The document discusses the principles and significance of mass spectrometry, detailing the ionization methods and mechanisms of fragment ion generation. It explains various ionization techniques, such as electron impact and chemical ionization, alongside their advantages and limitations. Additionally, it outlines the role of mass spectra in determining molecular weights and analyzing the composition of substances.

1. MASS SPECTROMETRY
HARSHPAL SINGH WAHI
SHIKHA D. POPALI
GURUNANAK COLLEGE OF PHARMACY, NAGPUR

2. SIGNIFICANCE OF MASS SPECTROmetrY
 To determine molecular weight
 To detect positions in molecule at which fragmentation occurs to
identify possible functionalities within molecule.
 Identification of analyte by comparison of mass spectrum of analyte
and that of known compound in digitalized libraries.
 To determine relative percentage of individual isotope in compound
under analysis.
STRUCTURAL ELUCIDATION
ISOTOPE IDENTIFICATION
What do you mean by
isotope

3. principlE OF MASS SPECTROmetrY
 Organic molecules (M) on electron bombardment are converted to
highly energetic positively charged ion [M + ֹ ] or negatively charge ion
[M - ֹ ] (Molecular / Parent ion)
M - e M + ֹ or M + e M - ֹ
 Positive ion MS is more probable than negative ion by 102 factor.
 The loss of electron from molecule gives molecular or parent ion.
 Molecular ion will then degraded in to its constituent ions known as
fragment ions or daughter ions at possible breaking points within
molecule.
What is ionization potential ?

4. 4
Why spectrometry but not spectroscopy ?

5. principlE OF MASS SPECTROmetrY (MS)
 The molecular ion usually decomposes into two components which
will be either radical (m2ֹ ֹ) plus ion (m1
+) or small molecule (m2ֹ) plus
radical cation (m1
+ ֹ ).
M + ֹ m1
+ + m2ֹ M + ֹ m1
+ ֹ + m2
 The radical (m2ֹ ֹ) or small molecule (m2ֹ) is stable and non-ionic and
hence are not detected by detector in mass spectrometry.
 Hence only molecular ion, radical cation or fragment ions will be
detected or deflected in mass spectroscopy.
 As in electron impact ionization, 70 eV potential is applied, double
charged ion (2m/2z ) will appear in MS at same value as single charge
ions of half mass (m/z) as 2m/2z = m/z.

6. 6

7. 7

8. INSTRUMENTATION OF MS
Single Focusing – No electric focusing but electric accelerating system

9. 9
Double Focusing – Electric and Magnetic Focusing

10. SEPARATION OF IONS IN ANALYZER
THE KINETIC ENERGY E, OF ION OF MASS, M TRAVELLING WITH
VELOCITY, V IS
E = ½ MV2 WHERE AS POTENTIAL ENERGY OF ION OF CHARGE, Z
BEING REPELLED BY AN ELECTROSTATIC FIELD OF VOLTAGE, V IS
ZV.
WHEN ION IS REPELLED BY ELECTRIC ACCELERATING SYSTEM,
POTENTIAL ENERGY IS CONVERTED INTO KINETIC ENERGY HENCE,
ZV = 1/2MV2 HENCE, V2 = 2ZV/M
WHEN THESE ARE DEFLECTED IN MAGNETIC FIELD, THEY ARE
DRAWN IN CIRCULAR MOTION (MV2/R)BY FIELD AND AT
EQUILIBRIUM, CENTRIFUGAL FORCE IS EQUALED BY CENTRIPETAL
FORCE (ZBV) (WHERE R IS RADIUS OF CIRCULAR MOTION AND B IS
MAGNETIC FIELD STRENGTH)
HENCE, MV2/R = ZBV HENCE, V = ZBR/M

11. RESOLUTION IN MS
THE RESOLUTION IS ABILITY OF MS TO SEPARATE TWO IONS BY MEASURING THE
DEPTH OF VALLEY BETWEEN PEAKS PRODUCED BY TWO IONS.
E.G. TWO IONS HAVING M/Z 999 AND 1000 CAN BE SAID TO HAVE RESOLUTION 1 IN
1000 IF RECORDER TRACE ALMOST REACHES BACK DOWN TO BASELINE BETWEEN
THEM, LEAVING VALLEY 10 % OF PEAK HEIGHT.
SINGLE FOCUSING HAS 1 IN 7500 RESOLUTION
DOUBLE FOCUSING HAS 1 IN 60000 RESOLUTION
EXPLANATION: IN SINGLE FOCUSING, IONS REPELLED BY ACCELERATING PLATES
MAY NOT HAVE IDENTICAL KINETIC ENERGY HENCE THIS ENERGY SPREAD
DECREASES RESOLUTION. HENCE IN DOUBLE FOCUSING, PRELIMINARY FOCUSING
WAS CARRIED OUT BY CURVED ELECTRIC PLATES (ELECTRIC ANALYZER). THIS
ANALYZER FOCUSES IONS OF IDENTICAL KINETIC ENERGY IN SLIT OPENING IN A
MAGNETIC ANALYZER WHATEVER MAY BE M/Z RATIO OF IONS. THE MAGNETIC
ANALYZER WILL THEN SEPARATES THEM ON BASIS OF M/Z RATIO. THIS IMPROVES
RESOLUTION.
E.G. RESOLUTION OF M/Z = 28 IONS I.E. CO+, N2
+ AND C2H4
+ IS NOT POSSIBLE BY LOW
RESOLUTION MS BUT DOUBLE FOCUSING MS WILL RESOLVE AS THEY HAVE
FOLLOWING EXACT FORMULA MASSES CO+(27.9949), N2
+ (28.0062) AND C2H4
+
(28.0312)

12. 12

13. Ionization of sample
There are various types of methods of ionization
1. Electron Impact Ionization –Most common method
2.Knudsen Cell
3.Surface Ionization
4.Spark Source Ionization
5. Chemical Ionization
6. Field Ionization and Desorption - Recently developed

14. Electron Impact Ionization
 Electrically heated filament produces thermal electrons------- These
electrons are accelerated towards anode in chamber-------Ionization of
molecule to positively charged ions--------These ions are accelerated by low
positively charged plates ------Finally with high negative charged plate
repels them into magnetic or electric analyzer------
 Method utilizes electric potential up to 70 eV for complete ionization of
all type of analytes even though most organic compounds ionized at 10-15.
 Electric accelerating system reduces contact time of molecular ion and
electron.
 Low pressure in chamber prevent contact of ions produced to recombine to
form substance, not present in original sample by decreasing collisions
between them.
 Disadvantages: Not efficient Method (complex fragmentation pattern)
Sample needs to be vaporized Background gas Ionization Molecule
don’t show molecular ion peak in MS spectrum if unstable to e impact
These limitations are overcame in following modifications,

15. Ionization of sample
 Knudsen Cell : Sample in crucible ------Heated by radiation or electron
bombardment to attain 2500OC constant temperature ---------Sample
Vaporized ------ Passed through small orifice to Ion source in MS -----------
Electron bombardment ---------------
 Method is combination of thermal and electronic bombardment
 Useful for thermodynamic studies
 Analysis of solids and low vapor pressure liquids.
 Surface Ionization: Solid sample coated on tungsten ribbon filament ------
Filament heated to 2000OC--------- Sample Vaporized ------ Passed through
small orifice to Ion source in MS -----------Electron bombardment ----------
 Useful for inorganic materials with 3-6 eV ionization potential.
 Advantageous as no ionization of background gas like other methods.
 Not useful for organic materials with 7-16 eV ionization potential.

16. Ionization of sample
 Spark Source Ionization: Two electrodes of (In-organic) analyte are
produced -------Held on movable vises ----------Potential 100kV is applied--
-----Positive ions are produced and evaporated-------- Passed through small
orifice to Ion source in MS -----------Electron bombardment ---------------
 Useful for inorganic materials with low ionization potential.
 Detection sensitivity is very high.
 Non-selective as analyze all elements in sample with same detection
sensitivity.

17. Chemical Ionization
 Mixing sample at 10-4 Torr with reacting gas at 1 Torr ---------Exposing this
mixture to electron bombardment.
 Mechanism: Methane / Ammonia/ Isobutane – Gas used.
On electron bombardment, CH4 CH4
+ ֹ
CH4
+ ֹ + CH4 CH5
+ + CH3 ֹ or CH4
+ ֹ + CH4 CH3
+ + CH5 ֹ
CH3
++ CH4 C2H5
+ + H2
The CH5
+ and C2H5
+ will then react with sample molecules and causes
ionization of molecules to produce ions which will be then electrically and
magnetically deflected in a similar manner .
 Disadvantage: The CH5
+ and C2H5
+ are not reactive with all organic
compounds, Base peak –Alkane M - 1 and bases (N containing ) M+1.
Lower sensitivity Low resolution
 Advantages Over Electron Impact:
More abundant peaks
Simple fragmentation patterns
Useful to study reaction kinetic study
Easy GC-MS – Methane as carrier and reacting gas respectively

18. Field ionization and desorption
 Sample Deposition on metal anode with electric field force of 1010 V/m----
--------Electrons from sample go to incomplete orbital in metal anode-------
positive charged ions ------ Transferred to focusing by desorption and by
electric accelerating system same as like electron impact ------
 Advantages Over Electron Impact:
1. More abundant peaks
2. Simple fragmentation patterns
3.Sensitive and selective
4. Useful for complex naturally occurring substances like carbohydrates, not
possible by Electron Impact.
Recent Development under investigations: Use of Lasers , nuclear fission
fragments or neutral atoms or molecules for ionization. Few methods are
successful for determining molecular masses of insulin (5733), chlorophyll
(6000) etc.
LD, LIMA, PD, SIMS and FAB are recent techniques which are able to
detect molecular ion up to 20000 daltons
Desorption means repelling from metal anode as positively charged

19. RECENT IONIZATION TECHNIQUES
LD AND LIMA: LASER DESORPTION & LASER IONIZATION MASS ANALYSIS
: IRRADIATION OF SAMPLE WITH PULSED LASER (105 W/CM2)……..
VAPORIZATION OF SMALL AMOUNT OF SAMPLE FROM SURFACE
…..PRODUCTION OF IONS AND NEUTRAL FRAGMENTS……..PASSED TO
FOCUSING CHAMBER ……. SECOND PULSE WITH HIGH POWER OR
ELECTRON IMPACT TO VAPORIZE AND IONIZE NEXT SURFACE LAYER OF
SAMPLE.
HENCE USEFUL FOR SURFACE ANALYSIS OF POLYMERS AND MICRO-
ELECTRONIC INDUSTRIES.
USEFUL FOR MASS ANALYSIS OVER SPECIFIC CROSS SECTION OF
MATERIAL (COUPLED WITH MICROSCOPE) AND FOR ELEMENTAL
ANALYSIS.
PD: PLASMA DESORPTION: IMPACTING NUCLEAR FISSION FRAGMENTS
WITH HIGH ENERGY (142BA18+ & 106TE22+) ,OBTAINED FROM 252CF WITH
SAMPLE……..GENERATES 1012 W OF POWER AND ABOUT 10000 K

20. MASS Spectrum
 The molecular ion, fragment ions and fragment radical ions are
separated by deflection in a variable magnetic filed according to their
mass to charge ratio (m/z) and generates current (ion current) at
collector in proportion to their relative abundances.
 Highest current produced by any fragment within molecule is called as
Base Peak. It indicates bond in molecule which is more prone to
decompose and hence produced in highest concentration hence carries
more ion current and hence its current is taken as abundance as
maximum i.e. 100 %.
 The current produced by other fragments or ions is recorded as relative
abundance i.e. relative to abundance of base peak.

21. MASS Spectrum
 Plot of relative abundance versus mass/charge ratio is known mass
spectrum.
 The mass spectrum of ethanol (C2H5OH)

22. TYPES OF IONS/PEAKS IN MS
FOLLOWING TYPE OF IONS/PEAKS ARE POSSIBLE IN MASS
SPECTROMETRY DUE IONIZATION OF MOLECULE AND
FRAGMENTATION OF MOLECULAR IONS.
1. MOLECULAR ION OR PARENT PEAK
2. BASE PEAK – (AS DESCRIBED IN PREVIOUS SLIDE NO. 20)
3. REARRANGEMENT IONS
4. MULTIPLY CHARGE IONS
5. NEGATIVE IONS
6. METASTABLE IONS

23. METASTABLE PEAK
 THE FATE OF MOLECULAR IONS IN MS: THE MOLECULES WILL BE
IONIZED IN IONIZATION CHAMBER ON ELECTRON IMPACTION OF 70
EV ENERGY TO GIVE MOLECULAR IONS. THESE WILL BE ACQUIRED
DIFFERENT AMOUNT OF ENERGY DURING IONIZATION AND HENCE
POSSESS DIFFERENT LIFETIME, HENCE VARYING STABILITY.
 LOW ENERGY- MAXIMUM STABLE-HIGH LIFETIME-DETECTED
MOLECULAR IONS AT COLLECTOR
 MODERATE ENERGY- MODERATE STABLE – OPTIMUM LIFETIME-
LEAVE IONIZATION CHAMBER AS MOLECULAR ION - MAY NOT
REACH DETECTOR INTACT- FRAGMENTED IN BETWEEN IONIZATION
CHAMBER AND COLLECTOR.
 HIGH ENERGY- MINIMUM STABILITY-LESS LIFETIME-DO NOT LEAVE
IONIZATION CHAMBER AS MOLECULAR ION- FRAGMENTED IN
IONIZATION CHAMBER – FRAGMENTS WITH HIGH STABILITY- (SAME
PROTOCOL FOR FRAGMENT IONS). M + ֹ A + + Bֹֹ
THESE LOW AND MODERATE ENERGY MOLECULAR IONS ACQUIRE
Why

24. 24

25. ION –TUBE REGIONS – METASTABLE IONS
THE IONIZATION CHAMBER TO DETECTOR, MOLECULAR ION HAS TO PASS FOLLOWING REGIONS,
1. FIRST FIELD FREE REGION: DOUBLE FOCUSING – IONIZATION CHAMBER TO ELECTROSTATIC
ANALYZER, NOT PRESENT IN SINGLE FOCUSING MS. METASTABLE IONS WITH ABNORMAL
KINETIC ENERGY-----FOCUSED OUT OF ANALYZER--- (UN) DETECTED AS BACKGROUND
CURRENT
2. ELECTROSTATIC ANALYZER: FOCUSED OUT OF ANALYZER-UNDETECTED OR DETECTED AS B.
C.
3. SECOND FIELD FREE REGION: IONIZATION CHAMBER & MAGNETIC ANALYZER IN SINGLE
AND ELECTROSTATIC & MAGNETIC ANALYZER IN DOUBLE FOCUSING. METASTABLE IONS WITH
SAME MASS AND HIGHER TRANSLATIONAL ENERGY THAN NORMAL FRAGMENT ION ----
DETECTED PREDOMINANTLY IN THIS REGION AT LOWER MASS NUMBER THAN NORMAL
FRAGMENT ION.
4. MAGNETIC ANALYZER: IONS PRODUCED IN THIS REGION ARE DETECTED ----- BUT WITH
SUBSTANTIAL ENERGY DIFFERENCE BETWEEN PRODUCED AT BEGINNING AND END OF
ANALYZER----- PRODUCES CONTINUUM OF WEAK SIGNAL BETWEEN NORMAL FRAGMENT MASS
AND METASTABLE PEAK MASS -------- HENCE TOO MUCH WEAK----REMAINS UNDETECTED
5. THIRD FIELD FREE REGION: MAGNETIC ANALYZER AND COLLECTOR IN BOTH SINGLE AND
DOUBLE FOCUSING MS. NO FOCUSING IS POSSIBLE IN THIS REGION BUT IF MOLECULAR ION IS
DECOMPOSED, METASTABLE PEAK IS OBSERVED AT M/Z OF PARENT PEAK AS METASTABLE
WILL ACQUIRE SAME PATH AS BY PARENT PEAK.

26. CALCULATION OF APPARENT M/Z OF METASTABLE IONS (M*):THE RELATIONSHIP BETWEEN
APPARENT M/Z OF MOLECULAR AND METASTABLE IONS IS GIVEN BY M + ֹ A+ + Bֹֹ
THE METASTABLE ION IS OBSERVED AT M* WHICH IS GIVEN BY M* = M2
2/M1 WHERE M1 IS
MASS OF MOLECULAR ION M + ֹ, M2 IS MASS OF FRAGMENT /METASTABLE ION A+ (AS REAL MASS
OF METASTABLE ION IS SAME AS THAT OF FRAGMENT ION, FRAGMENTED IN IONIZATION
CHAMBER).
THIS EQUATION GIVES APPARENT MASS 0.1 TO 0.4 UNITS LOWER THAN PRACTICALLY OBSERVED.
E.G. TOLUENE HAS STRONG PEAKS AT 91 AND 65 M/Z VALUES. THUS IT IS HAVING MOLECULAR ION
PEAK AT 91 M/Z AND FRAGMENT ION PEAK AT 65 (REAL MASS OF METASTABLE PEAK) HENCE
APPARENT MASS OF METASTABLE PEAK WILL BE, M*= 652/91 = 4225/91 = 46.4
SIGNIFICANCE OF METASTABLE PEAK: INITIALLY, IT WAS ASSUMED THAT METASTABLE PEAK
PRESENCE WILL CONFIRM ONE STEP DEGRADATION OF MOLECULAR ION TO DAUGHTER IONS.
BUT AFTERWARDS, IT HAS BEE OBSERVED THAT IT MAY NOT BE SINGLE STEP.
HENCE METASTABLE PEAKS ARE IMPORTANT TO STUDY FRAGMENTATION PATTERN OF
MOLECULAR IONS BUT NOT FOR STRUCTURE DETERMINATION. IT WILL HELP TO SOLVE
CONFUSION POSSIBLE IN MOLECULAR FORMULA DETERMINATION E.G. PEAK AT M/Z 46.4 WILL
NOT CORRESPONDS TO ANY FRAGMENT OF TOLUENE MOLECULE BUT IT IS METASTABLE PEAK.

27. 27

28. 28

29. TYPES OF IONS/PEAKS IN MS
REARRANGEMENT IONS: THESE ARE IONS, NOT A PART OF ORIGINAL
MOLECULE BUT FORMED FROM MOLECULAR ION BY
REDISTRIBUTION OF ATOMS OR GROUPS AT MOMENT OF
DECOMPOSITION OF MOLECULAR ION. THESE IONS ARE NOT
PREDICTABLE AND NONSPECIFIC IN HYDROCARBONS BUT
PREDICABLE AND SPECIFIC IN COMPOUNDS WITH HETERO ATOMS AS
THEY PRODUCE INTENSE PEAKS.
E.G. HYDROGEN AND METHYL GROUP MIGRATION DURING
MOLECULAR REARRANGEMENT.
CH3C+H2 + X
CH3CH2-X+
CH2=CH2
+ +HX
MULTIPLY CHARGED IONS: SOMETIMES MS WILL RECORD DOUBLE
OR TRIPLE CHARGED IONS AS 70 EV POTENTIAL IS APPLIED FOR
IONIZATION. SUCH IONS WILL APPEAR AT HALF OR 1/3 M/Z VALUES

30. MOLECULAR IONS/ PARENT PEAK
THE SMALL PEAK OR CLUSTER OF PEAKS AT HIGHEST M/Z VALUES
IN MASS SPECTRUM, AT ONE OR TWO MASS UNIT HIGHER (M+1 OR
M+2) THAN MOLECULAR MASS OF MOLECULE IS SAID TO BE
MOLECULAR ION OR PARENT ION.
 THIS IS APPEARED AT HIGHER MASS NUMBER DUE TO SMALL BUT
OBSERVABLE NATURAL ABUNDANCE OF 13C AND 2H IN THESE
MOLECULES. (ISOTOPIC ABUNDANCE)
 IF SAME MOLECULE HAS TWO HEAVY ISOTOPES, SMALL PEAK AT
M+2 IS OBSERVED E.G. CL OR BR CONTAINING COMPOUNDS.
 THE C-C S BOND IS MORE PRONE TO IONIZATION THAN C-H S
BOND IN AROMATIC COMPOUNDS, P ELECTRONS OF DOUBLE OR
TRIPLE BOND IN UNSATURATED COMPOUNDS AND NON-BONDED
ELECTRONS ON HETERO ATOMS ARE READILY REMOVED.
 THE MOLECULAR ION WITH LOSS OF ELECTRON FROM P BOND
WILL BE HIGH STABLE COMPARED TO SAME OF S BOND AS

31. MOLECULAR IONS/ PARENT PEAK
 AS LIKE UV SPECTROSCOPY, WE ARE NOT CONCERNED WITH
ELECTRONIC EXCITATIONS AS ENERGY OF ELECTRON IMPACT IS
70 EV WHICH LOSSES SPECIFICITY OF ATTACK ON MOLECULE I.E.
IT IS UNABLE TO JUDGE ORBITAL OF ELECTRON REMOVAL IN
MOLECULE.
 HENCE WHEN ELECTRONS IS REMOVED FROM HOMO, IT SHOULD
BE CONSIDERED THAT IT IS REMOVED FROM MOLECULE AS
WHOLE.
 HENCE TO REPRESENT MOLECULAR ION, EITHER OR BOTH OF
FOLLOWING METHODS, PARTIAL /COMPLETE SQUARE BRACKET,
[C2H5] + ֹ / C2H5˥ + ֹ OR
 FRAGMENTATION OF MOLECULAR ION IS NOT SIMPLE PROCESS. IT
NEEDS VARIOUS CONSIDERATIONS LIKE REACTIVITY OF

32. 32

33. RECOGNITION OF MOLECULAR IONS
 NEARLY 20 % COMPOUNDS HAVE WEAK OR UNDETECTED PARENT
PEAK IN MASS SPECTRUM DUE TO RAPID DECOMPOSITION DUE TO
ELECTRON IMPACTION OF 70 EV ENERGY.
 HENCE FOR UNKNOWN COMPOUNDS IONIC CLUSTER APPEARING AT
M+1 IS CONSIDERED TO BE MOLECULAR ION PEAK BUT IT SHOULD BE
VERIFIED BY SERIES OF TESTS.
 ABUNDANCE TEST : STRONG PEAK DUE TO HIGH ABUNDANCE –
ARYL AMINES, HALIDES, HETEROCYCLICS & AROMATIC
HYDROCARBONS WITH NO SIDE CHAIN LONGER THAN C2. WEAK
PEAK/ABSENCE OF PEAK - DUE TO LOW ABUNDANCE (EASILY
FRAGMENTABLE) – ARYL KETONES (WEAK) AND BENZYL
COMPOUNDS (WEAK) OR ARYL WITH MORE SUBSTITUTIONS (WEAK)
OR HIGHLY BRANCHED COMPOUNDS OF ANY FUNCTIONAL GROUP
E.G. ALCOHOLS (ABSENT)
 ISOTOPE ABUNDANCE: BY COMPARING MASS NUMBER OF
MOLECULAR IONS CLUSTER AT M, M+1, M+2, IT IS POSSIBLE TO

34. NITROGEN RULE
THIS RULE HELPS TO IDENTIFY NUMBER OF NITROGEN ATOMS IN
COMPOUND IF INTEGRAL MOLECULAR WEIGHT OF COMPOUND IS
KNOWN.
“ALL ORGANIC COMPOUNDS HAVING AN EVEN INTEGRAL
MOLECULAR WEIGHT MUST CONTAIN NO OR EVEN NUMBER OF
NITROGEN ATOMS AND THAT WITH ODD MOLECULAR WEIGHT
CONTAINS ODD NUMBER OF NITROGEN ATOMS”
E.G. C3H5NO2 – MOLECULAR WEIGHT – 87 – ODD – ONE NITROGEN
C2H7NS - MOLECULAR WEIGHT – 77 – ODD – ONE NITROGEN
C6H7BRN2 – MOLECULAR WEIGHT – 186 – EVEN – TWO
NITROGEN
BUT IT DEPENDS UP ON,
1.MASS NUMBERS AND NATURAL ISOTOPIC ABUNDANCE OF C, H,
N N
N

35. RING RULE
THIS RULE HELPS TO IDENTIFY NUMBER OF UNSATURATED SITES
IF MOLECULAR FORMULA IS KNOWN BY MASS SPECTROMETER.
“NUMBER OF UNSATURATED SITES ‘R’ IS EQUAL TO NUMBER OF
RINGS IN MOLECULE PLUS NUMBER OF DOUBLE BONDS PLUS
NUMBER OF TRIPLE BONDS. IF MOLECULE HAS CWHXNYPZ
(P-HALOGEN) THEN R IS GIVEN BY, R = W + 1 +( Y-X)/2- Z/2
E.G. 1. BENZENE, C6H6 THEN W = 6 X = 6 Y=0 AND Z = 0
R = 6 + 1 + 0-6/2 = 6+1-3=4,
THUS IT CONTAINS ONE RING AND THREE DOUBLE BONDS.
2. C8H8N2 HENCE W = 8 X = 8 Y= 2 AND Z=0,
R = 8 + 1 + 2-8/2 = 9-3 = 6,
THUS IT CONTAINS TWO RINGS AND FOUR DOUBLE BONDS
N
N
H
N
ClF
w = 11 x = 9 y= 1 and z=2,
R = 11 + 1 + 1-9/2-2/2 = 12-5 = 7,
Two rings and five double bonds

36. THE “RULE OF THIRTEEN” CAN BE USED TO IDENTIFY POSSIBLE MOLECULAR
FORMULAS FOR AN UNKNOWN HYDROCARBON, CNHM.
STEP 1: N = M+/13 (INTEGER ONLY, USE REMAINDER IN STEP 2)
E.G. MASS 120, 120/13 = 117/13, REMAINDER IS 3
STEP 2: M = N + REMAINDER FROM STEP 1
EXAMPLE: THE FORMULA FOR A HYDROCARBON WITH M+ =106 CAN BE
FOUND:
STEP 1: N = 106/13 = 8 (R = 2)
STEP 2: M = 8 + 2 = 10
FORMULA: C8H10
36
Rule of Thirteen for Hydrocarbons

37. RULE OF THIRTEEN
IF A HETEROATOM IS PRESENT,
SUBTRACT THE MASS OF EACH HETEROATOM FROM THE MW
CALCULATE THE FORMULA FOR THE CORRESPONDING
HYDROCARBON
ADD THE HETEROATOMS TO THE FORMULA
EXAMPLE: A COMPOUND WITH A MOLECULAR ION PEAK AT M/Z = 102
HAS A STRONG PEAK AT 1739 CM-1 IN ITS IR SPECTRUM.
DETERMINE ITS MOLECULAR FORMULA.
37
O
O

38. 38

39. DETERMINATION OF MOLECULAR FORMULA & WEIGHT
MOLECULAR WEIGHT =MOLECULAR ION PEAK M/Z VALUE - 1 (C,H, O ISOTOPE) OR 2 (HALIDE)
MOLECULAR FORMULA – LOW RESOLUTION – 100 , HIGH RESOLUTION – 100. 088 71
1. MOLECULAR WEIGHT HELPS TO KNOWN POSSIBLE COMPOUNDS WITH DIFFERENT
FORMULAE E.G. MOLECULAR WEIGHT – 100 COMPOUND A- C6H12O OR B- C4H4O3
2. MOLECULAR ION PEAK HEIGHT AND MASS VALUE WITH FOUR DECIMALS HELPS TO IDENTIFY
IT. E.G. HIGHER PEAK HEIGHT – 12C, 1H, 16O ISOTOPE, LESS PEAK HEIGHT- 13C, 2H, 17O ISOTOPE
AND MASS VALUE WITH FOUR DECIMALS HELPS TO KNOW EXACT MOLECULAR FORMULA
E.G. EXACT MOLECULAR WEIGHT IS 100.08871, WHICH ONE IS COMPOUND ?
AUTOMATIC MOLECULAR FORMULA INTERPRETATIONS ARE POSSIBLE USING HIGH RESOLUTION
INSTRUMENT INTERFACED TO COMPUTER WHICH BY PROGRAMMING USING REFERENCE PEAKS
INTERPOLATION HELPS TO GIVE LIST OF ION MASSES, ABUNDANCE AND COMPOSITIONS. THE
POSITION OF ACCURATE KNOWN MASSES CAN BE OBTAINED BY COMPARING PEAKS OBTAINED
USING ELECTRONIC MASS MARKER OR REFERENCE COMPOUND SUCH AS
PERFLOUOROKEROSENE (PFK) USING DOUBLE BEAM MASS SPECTROMETER.
ALTERNATE METHOD/ PEAK MATCHING: COUPLING OUTPUT OF ION WHOSE MASS IS TO BE
MEASURED AND ION OF KNOWN MASS FROM REFERENCE COMPOUND ON CATHODE RAY
OSCILLOSCOPE-----ACCELERATING VOLTAGE INCREASED UNTIL TWO MASSES OVERLAP ------
DIFFERENCE IN MASS CALCULATED AS FUNCTION OF CHANGE IN ACCELERATING VOLTAGE.
its compound A but not B

40. ISOTOPES AND ISOTOPIC ABUNDANCE
ISOTOPES: THE ELEMENT WITH SAME ATOMIC NUMBER WITH
DIFFERENT MASS NUMBER.
E.G. C– 12C OR 13C, H2 – 1H OR 2H, O2 – 16O OR 17O, CL – 35CL OR 37CL
COMMONLY SPECIFIED ATOMIC WEIGHT OR ATOMIC MASS IS RELATIVE
ONE AS IT IS WEIGHTED MEAN OF MASSES OF NATURALLY OCCURRING
ISOTOPES OF ELEMENT.
E.G. CARBON HAS 12.01 BUT IT IS MEAN OF 12C – 98.9 %- 12.000 000 AND 13C
–1.1 % -13.003 354.
BUT IN MASS SPECTROMETRY,
“EACH PEAK CORRESPONDS TO AN ION OF PARTICULAR ISOTOPIC
COMPOSITION AND ITS M/Z VALUE IS CALCULATED FROM THE ISOTOPIC
MASSES (CALCULATED W. R. T. 12C = 12.0000, AS SPECIFIED IN FOLLOWING
TABLE) AND NOT FROM RELATIVE ATOMIC MASSES OF ELEMENTS”
E.G. MASS SPECTRUM OF 2-METHYLPENTANE HAS MOLECULAR ION
PEAK, M AND M+1 PEAK OF INTENSITY 6.6 % WITH MOLECULAR ION. AS
M+1 PEAK IS OBSERVED , IT INDICATES THAT MOLECULAR ION PEAK
BEARS ALL 12C ATOMS WHERE AS M+1 PEAK BEARS 13C ATOMS
IMP- BR – TWO MOLECULAR ION PEAKS –EQUAL INTENSITY-
SEPARATED BY 2 MASS NUMBER

41. Table 14.1, p.548
Recall that the atomic weight is the average mass for all isotopes found in
nature.
35.453 = (100 * 34.9689 + 31.98 * 36.9659) /
131.98
41

43. 43

44. Further comments on presence of chlorine and bromine.
Both Cl and Br have two common isotopes separated by two mass units.
Given the natural abundances we may calculate the ratio of the M and M+2
peaks for various combinations of Cl and Br being present.
The presence of peaks at X,
X+2… for the molecular
ion or fragment hopefully
with close to the expected
ratio is taken as indication
of Cl or Br.
Ratio of peaks calculated as
35Cl2
35Cl37Cl & 37Cl35Cl 37Cl2
1.00*1.0
0
1.00*.324+.324*1.
00
.324*.324
Ratio of peaks calculated as
35Cl79Br 37Cl79Br & 35Cl81Br 37Cl81Br
1.00
*1.00
.324 *1.00+1.00
*.979
.324*.979
.767 1.00 .243
44

45. MOLECULAR PEAKS, M+1
HAVE SEEN THAT FOR CL AND BR, HAVING TWO COMMON
ISOTOPES, TWO RADICAL CATION PEAKS PRODUCED.
WHAT ABOUT OTHER ELEMENTS HAVING MORE THAN ONE
ISOTOPE?
WE KNOW WHAT THE ISOTOPES ARE AND THEIR NATURAL
OCCURRENCE.
FOR THE M+1 PEAK, ONE ATOM MUST BE USING AN ISOTOPE
HEAVIER BY ONE. 45

46. Here is the data. We will use isotopic occurrence data for H, C, O for the M + 1
peak.
46

47. THE M+2 PEAK
48
Recap: The M+1 peak has contributions from one atom being a heavier
isotope by 1.
The M+2 peak can have contributions from
•One atom being a heavier isotope by 2.
•Two atoms being heavier by 1 each.

48. M+2 PEAK, CONTRIBUTIONS FROM ONE ATOM
AND TWO ATOMS.
49
Recap:
The M+1 peak has contributions from one atom being a heavier isotope by 1.
(M+1)/M = ca. 1.1% * no. of C atoms + 0.36% * no. of N atoms
The M+2 peak can have contributions from two sources
•One atom being a heavier isotope by 2. Mainly O (excluding S, Cl and Br)
•Two atoms being heavier by 1 each. Mainly C atoms.
(M+2)/M = ca. (0.20% * no. of O atoms) + (1.1 * no. of C atoms)2/200%
Example 1: C5H5N
[(A + 1)+]/[A+] = 5 x 1.1% + 1 x 0.36%
= 5.9%
[(A + 2)+]/[A+] = 5.52/200 % = 0.15%
Example 2: C7H5O
[(A + 1)+]/[A+] = 7 x 1.1% = 7.7%
[(A + 2)+]/[A+] = 7.72/200 % + 0.20% =
0.50%

49. Technique to obtain molecular formula using intensities of M, M+1, M+2 peaks.
Consider the M+1 peak, nominal mass + 1.
If we know the formula we should be able to calculate the relative intensity of
that peak due to the contributions from each of the atoms present. Here are
the major contributors to M+1.
Here are major contributors to M+2.
Example. Given the
data.Peak Intensity
150 (M) 100
151
(M+1)
10.2
152
(M+2)
0.88Looking at M+2 there is
no Br, Cl or S. There
could be oxygen.
Even mass for M means
there could only be
even number of
Nitrogen
50

50. Technique to obtain molecular formula using intensities of M, M+1, M+2 peaks.
Example. Given the
data.Peak Intensity
150 (M) 100
151
(M+1)
10.2
152
(M+2)
0.88
Equations
M+1: (1.11% x # of C) +
(0.38 x # of N+ small
contributions from O
M+2: (0.20 x # of O) +
(1.1 x # of C)2/200
We can have 0 or 2 nitrogens. Even number.
We can have 0,1,2,3,4 oxygens. 0.88/0.2 < 5
Can have 0,1,2,3,4,5,6,7,8,9 carbons. 10.2/1.11
<10Find molecular formulas having reasonable M+1
M+1 M+2
C7H10N4 9.25 0.38
C8H10N2O 9.61 0.61
C9H10O2 9.96 0.84
C9H14N2 10.7 0.52
Examine reasonable
formulae. Calculate
M+1, M+2 peaks
51

51. Example. Identify this molecule
m/e Abundance
1 <0.1
16 1.0
17 21
18 100
19 0.15
20 0.22
Due to heavier
isotopes
Molecular radical ion
Ejection of an H
H2O
52

52. Example 2
m/e Abundance
12 3.3
13 4.3
14 4.4
15 0.07
16 1.7
28 31
29
30
31
32
100
89
1.3
0.21
Heavier isotopes
parent
H ejection
Oxygen
carbon
CH2O 53

53. EASILY RECOGNIZED ELEMENTS IN MS
2-
BROMOPROPANE
54
 Bromine:
 M+ ~ M+2 (50.5% 79Br/49.5% 81Br)
M+ ~ M+2

54. EASILY RECOGNIZED ELEMENTS IN
MS
• CHLORINE:
• M+2 IS ~ 1/3 AS LARGE AS M+
55
Cl
M+2
M+

55. EASILY RECOGNIZED ELEMENTS IN
MS
• SULFUR:
• M+2 LARGER THAN USUAL (4% OF M+)
56
M+
Unusually
large M+2
S

56. EASILY RECOGNIZED ELEMENTS IN
MS
• IODINE
• I+ AT 127
• LARGE GAP
57
Large gap
I+
M+
ICH2CN

57. 58
FRAGMENTATION IN MS

58. 59

59. 60

60. 61
FRAGMENTATION NOTES

61. 62

62. 63

63. 64

64. 65

65. 66

66. 67

67. 68
MacLafferty Rearrangement
It has been observed in following types of compounds i.e. carbonyl group
and γ proton
containing compounds like,
1. Aldehyde
2. Ketones
3. Acids
4. Esters
5. Amides

68. 69
MacLafferty Rearrangement in Aldehydes

69. 70
MacLafferty Rearrangement in Ketones

70. 71
MacLafferty Rearrangement in Carboxylic Acids

71. 72
MacLafferty Rearrangement in Esters

72. 73
MacLafferty Rearrangement in Amides

73. 74
MacLafferty Rearrangement Limitation

74. 75
MASS SPECTRA OF
ORGANIC
COMPOUNDS

75. 76

76. 77

77. 78

78. 79

79. 80

80. 81

81. 82

82. 83

83. 84

84. 85

85. 86

86. 87

87. 88

88. 89

89. 90

90. 91

91. 92

92. 93

93. 94

94. 95

95. 96

96. 97

97. 98

98. 99

99. 100

100. 101

101. 102

102. 103

103. 104

104. 105

105. 106

106. 107

107. 108

108. 109

109. 110

110. 111

111. 112

112. 113

113. 114

114. 115

115. 116

116. 117

117. 118

118. 119

119. 120
EXAMPLES

120. 121

121. 122

122. 123

123. 124

124. 125

125. 126

126. 127
APPLICATIONS
1. Molecular Mass Determination: Molecular ion peak
2. Isotopic Abundance: M, M+1, M+2, M+4, M+6 peaks and intensities are
useful
3. Isotopic Dilution Method: Addition of selective isotope and its estimation .
4. Quantitative Analysis of Mixture : Standard Addition Method as peak height
contribution is concentration dependant in mixture of compounds.
5. Distinction Between Cis and Trans forms: Molecular ion peak for trans isomer
is more intense than cis one. e.g. Hex-2-ene-1-ol isomers
6. Evaluation of Heat of Sublimation: Peak height is measured at various
temperature as vapor pressure is proportional to change in peak intensity.
7. Determination of Ionization Potential: Changing electron beam energy in eV,
it is possible to measure it.

127. 128
8. Bonding: Fragments obtained in mass spectrum of compound assist to identify
types of bonds in molecules of compound.
9. Determination of Bond Dissociation Energies: Changing electron beam energy
in eV, it is possible to measure it.
10. Reaction Kinetics: As mass spectrum identifies and quantifies most of
unstable intermediate in reaction of substance, it is possible to study its kinetics.
11. Latent Heat of Vaporization of Liquids: As energy required for vaporization
is possible to determine by Mass spectroscopy, It is possible to calculate it.
12. Reaction Mechanism Study: Fragmentation patterns helps to study it.
13. Impurity Detection: Identify undesired mass peaks in Mass spectrum .
14. Identification of unknown compound: Comparison of mass spectrum of
substance under study with that in literature.
15. Characterization of polymers: Identification of halogenated polymers is
possible by mass spectroscopy. Whether halogens are present in polymer
randomly or in blocks ? It is possible to identify by MS.