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Definition of Mass Spectrometry

Mass spectrometry (MS) :

An analytical technique by using mass spectrometry for the determination of the composition of a sample or molecule and elucidation of the chemical structures of molecules, such as peptides and other chemical compounds.

Mass spectrometry has been described as the smallest scale in the world, not because of the mass

spectrometer’s size but because of the size of what it

weighs -- molecules.

What information can be determined?

Molecular weight Molecular formula (HRMS) Structure (from fragmentation fingerprint) Isotopic incorporation / distribution Protein sequence (MS-MS)

Rule of Thirteen

Calculating Mass

The “Rule of Thirteen” can be used to identify possible molecular formulas for an unknown hydrocarbon, C n H m .

Step 1: n = M + /13 (integer only, use remainder in step 2)

Step 2:

m = n + remainder from step 1

Rule of Thirteen

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: C 8 H 10

Nitrogen Rule

This rule states that if a compound has an even no.of N

atoms (or no N atom) , its molecular ion will appear at

an even mass value.

On the other hand , a molecule with an odd no.of N atom will form a molecular ion with an odd mass.

The N rule stems from the fact that N , although it has

an even mass , has an odd numbered valence. Consequently , an extra hydrogen atom is included as a

part of a molecule , giving it an odd mass.

Nitrogen Rule

To picture this effect, consider Ehtylamine,

C2H5NH2.

This substance has one N atom, and its mass is an odd number (45), whereas EhtyleneDiamine H2N-CH2-CH2-NH2, has 2 N atoms, and its mass is an even number (60).

Basic principle of working

Find a way to “charge” an atom or molecule (ionization)

Place charged atom or molecule in a magnetic field or subject it to an electric

field and measure its speed or radius of curvature relative to its mass-to-charge ratio

(mass analyzer) Detect ions using microchannel plate

Mass Spectrometry

An outline of what happens in a mass spectrometer

Atoms can be deflected by magnetic fields - provided the atom is first turned into an ion. Electrically charged particles are affected by a magnetic field although electrically neutral ones aren't.

»The sequence is :

Mass Spectrometry Ionization Slightly +ve To repel +ve ions Usually carry +1 chrge Difficult to remove
Mass Spectrometry
Ionization
Slightly +ve
To
repel +ve ions
Usually carry +1 chrge
Difficult to remove
another electron

Mass Spectrometry

Mass Spectrometry

Mass Spectrometry

Assume all streams having +1 charge Lightest m/z ratio Bulkiest m/z ratio Optimum m/z ratio
Assume all streams having +1 charge
Lightest m/z ratio
Bulkiest m/z ratio
Optimum m/z ratio

Mass Spectrometry

Detection

Creating electron vacancy Causing current to flow which is measured
Creating electron vacancy
Causing current to flow which is measured

Elements to Mass Spectrometry (J.J. Thomson ~ 1910)

Elements to Mass Spectrometry (J.J. Thomson ~ 1910) • • • • • • Sample Gas

Sample

Gas Phase/

Ionize

Elements to Mass Spectrometry (J.J. Thomson ~ 1910) • • • • • • Sample Gas

Electron Impact (EI)

Chemical Ionization (CI) Electrospray (ESI)

Atmospheric Pressure Chemical

Ionization (APCI)

Photo-ionization (APPI)

Matrix Assisted Laser Desorption and Ionization (MALDI)

Elements to Mass Spectrometry (J.J. Thomson ~ 1910) • • • • • • Sample Gas
Elements to Mass Spectrometry (J.J. Thomson ~ 1910) • • • • • • Sample Gas

Separate Based on Mass/Charge

Detector
Detector

Mass Spectrometry

The Mass Spectrometer

Fundamental operating principle

Determine mass by manipulating flight path of an ion in a magnetic field

Electron gun Ionization: X + e -  Magnet Ionization m/z too large sample introduction +
Electron gun
Ionization: X
+
e -
Magnet
Ionization
m/z too large
sample
introduction
+
-
Accelerator
plates
Detector quiet

Detector quiet

X +.

+

2 e -

Mass Spectrometry The Mass Spectrometer Fundamental operating principle Determine mass by manipulating flight path of an

Measure ion

mass-to-charge ratio

(m/z)

m/z just right

Detector fires

Detector

Mass Spectrometry The Mass Spectrometer Fundamental operating principle Determine mass by manipulating flight path of an

Isotopes

Isotopes: atoms with same number of protons and same number of electrons but different numbers of neutrons

Aston mass spectrum of neon (1919) Ne empirical atomic weight = 20.2 amu Ne mass spectrum: predict single peak at m/z = 20.2

Results m/z

relative intensity

  • 20.2 no peak

  • 20.0 90%

  • 22.0 10%

Isotopes Isotopes : atoms with same number of protons and same number of electrons but different

Conclusions Neon is a mixture of isotopes Weighted average: (90% x 20.0 amu) + (10.0% x 22.0 amu) = 20.2 amu

Nobel Prize in Chemistry 1922 to Aston for discovery of stable element isotopes

The Mass Spectrum

Origin of Relative Ion Abundances

 

M contributors

M+1 contributors

 

M+2 contributors

 

Natural

 

Natural

 

Natural

Isotope

Abundance

Isotope

Abundance

Isotope

Abundance

1

H

99.9855%

2

H

0.015%

3

H

ppm

12

C

98.893

13

C

1.107

14

C

ppm

14

N

99.634

15

N

0.366

   

16

O

99.759

17

O

0.037

18

O

0.204

19

F

100.0

       

32

S

95.0

33

S

0.76

34

S

4.22

35

Cl

75.77

   

37

Cl

24.23

79

Br

50.69

   

81

Br

49.31

 

127

I

100.0

       
Ionization Methods • Electron bomb Ionization (EI) • Chemical Ionization (CI) • Field ionization (FI) •

Ionization Methods

Electron bomb Ionization (EI)

Chemical Ionization (CI)

Field ionization (FI)

Matrix Assisted Laser Desorption Ionization (MALDI)

Fast atom bombardment (FAB)

Electro Spray Ionization (ESI)

But when to use which ionization technique?

Ion Source Depends on Sample

Solid Sample

Liquid Sample

Gas Sample

Ion Source Depends on Sample Solid Sample Liquid Sample Gas Sample Make into Solid ? Make

Make into Solid ?

Make into Solution ?

MALDI
MALDI

Chemical Properties of analyte in solution phase ?

Ion Source Depends on Sample Solid Sample Liquid Sample Gas Sample Make into Solid ? Make
Ion Source Depends on Sample Solid Sample Liquid Sample Gas Sample Make into Solid ? Make
APCI
APCI
APPI
APPI
ESI
ESI

Turn into Gas?

Chemical Properties of analyte in gas phase ?

CI EI
CI
EI
Electron Impact e- e- e- M
Electron Impact
e-
e-
e-
M

M (g) + e - M + (g) + 2e -

This reaction creates the molecular ion so is very useful. However, the excess energy from the electron can cause the molecular ion to fall apart:

Neutral

Molecule

IP 2

Neutral Molecule IP IP s 1 s 0 s 1 Ionized Molecule s 0 Excess Energy

IP

s 1

s 0

Neutral Molecule IP IP s 1 s 0 s 1 Ionized Molecule s 0 Excess Energy

s 1

Neutral Molecule IP IP s 1 s 0 s 1 Ionized Molecule s 0 Excess Energy
Neutral Molecule IP IP s 1 s 0 s 1 Ionized Molecule s 0 Excess Energy
Neutral Molecule IP IP s 1 s 0 s 1 Ionized Molecule s 0 Excess Energy

Ionized

Molecule

s 0

Neutral Molecule IP IP s 1 s 0 s 1 Ionized Molecule s 0 Excess Energy
Neutral Molecule IP IP s 1 s 0 s 1 Ionized Molecule s 0 Excess Energy

Excess Energy get

redistributed throughout ion to cause

fragmentation.

Electron Impact

e- e- e- M
e-
e-
e-
M

M (g) + e - M + (g) + 2e -

Electron Impact e- e- e- M M + e  M + 2e A + (g)
A +
A +
Electron Impact e- e- e- M M + e  M + 2e A + (g)

M + (g) A + Fragment 1 (g) + B Fragment 2 (g)

Electron energy is chosen by compromise. Fragment Information is useful. It can help structural determination. However, many ions produce only fragments with no molecular ion remaining. Molecular ion are often very unstable. 70 eV “Classical Spectra” to be used for comparisons

Properties of EI Hard ionization Gas-phase molecules enter source through heated probe or GC column 70

Properties of EI

Hard ionization

Gas-phase molecules enter source through heated

probe or GC column

70 eV electrons bombard molecules forming M+*

ions that fragment in unique reproducible way to

form a collection of fragment ions

EI spectra can be matched to library stds CI (soft

ionization)

Chemical Ionization

EI is not appropriate for some molecules (it causes too much fragmentation)

Instead, ionize a reagent gas (by EI) then react it with a analyte molecules

Typically use methane or ammonia for reagent gas

Properties of CI Advantages Parent Ion Interface to GC Insoluble Samples CI is lower energy process

Properties of CI

Advantages Parent Ion Interface to GC Insoluble Samples CI is lower energy process than EI
Advantages
Parent Ion
Interface to GC
Insoluble Samples
CI is lower energy
process than EI
Disadvantages No Fragment Library Need Volatile Sample Need Thermal Stability Low Mass Compounds (<1000 amu)
Disadvantages
No Fragment Library
Need Volatile Sample
Need Thermal Stability
Low Mass Compounds
(<1000 amu)

CI: Form Reagent Ions First

For Example - Methane CI

  • 1. electron ionization of CH4:

CH 4 + e- CH 4 + + 2e -

Fragmentation forms CH 3 + , CH 2 + , CH +

  • 2. ion-molecule reactions create stable reagent ions:

CH 4 + + CH 4 CH 3 + CH 5

+

CH 3 + + CH 4

H 2 + C 2 H 5

+

CH 5 + and C 2 H 5 + are the dominant methane CI reagent ions

Methane CI Reagent Ions

Ions at m/z 17, 29, and 41 are from methane;

H 3 O + is also formed from water vapor in the vacuum system

Methane CI Reagent Ions – Ions at m/z 17, 29, and 41 are from methane; •

Field ionization (FI)

In field ionization, a high-potential electric field is applied to an emitter with a sharp surface, such

as a razor blade, or more commonly, a filament from which tiny "whiskers" have formed. This

results in a very high electric field which can result in ionization of gaseous molecules of the

analyte. Mass spectra produced by FI have little or no fragmentation. They are dominated by

molecular radical cations M+. and less often, protonated molecules.

Probe

Field ionization (FI) In field ionization, a high-potential electric field is applied to an emitter with
+ +
+
+
+ + Probe + + + + d<1mm
+
+
Probe
+
+
+
+
d<1mm
+ + + + + Ionization
+
+
+
+
+
Ionization
Field ionization (FI) In field ionization, a high-potential electric field is applied to an emitter with

Field ionization (FI)

Application:

FD/FI being used for analysis of polar and nonvolatile analytes such as

polymers and biological molecules.

However, FD/FI remains one of the only ionization techniques that can produce simple mass spectra with molecular information from hydrocarbons and other particular analytes.

The most commonly encountered application of FD/FI at the present time is the analysis of complex mixtures of hydrocarbons such as that found in petroleum fractions.

Difference

There are three practical differences between CI and FI: there is less

fragmentation in FI

There is no high-resolution FI, and FI is less sensitive.

Sensitivity is not an issue unless there is an extremely small amount of sample. FI can be performed by direct probe and GC/MS.

Field ionization (FI)

Field ionization (FI)

Matrix Assisted Laser Desorption

Ionization (MALDI)

MALDI is achieved in two steps. In the first step, the compound to be analyzed

is dissolved in a solvent containing in solution small organic molecules, called

the matrix. The second step occurs under vacuum conditions inside the source of the mass spectrometer.

The use of a chemical matrix in the form of small, laser-absorbing organic molecules in large excess over the analyte is at the core of the MALDI principle. One important feature is the way in which the matrix and analyte interact in the MALDI sample. In a typical UV-MALDI sample preparation small volumes of an analyte is mixed with amount 10 6 of matrix. Upon solvent evaporation, the matrix crystallizes to form a bed of small crystals that range in size from a few to a few hundred micrometers, depending on the matrix and the details of the

preparation.

Properties of MALDI

Good solubility

Vapour pressure must be sufficiently low to maintain vacuum conditions

Viscosity must allow diffusion of the analyte from the bulk to the surface

Lower PRACTICAL detection limits

Easier to interpret spectra (less multiple charges)

Quick and easy

Higher mass detection

Higher Throughput (>1000 samples per hour)

Low levels of some salts, buffers, and detergents can be tolerated as well as less than 2% of glycerol.

Principle of MALDI

Principle of MALDI MALDI mass spectrometry has become a powerful analytical tool for both synthetic polymers

MALDI mass spectrometry has become a powerful analytical tool for both synthetic polymers and biopolymers.

ElectroSpray Ionization (ESI) Electrospray is abbreviated to ESI , sample is sprayed out of a narrowmacromolecules because it overcomes the propensity of these molecules to fragment when ionized. " id="pdf-obj-32-2" src="pdf-obj-32-2.jpg">

ElectroSpray Ionization (ESI)

ElectroSpray Ionization (ESI) Electrospray is abbreviated to ESI , sample is sprayed out of a narrowmacromolecules because it overcomes the propensity of these molecules to fragment when ionized. " id="pdf-obj-32-6" src="pdf-obj-32-6.jpg">

Electrospray is abbreviated to ESI , sample is sprayed out of a narrow nozzle in a high potential field. Generates positive

(M+nH) n + and negative (M - nH) n - ions and almost no fragmentation. Generates multiple charged ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when

ionized.

ElectroSpray Ionization (ESI) Electrospray is abbreviated to ESI , sample is sprayed out of a narrowmacromolecules because it overcomes the propensity of these molecules to fragment when ionized. " id="pdf-obj-32-23" src="pdf-obj-32-23.jpg">
ElectroSpray Ionization (ESI) Electrospray is abbreviated to ESI , sample is sprayed out of a narrowmacromolecules because it overcomes the propensity of these molecules to fragment when ionized. " id="pdf-obj-32-25" src="pdf-obj-32-25.jpg">
2. Principle
2. Principle
Properties of ESI Advantages Electrospray Ionization can be easily interfaced to LC. Absolute signals from Electrospray

Properties of ESI

Properties of ESI Advantages Electrospray Ionization can be easily interfaced to LC. Absolute signals from Electrospray
Advantages Electrospray Ionization can be easily interfaced to LC. Absolute signals from Electrospray are more easily
Advantages
Electrospray Ionization can be
easily interfaced to LC.
Absolute signals from
Electrospray are more easily
reproduced, therefore, better
quantitation.
Mass Accuracy is considered
better.
Multiple charging is more
common then MALDI.
Disadvantages No Fragmentation Need Polar Sample Need Solubility in Polar Solvent (MeOH, ACN, H 2 O,
Disadvantages
No Fragmentation
Need Polar Sample
Need Solubility in Polar
Solvent (MeOH, ACN, H 2 O,
Acetone are best)
Sensitive to Salts
Suppression

Relative ion abundance (%)

The Mass Spectrum

Example: methane

CH 4

+

e - CH 4 +.

+

2 e -

C H
C
H

m/z = (1 x 12) + (4 x 1) = 16

Relative ion abundance (%) The Mass Spectrum Example : methane CH + e  CH +

Base peak: most abundant ion

Relative ion abundance (%) The Mass Spectrum Example : methane CH + e  CH +
Relative ion abundance (%) The Mass Spectrum Example : methane CH + e  CH +

mass-to-charge ratio (m/z)

Tandem Mass Spectrometry Tandem mass spectrometry, also known as MS/MS, involves multiple steps of mass spectrometry

Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.

Tandem Mass Spectrometry Tandem mass spectrometry, also known as MS/MS, involves multiple steps of mass spectrometry

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. Stability of the radical is less important.

Fragmentation Patterns

Alkanes

Fragmentation often splits off simple alkyl groups:

Loss of methyl Loss of ethyl Loss of propyl Loss of butyl

M + - 15 M + - 29 M + - 43 M + - 57

Branched alkanes tend to fragment forming the most stable carbocations.

Fragmentation Patterns

Mass spectrum of 2-methylpentane

Fragmentation Patterns • Mass spectrum of 2-methylpentane

Fragmentation Patterns

Alkenes:

Fragmentation typically forms resonance stabilized allylic carbocations

Fragmentation Patterns • Alkenes: – Fragmentation typically forms resonance stabilized allylic carbocations

Fragmentation Patterns

Aromatics:

Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the

tropylium ion)

H H C Br
H
H
C
Br
H H H C H C or M +
H
H
H
C
H
C
or
M
+

Fragmentation Patterns

Alcohols

Fragment easily resulting in very small or missing parent ion peak Commonly losses H2O or OH 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 +

Fragmentation Patterns

• MS for 1-propanol CH 3 CH 2 CH 2 OH H 2 C OH M
• MS for 1-propanol
CH 3 CH 2 CH 2 OH
H 2 C
OH
M
+ -18
M
+
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced

Fragmentation Patterns

Amines

Odd M + (assuming an odd number of nitrogens are present)

a-cleavage dominates forming an iminium ion

CH 3 CH 2

CH 2

N

CH 2

H

Fragmentation Patterns • Amines – Odd M (assuming an odd number of nitrogens are present) –

CH 2 CH 2 CH 3

Fragmentation Patterns • Amines – Odd M (assuming an odd number of nitrogens are present) –
Fragmentation Patterns • Amines – Odd M (assuming an odd number of nitrogens are present) –

CH 3 CH 2 CH 2 N H

Fragmentation Patterns • Amines – Odd M (assuming an odd number of nitrogens are present) –

CH 2

m/z =72

iminium ion

Fragmentation Patterns

CH 3 CH 2

86 CH 2 N CH 2 H
86
CH 2
N
CH 2
H

CH 2 CH 2 CH 3

72

Fragmentation Patterns CH CH 86 CH 2 N CH 2 H CH CH CH 72

Fragmentation Patterns

Aromatics may also have a peak at m/z = 77 for the benzene ring.

77 NO 2 M + = 123 77
77
NO 2
M + = 123
77

Fragmentation Patterns

Aldehydes (RCHO)

Fragmentation may form acylium ion

Fragmentation Patterns • Aldehydes (RCHO) – Fragmentation may form acylium ion RC O – Common fragments:
 
RC O – Common fragments: RC O • M - 1 for R (i.e. RCHO -
 

RC

O

RC O

Common fragments:

 
– Common fragments:
 

RC

O

RC O
RC O – Common fragments: RC O • M - 1 for R (i.e. RCHO -

M + - 1 for

 

R

R (i.e. RCHO - CHO)

(i.e. RCHO - CHO)

M + - 29 for

Fragmentation Patterns

MS for hydrocinnamaldehyde

105 91 H H O C C C H M + = 134 H H 105
105
91
H
H
O
C
C
C H
M + = 134
H
H
105
133
91

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced

Fragmentation Patterns

Ketones

O RCR'
O
RCR'

Fragmentation leads to formation of acylium ion:

Loss of R forming

Loss of R’ forming

 
R'C O

R'C

O

R'C O
R'C O
 
R'C O

RC

O

R'C O
R'C O

Fragmentation Patterns

O

MS for 2-pentanone

CH 3 CCH 2 CH 2 CH 3

CH 3 C O CH 3 CH 2 CH 2 C O M + SDBSWeb :
CH 3 C
O
CH 3 CH 2 CH 2 C O
M
+
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced

Fragmentation Patterns

Esters (RCO 2 R’)

Common fragmentation patterns include:

Loss of OR’

peak at M + - OR’

Loss of R’

peak at M + - R

Frgamentation Patterns 105 77 O C O CH 3 77 M + = 136 105
Frgamentation Patterns
105
77
O
C
O
CH 3
77
M + = 136
105

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced

2. Rearrangement
2. Rearrangement
2. Rearrangement McLafferty rearrangement Pattern I H H H A E A E A E +

McLafferty rearrangement

Pattern I
Pattern I
H H H A E A E A E + B D B D B D
H
H
H
A
E
A
E
A
E
+
B
D
B
D
B
D
H 2 C
C
C
H
H
H
A
E
A
E
E
+
B
D
B
D
D
C
C
C
55
Retro Diels-Alder rearrangement R - e R Examples: CH 3 R + + R R CH

Retro Diels-Alder rearrangement

R

  • - e R

Retro Diels-Alder rearrangement R - e R Examples: CH 3 R + + R R CH
Examples:
Examples:
CH 3
CH 3
Retro Diels-Alder rearrangement R - e R Examples: CH 3 R + + R R CH
R
R
+ +
+
+

R

Retro Diels-Alder rearrangement R - e R Examples: CH 3 R + + R R CH
R CH 3
R
CH 3

R

Retro Diels-Alder rearrangement R - e R Examples: CH 3 R + + R R CH

+

Loss of small molecules, such as H O, CO, C H HO H C 6 H

Loss of small molecules, such as H 2 O, CO, C 2 H 4

HO H
HO
H

C 6 H 13

Loss of small molecules, such as H O, CO, C H HO H C 6 H

H 2

O

+ C 6 H 13

Loss of small molecules, such as H O, CO, C H HO H C 6 H
OH H
OH
H
Loss of small molecules, such as H O, CO, C H HO H C 6 H

CH 3

H

2 C

Loss of small molecules, such as H O, CO, C H HO H C 6 H

CHCH 3

+

H

  • 2 O

+

CH 2 =CH 2

O O - CO - CO
O
O
- CO
- CO
O O O H O H
O
O
O
H
O
H

+

H 2 O

  • Four-member ring rearrangement

CH 3

CH 2

Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =

O

CH 2

CH 3

- CH 3

Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =

CH 3

CH 2

O
O

CH 2

=

- C 2 H 4

Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =
Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =

HO

CH 2

C H H 2 C O H 2
C
H
H 2
C
O
H 2

CH 2

Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =

Other rearrangement

R

X
X
Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =

R

Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =

+

Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =

X

Four-member ring rearrangement CH CH O CH CH - CH CH CH O CH =

oConcluding remarks

oSoft techniques produces Molecular ion peaks

whereas hard techniques produces Fregmentation. oBy Soft techniques exact molecular ion peak can be obtained for large and small molecules. oBy Fregmentation connection pattern of a molecule can be concluded, ehich helps in structure

elucidation.