : Ethyl chloride.  CH 3 -CH 2 -Cl + e- = CH 3 -CH 2 -Cl + + 2e-  CH 3 -CH 2 -Cl + = CH 3 -CH 2 + + Cl. Or CH 2 -CH 2 + + HCl. (Fragment ion) 16

17. M + e- M+* + 2e- + M+*1M+*1 M2M2 M4+M4+ Fragmentation Process OR M3*M3* +

18. Metastable ion  The ions in a mass spectrometer that have sufficient energy to fragment sometime after leaving the ion source but before arriving at the detector.  M + A + + N (m 1 /z) (m 2 /z) (m 1 -m 2 )  M + with large amount of internal energy will fragment in the ionization source, producing “normal” A + ions. These A + ions will be seen as narrow peaks at m/z values correct for the mass and charge on the ion A +.  M + having only a small excess of internal energy, reach detector before decomposition can occur. Narrow peaks for “normal” M + appear 18

19. Metastable ion  M + which posses excesses of internal energy that are in between the those in above two cases, may fragment after leaving the ion source and before reaching the detector. The product ions, A+, are seen in the mass spectrum as broad peaks, centered at m/z values that are nor correct for the mass and charge on the ion A +.  These broad peaks are called “metastable ion peaks”  These ““metastable ion peaks” do not represent metastable M + ions, but represent products of decomposition of metastable ions.  The cause of A + ions from metastable ion decomposition being detected differently form “normal” A+ ions is due to their different momentum. 19

20. FRAGMENTATION MODES The RA of fragment ion formed depends upon’ 1)The stability of the ion 2)Also the stability of radical lost. The radical site is reactive and can form a new bond. The formation of new bond is a powerful driving force for ion decompositions. The energy released during bond formation is available for the cleavage of some bonds in the ion. Some imp. Fragmentation modes are described below 1)Simple cleavage : Involves i) Homolytic or ii) Heterolytic cleavage of a single covalent bond. 20

21. Fragmentation modes  1) Homolytic cleavage : odd electron ions have unpaired electron which is capable of new bond formation. Bond is formed, energy is released, help offset the energy required for the cleavage of some other bond in the ion. Homolytic cleavage reactions are very common. 2) Heterolytic cleavage : It may be noted the cleavage of C-X (X= 0,N,S,Cl) bond is more difficult than that of C-C bond. In such cleavage, the positive charge is carried by the carbon atom and not by the heteroatom. R-CH 2 -Cl.+ = Cl. + RC + H 2 21

22. Fragmentation modes 2) Retro –Diels –Alder reaction The reaction is an example of multicentre fragmentation which is characteristic of cyclic olefins. It involves the cleavage of two bonds of a cyclic system, result the formation of 2 stable unsaturated fragment in which 2 new bonds are formed. This process is not accompanied by any hydrogen transfer rearrangement. The charge can be carried by any one of the fragment. 22

23. 3)Mc Lafferty Rearrangement: This involves migration of hydrogen atom from one part of the ion to another. To undergo a Mc Lafferty Rearrangement a molecule must possess a)An appropriately located heteroatom e.g. O, N b)A pi electron system ( usually a double bond) & c)An abstractable hydrogen atom gamma to the C = X system Gamma hydrogen atom is transferred through a six membered transition state to an electron deficient centre followed by cleavage at beta bond. The reaction results in the elimination of a neutral molecule. 23

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26. Rules  A number of general rules for predicting prominent peak in electron impact spectra are recorded and can be summarized below  1) most compound give molecule ion peak but some do not. Existence of molecular ion peak in the spectrum is dependent on the stability of molecule  2)In case of alkenes, the relative intensity of the molecule ion peak is greatest for the straight chain compound but, a)The intensity decreases with increases degree of branching. b)The intensity decreases with increasing molecular weight in a homologous series. 26

27. Rules  3) cleavage is favored at alkyl substituted carbons,the more substituted,the more likely is the cleavage.Hence the tertiary carbocation is more suitable than secondary, which is more turn stable then primary. The cation stability order is CH3 < R-CH2 <R2 CH+ < R3C+.Generally the largest substituent at a branch is eliminated most readily as a radical, presumably because a long chain radical can achieve some stability by delocalization of the lone electrons. 4)In alkyl substituted ring compounds, cleavage is favoured at the bound at the bond beta to the ring giving the resonance stabilized benzyl ion. 5)Saturated rings containing side chain, lose the side chains at the alpha bond. the ve+ charge tend to stay with ring fragment. 27

28. Rules 6)The cleavage of a C-X bond is more difficult than that of a C-C bond (X=O, N, S, F, CI, etc). If occurred,the positive charge is carried by the carbon atoms, and not to the heteroatom.the halogens having great electron affinity do not have tendency to carry the positive charge. 7)Double bonds favour allylic cleavage and give the resonance stabilized allylic carbonium ion. 8)Compounds containing a carbonyl group tend to break at this group with positive charge remaining with the carbonyl portions. 28

29. Rules 9)During fragmentation, small, suitable neutral molecules e.g. water, carbon monoxide, alcohol, ammonia, hydrogen cyanide, carbon dioxide, ethylene etc, are eliminated from appropriate ions. 29

30. Fragmentation Pattern for org. comp. Organic molecules will fragments in very specific ways depending upon what functional groups are present in the molecule. These fragments (if positively charged are detected in mass spectroscopy) The presence or absence of various mass peaks in the spectrum can be used to deduce the structure of the compound in question. 30

31. Fragmentation rules in MS M. + Larger for linear chain 1.Intensity of M. + is Larger for linear chain than for branched compound M. + decreaseIncreasingMW. 2.Intensity of M. + decrease with Increasing MW. (fatty acid is an exception) favored at branching Increased stability of the ion 3.Cleavage is favored at branching  reflecting the Increased stability of the ion Stability order: CH 3 + < R-CH 2 + < R R CH + < C + R R R R R” CH R’ Loss of Largest Subst. is most favored 31

32. Fragmentation Patterns  The impact of the stream of high energy electrons often breaks the molecule into fragments, commonly a cation and a radical. - Bonds break to give the most stable cation.  Alkanes - Fragmentation often splits off simple alkyl groups :  Loss of methyl M + - 15 Loss of ethylM + - 29 Loss of propylM + - 43 Loss of butylM + - 57 -Branched alkanes tend to fragment forming the most stable carbocation's. 32

33. Fragmentation Patterns  Mass spectrum of 2-methylpentane 33

34. Fragmentation Patterns  Alkenes: - Fragmentation typically forms resonance stabilized allylic carbocation. 34

35. Fragmentation Patterns  Aromatics: - Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation. (which rearranges to the tropylium ion) M+M+ 35

36. Fragmentation Patterns  Aromatics may also have a peak at m/z = 77 for the benzene ring. 77 M + = 123 36

37. Fragmentation Patterns  Alcohols : - Fragment easily resulting in very small or missing parent ion peak -May lose hydroxyl radical or water -M + - 17 or M + - 18 - Commonly lose an alkyl group attached to the carbinol carbon forming an oxonium ion. -1 o alcohol usually has prominent peak at m/z = 31 corresponding to H 2 C=OH + 37

38. Fragmentation Patterns  MS for 1-propanol M + -18M+M+ 38

39. Fragmentation Patterns  Amines: - Odd M + (assuming an odd number of nitrogen are present)   -cleavage dominates forming an iminium ion 39

40. Fragmentation Patterns 40

41. Fragmentation Patterns  Ethers -  -cleavage forming oxonium ion - Loss of alkyl group forming oxonium ion - Loss of alkyl group forming a carbocation 41

42. Fragmentation Patterns  MS of diethyl ether (CH 3 CH 2 OCH 2 CH 3 ) 42

43. Fragmentation Patterns  Aldehydes (RCHO) - Fragmentation may form acylium ion - Common fragments M + - 1 for M + - 29 for 43

44. Fragmentation Patterns  MS for hydrocinnamaldehyde 91 105 M + = 134 44

45. Fragmentation Patterns  Ketones : - Fragmentation leads to formation of acylium ion: -Loss of R forming -Loss of R’ forming 45

46. Fragmentation Patterns  MS for 2-pentanone M+M+ 46

47. Fragmentation Patterns  Esters (RCO 2 R’) - Common fragmentation patterns include : Loss of OR’ - peak at M + - OR’ Loss of R’ -peak at M + - R ’ 47

48. Fragmentation Patterns 77 105 M + = 136 48

49. Summary of Fragmentation pattern: 49

50. Alkanes good M+ 14-amu fragments Alkenes distinct M+ m/e = 27CH 2 =CH+ m/e = 41CH 2 =CHCH 2 + M-15, M-29, M-43, etc...loss of alkyl Cycloalkanes strong M+ M-28loss of CH 2 =CH 2 M-15, M-29, M-43, etc...loss of alkyl Aromatics strong M+ m/e = 105C8H9+C8H9+ m/e = 91C7H7+C7H7+ m/e = 77C6H5+C6H5+ m/e = 65 (weak)C5H5+C5H5+ Halides M+ and M+2Cl and Br m/e = 49 or 51CH 2 =Cl+ m/e = 93 or 95CH 2 =Br+ M-36, M-38loss of HCl M-79, M-81 loss of Br · M-127 loss of I ·

51. Alcohols M+ weak or absent M-15, M-29, M-43, etc...loss of alkyl m/e = 31CH 2 =OH+ m/e = 45, 59, 73,...RCH=OH+ m/e = 59, 73, 87,...R 2 C=OH+ M-18loss of H 2 O M-46loss of H 2 O and CH 2 =CH 2 Phenols strong M+ strong M-1 loss of H · M-28loss of CO Ethers M+ stronger than alcohols M-15, M-29, M-43, etc...loss of alkyl M-31, M-45, M-59, etc...loss of OR m/e = 45, 59, 73,...CH 2 =OR+ Amines M+ weak or absentNitrogen rule m/e = 30CH 2 =NH 2 + (base peak) M-15, M-29, M-43, etc...loss of alkyl 51

52. Aldehydes weak M+ m/e = 29HCO+ M-29loss of HCO M-43loss of CH 2 =CHO m/e = 44, 58, 72, 86,...McLafferty rearrangement strong M+aromatic aldehyde M-1 aromatic aldehyde loss of H · Ketones M+ intense M-15, M-29, M-43, etc...loss of alkyl m/e = 43CH 3 CO+ m/e = 55+CH 2 CH=C=O m/e = 42, 83in cyclohexanone m/e = 105, 120in aryl ketones Carboxylic Acids M+ weak but observable M-17loss of OH M-45loss of CO 2 H m/e = 45CO 2 H+ m/e = 60 · CH 2 C(OH) 2 + M+ largearomatic acids M-18ortho effect 52

53. Esters M+ weak but observablemethyl esters M-31methyl esters loss of OCH 3 m/e = 59methyl esters CO 2 CH 3 + m/e = 74 methyl esters CH 2 C(OH)OCH 3 + M+ weakerhigher esters M-45, M-59, M-73, etc...loss of OR m/e = 73, 87, 101CO 2 R+ m/e = 88, 102, 116 · CH 2 C(OH)OR+ m/e = 61, 75, 89RC(OH) 2 + (long alkyl ester) m/e = 108 loss of CH 2 =C=O (benzyl, acetate) m/e = 105C 6 H 5 CO+ (benzoate) M-32, M-46, M-60loss of ROH (ortho effect) 53

54. RA of Isotopes  RELATIVE ABUNDANCES OF ISOTOPES  Isotope peak : The isotope peak are obtained when a molecule contains heavier isotope of certain atoms than the common isotopes.  Commonly seen isotope peak are (M+1) + peaks or (M+2) + peaks  Intensity of an isotope peak is much lesser than that of the (M) + peak except when Cl or Br is present in the molecule. 54

55. ISOTOPES  Most elements occur naturally as a mixture of isotopes. - The presence of significant amounts of heavier isotopes leads to small peaks that have masses that are higher than the parent ion peak.  M+1 = a peak that is one mass unit higher than M +  M+2 = a peak that is two mass units higher than M + 55

56. RA of Isotopes  RELATIVE ABUNDANCES OF ISOTOPES  intensity of an isotope peak depends on the relative abundance of that isotope in nature.  The relative abundance of an isotope is calculate on the basis of 100molecules.  From RA, the intensity of (M+1) +, (M+2) + peaks can be determined.  For a compound containing one carbon atom, out of every 100 molecules, 98.892 molecule contain C 12 isotope and 1.108 molecule contain C 13 isotope 56

57. RA of Isotopes  RELATIVE ABUNDANCES OF ISOTOPES  Hence, the intensity of (M+1) + peak is about 1.1% of the intensity of the (M) + peak and the ratio of the intensities of M + and (M+1) + peak is 98.892:1.108.  For compound containing silicon, the intensities of (M) + peak corresponding to Si28 isotope, (M+1) + peak corresponding to Si 29 isotope and (M+2) + peak corresponding to Si 30 isotope are in proportion of their relative abundance in the nature, i.e. 92.18:4.71:3.12. 57

58. RA of Isotopes  RELATIVE ABUNDANCES OF ISOTOPES  For compound containing sulphur, the ratio of intensities of (M) + : (M+2) + peaks, corresponding to S 32 and S 34 isotopes is 95.018:4.215.  The height of the peak is the measure of intensity of that peak.  Fluorine and iodine have only one naturally occurring isotope corresponding to atomic mass of 19 and 127, resp.  Hence they produced only one peak corresponding to (M) + ion. 58

59. RA of Isotopes  RELATIVE ABUNDANCES OF ISOTOPES S.IsotopeRelative abundancepeak 11H:2H99.985:0.015(M+1) 212C:13C98.892:1.108(M+1) 314N:15N99.635:0.365(M+1) 416O:17O:18O99.759:0.037:0.204(M+1),(M+2) 528Si:29Si:30Si92.18:4.71:3:12(M+1), (M+2) 632S:33S:34S95.018:0.75:4.215(M+1), (M+2) 735Cl:36Cl75.529:24.471(M+2) 879Br:81Br50.52:49.48(M+2) 59

60. References: 1.Silverstein R.M., & Webster F.X, Spectrometric Identification of Organic Compounds, Sixth edition 2006, Page no. 2 – 28. 2.Sharma Y. R. Elementary Organic Spectroscpoy Principles and Chemical Application, Fourth edition 2007, S. Chand & Company, Page no. 280 – 339. 3.Chatwal G.R., Aanand S.K., Instrumental Methods ofAnalysis, Himalaya Publishing House, 5 th Edition, Page no. 2.272-2.302 4.http://www.chemistry.ccsu.edu/glagovich/teaching/316/index.ht ml access date - 19 sept 2013 5.http://en.wikipedia.org/wiki/Mass_spectrometry access date – 19 sept 2013 6.Dr. supriya s. mahajan,Instuumental methods of analysis, page no. 125 -154 60

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