MASS SPECTROMETRY

Mass Spectrometry (MS)

• An analytical technique for measuring the
mass-to-charge ratio (m/z) of ions, most
commonly positive ions, in the gas phase

• Today, mass spectrometry is our most

valuable analytical tool for the
determination of precise molecular weights
(= High Resolution Mass Spectrometry)
Mass spectrometry (MS) differs from other common
forms of organic spectral analysis in that the sample does
not absorb radiation such as infrared, ultraviolet, or radio
waves from the electromagnetic spectrum.

In contrast to infrared (IR) or nuclear magnetic resonance

(NMR) spectrometry, both of which identify compounds
with specificity comparable to that of mass spectrometry,
MS is a destructive method of analysis—that is, the
sample cannot be recovered after mass spectral analysis.

On the other hand, MS is highly sensitive and requires

less sample than either IR or NMR in order to provide
more information about the structure of the analyte.
Mass spectrometers are typically not stand alone instruments.

Most often they are connected physically and electronically

to a chromatograph as well as a computer.

The chromatograph separates mixtures and introduces the

sample into the mass spectrometer.

The mass spectrometer ionizes analyte molecules, then

separates and detects the resulting ions.
In order to be analyzed by mass spectrometry, sample
molecules must be ionized.

In the case of electron ionization mass spectrometry

(EIMS), electrically neutral molecules are converted to
molecular ions (M+) by means of a beam of high-energy
electrons.

Ionization is followed almost immediately by

fragmentation of the M+ in which some bonds break, and
in many instances new bonds form, in ways that are
characteristic of the structure of the fragmenting ion.
 MASS SPECTROMETRY
• Molecular Mass
Odd molecular weight (number of odd N) or Even molecular weight
(number of even N)
• Atomic composition
(Cl and Br)
• Molecular formula
High resolution MS, there is molecular formula in each peaks

• Functional group
Based on its fragmentation pattern

• Molecular structure
Based on its fragmentation pattern
 A Mass Spectrometer
A mass spectrometer is designed to do
three things
1. convert neutral atoms or molecules into a
beam of positive (or negative) ions

2. separate the ions on the basis of their mass-

to-charge ratio (m/z)

3. measure the relative abundance of each ion

 Ionization Method

• EI – electron ionization (gas)

• CI – chemical ionization (gas)

• APCI – atmospheric pressure chemical ionization (liquid)

• ESI – electrospray ionization (liquid)

• MALDI – matrix-assisted laser desorption ionization

(solid)

• FAB – fast atom bombardment (solid)

Comparing
different
techniques
 Ionization Techniques
Electron Impact (EI)

Under these conditions, very energetic Ions are produced

=> Fragmentation M
.+ is often weak

e.g. ROH often loose H2O

Intensity of M
.+ Depends on the stability of the Ion

Most Stable
Ketones
Aromatic
Amines
Conjugated Alkenes
Ester
Cyclic compounds
Ether
Sulfides
Acid ~ Amides ~ Aldehydes
Short Linear Alkanes
Mercaptans
Least Stable

.+ is often absent in: Alcohols,

M { Nitriles, Nitrates, Nitro, Nitriles
 Other Ionization Techniques
Under EI (Electron Impact), the molecular ion may be weak or absent

In such case, the best solution is to run CI – Chemical Ionization

Which result in intense M+1 ion with little fragmentation
CI – Chemical Ionization

• Vaporized Sample is introduced with excess gas (usually methane)

• The gaz is ionized producing Primary ions that react with excess gaz
to produce secondary ions

.+ + CH .
4 → CH5 + CH3
CH4 +

CH3+ + CH4 → C2H5+ + H2

• Secondary ions react with sample M:

CH5+ + M → [M+H]+ + CH4

C2H5+ + M → [M+H]+ + C2H4

Less energy than CI => less fragment

 Chemical Ionization techniques
In Electron Impact (EI) and chemical ionization,
vaporization of the Sample is a prerequisite.

Therefore: the study of non-volatile or thermally fragile compound is not possible

with these techniques.

Several methods have been developed to go around these limitation

(methods that enable ions to be obtained from liquid/solid)

1- Field Desorption: ions are desorbed by strong electric field

In this and other techniques,, the aim is to transfer energy to the Sample,
causing transfer of molecules/ions to gas phase
2- SIMS : other source of energy is a beam of ions (Secondary Ion
Mass Spectroscopy): Typically Argon (FAB)

Field Desorption methods give a high concentration of Molecular ion

=> Particularly useful for identification of unknown
Classification according to method of separating charged
particle

Magnetic Field Deflection

– Magnetic Field only: Mass 12-500 in seconds
– Double Focussing:
High resolution to 4 decimals
r = 60,000

Quadrupole Mass Spectrometer

• Quadrupole Mass Filter: Mass Scanning by varying RF
& DC Frequencies
• Quadrupole Ion Storage (Ion Trap)
- Compact
-Inexpensive
-Very Sensitive
-GC/MS
3. Time of Flight
- Need Fast Electronic (10-7 s)
- Used With Sophisticated Ionization Methods
(FAB, Laser Desorption ….)
4. FT-ICR (Fourier Transform Ion Cyclotron
Resonance)
- Very High Precision – most expensive
5. MS/MS (Tandem Mass Spectro.)
-Specific Ions are Separated in First MS
- Pass one at a time in a collision chamber
- Second MS produce “Daughter Ions”
-used for large molecule and resolution of
mixture
Molecular formula M+ (molecular ion) ➔ m/z
M ➔ mass of the most abundant isotope

e.g C7H7NO : 7 x 12C = 84

7 x 1H = 7
1 x 14N = 14
1 x 16O = 16

m/z ➔ 121

Isotope peaks: (% respect to M)

M+1 due to (13C) or 15N (17O) or 2H are negligeable)

%(M+1) = (1.1 * #C) + (.38 * #N) = 8.08 %

%(M+2) = (1.1 * #C)2/200 + (.2 * #O) = 0.5%

For C, H, N,O, F, P composition

 Molecular formula in general
Molecular ion:

Even Mass : can contain C, H, O , halogen, even # N

Odd Mass : can contain C, H, O , halogen, odd # N

Fragmentation ions:

Even Mass : From even mass M+

comes from rearrangement or 2 bond breaking

Odd Mass : From even mass M+

comes from single bond breaking

even Mass : from Odd Mass M+

comes from single bond breaking
 Molecular formula: RULE of 13
Consider CH unit ➔ 13 amu

If we divide the mass by 13 we can establish easily possible formula:

Example: Molecular ion => 152

152 / 13 = # carbons = 11

Mass = 12 * 11 = 132 therefore H = 152 – 132 = 20

Basic formula C11 H20

If Oxygen is present : mass = 16 ➔ remove CH4
If Nitrogen is present : mass = 14 ➔ remove CH2

If one oxygen : C11 H20 – CH4 + O = C10 H16 O

If second oxygen : C10 H16 O – CH4 + O = C9 H12 O2
If third oxygen : C9 H12 O2 – CH4 + O = C8 H8 O3
 Molecular formula: RULE of 13
After establishing the basic formula with only Carbon/hydrogen,
Other element can be introduced by substracting the proper hydrocarbon
value

16O => CH4

14N => CH2
19F => CH7
1H => C
12
28Si => C2H4

31P => C2H7

32S => C2H8

35Cl => C2H11

79Br => C6H7

127I => C10H7

 Nitrogen rule

Molecular ion => 26 Molecular ion => 27 odd!

26 / 13 = # carbons = 2 27 / 13 = # carbons = 2

Mass = 12 * 2 = 24 therefore H = 2 Basic formula C2 H3

Basic formula C2 H2 One Nitrogen C2 H3 - CH2 + N
H C C H H C N
 Nitrogen
OH
rule NH2

Molecular ion => 99 odd!

Molecular ion => 100
100 / 13 = # carbons = 7 Basic formula C7 H16
Mass = 12 * 7 = 84 therefore H = 16 1 Nitrogen: C7 H16 – CH2 + N
Basic formula C7 H16
C6 H14 N
1 Oxygen: C7 H16 - CH4 + O

C6 H12 O
3-methyl-6-i-PropylCyclohex-2-ene-1-one

MW = 152

Isotopic cluster M, M+1, M+2

 Neutral losses and Ion series
M-1 H• M-30 NO • (Nitro compounds),
H2 CO • (anisoles)
M-15 CH3 •

M-16 O • (rare) , NH2 • M-31 CH3O •

M-17 OH •, NH3 (rare)
M-32 CH3OH
M-18 H2O
M-35 Cl •
M-19 F•
M-36 HCl
M-20 HF (very rare)
M-42 CH2=C=O, CH2=CH-CH3
M-26 HCCH , CN
M-43 CH3CO • , C3H7 •
M-27 HCN
M-44 CO2
M-28 H2C=CH2 , CO

M-29 CH3CH2 • , HCO • M-45 CH3CH2O • , CO2H •

 Neutral losses and Ion series
Figuring out which peak is molecular ion can be supported by identifying what fragment
is lost.

There can be sometimes 2 consecutive loss:

In steroid, M-33 is often observed: comes from the loss of Me and H2O

The ions loss are only useful from molecular ion

There is no fragment in organic compounds between M-1 and M-15

Loss of M-14 is never observed!

Other gaps in mass loss are: between 21-25, 33-34, 37-41

Ions in these areas should be viewed suspiciously: either compound is not pure or
postulated molecular ion is wrong
 Neutral losses and Ion series

Among the losses: most common are

Loss of H•, CH3 • , H2O (from some oxygenated compounds),
HCCH (from aromatic compounds),
HCN (from aromatic compounds containing Nitrogen),
CO and CH2=CH2 (both at 28! Difficult to tell which one is lost)
Ethyl radical (29)
Methoxy radical (31)
Cl and HCl (35, 36)
Acetyl (43) accompanied by m/z 43 prominent and propyl (43) radical
Isotopic Abundance
 Isotopic Abundance

• Some distinguishing characteristics of certain elements in a

mass spectrum.

Element Distinguishing Characteristic

N Odd M+
S Large M+2 (about 4 %)
Cl M+2 one-third as large as M+
Br M+2 as large as M+
Mass Spectrum with Chlorine
Mass Spectrum with Bromine
Exact Masses
of isotopes
 Exact Mass can provide Molecular Formula
Consider the following 2 formulas which have m/z 287:
C15H10NO3Cl C14H8N2O3Cl

Mass Defect of C15H10NO3Cl:

(10 x .0078) + (.0031) + (3 x -.0051 ) + (-.0311 ) = .0347

Mass Defect of C14H8N2O3Cl:

(8 x .0078) + (2 x .0031) + (3 x -.0051 ) + (-.0311 ) = .0202

With sufficiently high resolution MS, it is possible to propose a unique empirical formula
for an ion.
 High resolution:
Exact Mass can provide Molecular Formula

With sufficient accuracy, unique molecular formula can be determine

e.g. Distinguish CO, N2, CH2N and C2H4 (all having m/z 28)

12C 12.0000 14N

CO N2 14.0031
16O 15.9949 14N 14.0031
27.9949 28.0062

12C 12.0000
CH2N C2H4 12C x2 24.0000
1Hx2 2.0156 1H x4 4.0312
14N 14.0031 28.0312
28.0187
 M+2 and M+1 Peaks
• The most common elements giving rise to M + 2 peaks are
chlorine and bromine

• Chlorine in nature is 75.77% 35Cl and 24.23% 37Cl

– a ratio of M to M + 2 of approximately 3:1 indicates the
presence of a single chlorine in a compound

• Bromine in nature is 50.7% 79Br and 49.3% 81Br

– a ratio of M to M + 2 of approximately 1:1 indicates the
presence of a single bromine in a compound
 Interpreting MS
• The only elements to give significant M + 2
peaks are Cl and Br. If no large M + 2 peak
is present, these elements are absent

• Is the mass of the molecular ion odd or even?

• Nitrogen Rule: if a compound has

– zero or an even number of nitrogen atoms, its
molecular ion will appear as a even m/z value
– an odd number of nitrogen atoms, its molecular
ion will appear as an odd m/z value
 Isotope peaks : CH3Br
[12CH379Br]+
[12CH381Br]+

M-H
[13CH379Br]+
[12CH279Br]+
[12CH281Br]+

[13CH381Br]+
Calculating M+1 and M+2
Good-quality spectra:

1. Should show appropriate isotope peaks (e.g., should

not be missing expected peaks due to 13C), especially at
high m/z values;

2. Should not exhibit excessive background noise (e.g.,

spurious peaks of instrument origin throughout the mass
range) or the obvious presence of extraneous materials
(column bleed, coeluting GC peaks, contaminants in the
ion source); and
3. Must be consistent with the known or proposed
structure—that is,
a. all m/z values must be correct;
b. the M+ peak, if present, must correspond to the
molecular mass;
c. important peaks at high m/z values that reflect the
loss of neutral fragments must correspond to
functional groups or structural arrangements
present in the molecule; and
d. the base peak must be consistent with the structure.
 Steps in the Identification of Unknown
• Identify Molecular ion M.+
• Determine Molecular Formula (odd / even mass)
• Analyze heteroatom (M+1 and M+2 …)
– S, Si, Cl, Br, ….
• Use rule of 13 to determine # Carbons (M+1 and M+2 …)
• Compare with 13C-NMR (# carbons) with APT experiment (J-
MOD) ( # protons)
• Compare with proton NMR ( # protons)
• Identify base peak (note if even / odd)
– One or two bond fragmentation
• Test your conclusions: in lab make derivatives (TMS … or
Na or K complexes ➔ mass shift)
 Solving problems in MS
• Try to identify the Molecular Ion or decide if it is present (most
critical step in solving a structure)
– Check if [M+1]+ ion is too large to accommodate reasonable
number of carbons. (the [M+1]+ ion might be the very small M+
instead!)
– Determine the first loss from proposed molecular ion. Some
loss are impossible (e.g 12, 14, 23 daltons)
– Does the spectrum appear dirty? (lots of small peaks even at high
mass)
– If GC of the comopund Is available, compare retention time
• Is the molecular weight even or odd?
– An odd mass can be associated with an odd number of Nitrogen
– An even mass means no Nitrogen or an even number of Nitrogen
– This Rule is applicable only to Molecular ion and to odd-electron ions
• Examine ion cluster for isotopic natural abundance (look for special
heteroatom pattern). Try to calculate number of carbons
 Solving problems in MS
• From the overall appearance: is it a fragile compound?
Is it likely to be aromatic or aliphatic?
• Look in the low mass ions. Do you see any clues of the
family of compounds that you might be dealing with?
• Make a list of suggested losses from the molecular ion and
try to make a pattellsrn from them.
• Look for intense odd-electron ions in the spectrum: this is
almost impossible in compounds containing Nitrogen! These
provides clues for rearrangements (retro Diels Alder,
McLafferty…)
• Speculate on the structure using all that information
Index of Hydrogen deficiency

CxHyNzOn Index = x – ½ y + ½ z +1
 Comparing Mass Spectrum of a compound in
different type of mass spectrometers
Magnetic sector Quadrupole

Time of Flight

105
MW = 164