NMR Spectroscopy

Nuclear magnetic resonance (NMR)

spectroscopy: A spectroscopic
technique that gives us information
about the number and types of atoms in
a molecule, for example, about the
number and types of
hydrogen atoms using 1H-NMR
spectroscopy.
carbon atoms using 13C-NMR
spectroscopy.
phosphorus atoms using 31P-NMR
spectroscopy.
1

Nuclear Spin States

Many atomic nuclei have a property called
spin: the nuclei behave as if they were
spinning
Any atomic nucleus that posses either odd
mass, odd atomic number, or both has a
quantized spin angular momentum and a
magnetic moment
An electron has a spin quantum number of 1/2
with allowed values of +1/2 and -1/2.
This spinning charge has an associated
magnetic field.
In effect, an electron behaves as if it is a
tiny bar magnet and has what is called a
magnetic moment.
The same effect holds for certain atomic
nuclei.
Any atomic nucleus that has an odd mass

Nuclear Spin States

For each nucleus, the number l is a physical
constant, and there are 2I + 1 allowed spin
states with integral differences ranging from
+l to -l; if I = 1/2, there are two allowed spin
states.
Spin quantum numbers and allowed nuclear
spin states for atoms common to organic
1
2
12
13
14
15
16
19
31
32
compounds.
H
H
C
C
N
N
O
F
P
S
Element
Nuclear spin
quantum
1/2
number (I )
Number of
spin states

1/2

1/2

0 1/2 1/2

Nuclear Spins in B0
In the absence of applied magnetic field. All spin
states of a given nucleus are of equivalent
energy(degenerate), and in a collection of
atoms all of the spin states should be almost
equally populated, with the same number of
atoms having each of the allowed spins
E.g. within a collection of 1H and 13C atoms,
nuclear spins are completely random in
orientation.
When placed in a strong external magnetic field
of strength B0, however, interaction between
nuclear spins and the applied magnetic field is
quantized. The result is that only certain
orientations of nuclear magnetic moments are 4

Nuclear Spins in B0
for 1H and 13C, only two orientations
are allowed.

Nuclear Spins in B0
The applied magnetic resonance phenomenon
occurs when nuclei with an applied field are
induced to absorb and change their spin
orientation with respect to the applied field
In an applied field strength of 7.05T the
difference in energy between nuclear spin
states for
1H is approximately 0.120 J (0.0286 cal)/mol,
which corresponds to a frequency of 300
MHz (300,000,000 Hz).
13C is approximately 0.030 J (0.00715
cal)/mol, which corresponds to a frequency
of 75MHz (75,000,000 Hz).

Nuclear Spin in B0
The energy difference between allowed
spin states increases linearly with applied
field strength.
Values shown here are for 1H nuclei.

Nuclear Magnetic Resonance

When nuclei with a spin quantum number
of 1/2 are placed in an applied field, a
small majority of nuclear spins are aligned
with the applied field in the lower energy
state.
The nucleus begins to precess and traces
out a cone-shaped surface, in much the
same way a spinning top or gyroscope
traces out a cone-shaped surface as it
precesses in the earths gravitational
field.
8

Nuclear Magnetic
Resonance
If the precessing nucleus is
irradiated with electromagnetic
radiation of the same frequency as
the rate of precession,
the two frequencies couple
energy is absorbed
the nuclear spin is flipped from spin
state +1/2 (with the applied field) to
-1/2 (against the applied field).
9

Energy Absorption
The nuclear magnetic resonance phenomenon occurs when
nuclei aligned with an applied field are induced to absorb
energy and change their spin orientation with respect to the
applied field.
The energy absorption is quantized process, and the energy
absorbed must equal the energy difference between the two
states involved.
Eabsorbed = (E-2 state E+2 state ) = h
In practice, this energy difference is a function of the strength
of the applied magnetic field, Bo . The stronger the applied
magnetic field, the greater the energy difference between the
two states.

10

Energy Absorption
The magnitude of the energy- level separation also
depends on the particular nucleus involved. Each
nucleus (hydrogen, chlorine, and so on) has a
different ratio of magnetic to angular momentum,
since each has different charge and mass. This ratio,
called the magnetogyric ratio, , is a constant for
each nucleus and determines the energy
dependence on the magnetic field:
E = f(Bo ) = h
Since the angular momentum of the nucleus is
quantized in units of h/2, the final equation takes
the form
E = (h/2)Bo = h
= ( /2)Bo
11

Nuclear Magnetic Resonance

(a) Precession and (b) after absorption of
electromagnetic radiation.

12

Nuclear Magnetic Resonance

Resonance: In NMR spectroscopy,
resonance is the absorption of energy by a
precessing nucleus and the resulting flip
of its nuclear spin from a lower energy
state to a higher energy state.
The precessing spins induce an oscillating
magnetic field that is recorded as a signal
by the instrument.
Signal: A recording in an NMR spectrum of a
nuclear magnetic resonance.
13

Nuclear Magnetic
Resonance
If we were dealing
with H nuclei isolated from
1

all other atoms and electrons, any combination

of applied field and radiation that produces a
signal for one 1H would produce a signal for all
1
H. The same is true of 13C nuclei.
Hydrogens in organic molecules, however, are
not isolated from all other atoms. They are
surrounded by electrons, which are caused to
circulate by the presence of the applied field.
The circulation of electrons around a nucleus in
an applied field is called diamagnetic current
and the nuclear shielding resulting from it is
called diamagnetic shielding.
14

Nuclear Magnetic Resonance

The difference in resonance frequencies
among the various hydrogen nuclei within a
molecule due to shielding/deshielding is
generally very small.
The difference in resonance frequencies for
hydrogens in CH3Cl compared to CH3F
under an applied field of 7.05T is only 360
Hz, which is 1.2 parts per million (ppm)
compared
frequency.
1.2
360 with
Hz the irradiating
6

300 x 10 Hz

10

= 1.2 ppm

15

Nuclear Magnetic Resonance

Signals are measured relative to the signal
of the reference compound
tetramethylsilane (TMS).
CH
3

CH3 Si CH3
CH3
Tetramethylsilane (TMS)

For a 1H-NMR spectrum, signals are reported

by their shift from the 12 H signal in TMS.
For a 13C-NMR spectrum, signals are
reported by their shift from the 4 C signal in
TMS.
Chemical shift (): The shift in ppm of an
16

NMR Spectrometer
Essentials of an NMR spectrometer are a
powerful magnet, a radio-frequency
generator, and a radio-frequency
detector.
The sample is dissolved in a solvent,
most commonly CDCl3 or D2O, and
placed in a sample tube which is then
suspended in the magnetic field and set
spinning.
Using a Fourier transform NMR (FT-NMR)
spectrometer, a spectrum can be
recorded in about 2 seconds.
17

NMR Spectrometer
Schematic diagram of a nuclear
magnetic resonance spectrometer.

18

19

NMR Spectrum
1H-NMR spectrum of methyl acetate.

High frequency: The shift of an NMR signal

to the left on the chart paper.
Low frequency: The shift of an NMR signal to
the right on the chart paper.
20

Equivalent Hydrogens
Equivalent hydrogens: Hydrogens that
have the same chemical environment.
A molecule with 1 set of equivalent
hydrogens gives 1 NMR signal.
O
CH3 CCH3

ClCH2 CH2 Cl

C
H3 C

Propanone
(Acetone)

CH3

H3 C

1,2-Dichloroethane

Cyclopentane

C
CH3

2,3-Dimethyl2-butene
21

Equivalent Hydrogens

A molecule with 2 or more sets of

equivalent hydrogens gives a
different NMR signal for each set.
Cl
CH3CHCl
1,1-Dichloroethane
(2 signals)

Cl
O
Cyclopentanone
(2 signals)

CH3

C C
H
H
(Z)-1-Chloropropene
(3 signals)

Cyclohexene
(3 signals)

22

Chemical Shift - 1H-NMR

23

Chemical
Shifts
1
H-NMR

Chemical
Type of
Hydrogen
Shift ()
(CH3 ) 4 Si
0 (by definition)
RCH3
0.8-1.0
RCH2 R
1.2-1.4
R3 CH
1.4-1.7
R2 C=CRCHR2 1.6-2.6
RC CH
2.0-3.0
ArCH3
2.2-2.5
ArCH2 R
2.3-2.8
ROH
0.5-6.0
RCH2 OH
3.4-4.0
RCH2 OR
3.3-4.0
R2 NH
0.5-5.0
O
RCCH3
2.1-2.3
O
RCCH2 R
2.2-2.6

Type of
Hydrogen
O
RCOCH3
O
RCOCH2 R
RCH2 I
RCH2 Br
RCH2 Cl
RCH2 F
ArOH
R2 C=CH2
R2 C=CHR
ArH
O
RCH
O
RCOH

Chemical
Shift ()
3.7-3.9
4.1-4.7
3.1-3.3
3.4-3.6
3.6-3.8
4.4-4.5
4.5-4.7
4.6-5.0
5.0-5.7
6.5-8.5
9.5-10.1
10-13

24

Chemical Shift
Chemical shift depends on the (1)
electronegativity of nearby atoms, (2)
hybridization of adjacent atoms, and (3)
diamagnetic effects from adjacent pi bonds.
Electronegativity
CH3-X

Electronegativity of X

Chemical
Shift ()

CH3F
CH3OH
CH3Cl
CH3Br
CH3I

4.0
3.5
3.1
2.8
2.5

4.26
3.47
3.05
2.68
2.16

(CH3) 4C
(CH3) 4Si

2.1
1.8

0.86
0.00

25

Chemical Shift
Hybridization of adjacent atoms.
Type of Hydrogen
(R = alkyl)

Name of
Hydrogen

Chemical
Shift ()

RCH3 , R2 CH2 , R3 CH

Alkyl

0.8 - 1.7

R2 C=C(R)CHR2

Allylic

1.6 - 2.6

RC CH

Acetylenic

2.0 - 3.0

R2 C=CHR, R2 C=CH2

Vinylic

4.6 - 5.7

RCHO

Aldehydic

9.5-10.1
26

Chemical Shift
Diamagnetic effects of pi bonds
A carbon-carbon triple bond shields an
acetylenic hydrogen and shifts its signal to
lower frequency (to the right) to a smaller
value.
A carbon-carbon double bond deshields
vinylic hydrogens and shifts their signal to
higher frequency (to the left) to a larger
Chemical
value.Type of H
Name
Shift ()
RCH3
RC CH
R2 C=CH2

Alkyl
Acetylenic
Vinylic

0.8- 1.0
2.0 - 3.0
4.6 - 5.7

27

28

29

30

Signal Splitting; the (n + 1)

Peak: The units intoRule
which an NMR signal is

split; doublet, triplet, quartet, multiplet, etc.

Signal splitting: Splitting of an NMR signal
into a set of peaks by the influence of
neighboring nonequivalent hydrogens.
(n + 1) rule: If a hydrogen has n hydrogens
nonequivalent to it but equivalent among
themselves on the same or adjacent atom(s),
its 1H-NMR signal is split into (n + 1) peaks.

31

Signal Splitting (n + 1)
1H-NMR spectrum of 1,1dichloroethane.

For these hydrogens, n = 1;

their signal is split into
(1 + 1) = 2 peaks; a doublet

For this hydrogen, n = 3;

CH3 -CH-Cl its signal is split into
(3 + 1) = 4 peaks; a quartet
Cl

32

Origins of Signal Splitting

Signal coupling: An interaction in which the
nuclear spins of adjacent atoms influence
each other and lead to the splitting of NMR
signals.
Coupling constant (J): The separation on an
NMR spectrum (in hertz) between adjacent
peaks in a multiplet.
A quantitative measure of the spin-spin
coupling with adjacent nuclei.
33

Problem: Predict the number of 1H-NMR

signals and the splitting pattern of each.
O
(a) CH3CCH2 CH3
O
(b) CH3CH2CCH2 CH3
O
(c) CH3CCH(CH3)2

34

Origins of Signal Splitting

Signal coupling: An interaction in which
the nuclear spins of adjacent atoms
influence each other and lead to the
splitting of NMR signals.
Coupling constant (J): The separation on
an NMR spectrum (in hertz) between
adjacent peaks in a multiplet.
A quantitative measure of the spinspin coupling with adjacent nuclei.
35

 Illustration of spin-spin coupling that gives

rise to signal splitting in 1H-NMR spectra.

36

Origins of Signal Splitting

37

Coupling Constants
Coupling constant (J): The distance between
peaks in a split signal, expressed in hertz.
The value is a quantitative measure of the
magnetic interaction of nuclei with coupled
spins.
Ha

Ha Hb
C C
6-8 Hz
Ha

Hb
11-18 Hz

Ha

H
Hb a

Hb
Hb
8-14 Hz
Ha

0-5 Hz
Ha

Ha

Hb
0-5 Hz

Hb
8-11 Hz

Hb
C

5-10 Hz

0-5 Hz

38

Origins of Signal Splitting

The origins of signal splitting patterns. Each
arrow represents an Hb nuclear spin
orientation.

39

40

Physical Basis for (n + 1)

Rule

Coupling that arises when Hb is

split by two different
nonequivalent H atoms, Ha and Hc.

41

Coupling Constants
An important factor in vicinal coupling is
the angle between the C-H sigma bonds
and whether or not it is fixed.
Coupling is a maximum when is 0 and
180; it is a minimum when is 90.

42

More Complex Splitting

Patterns
Since the angle between C-H bond determines the

extent of coupling, bond rotation is a key

parameter.
In molecules with free rotation about C-C sigma
bonds, H atoms bonded to the same carbon in CH3
and CH2 groups are equivalent.
If there is restricted rotation, as in alkenes and
cyclic structures, H atoms bonded to the same
carbon may not be equivalent.
Nonequivalent H on the same carbon will couple
and cause signal splitting.
This type of coupling is called geminal coupling.

43

More Complex Splitting

Patterns

In ethyl propenoate, an
unsymmetrical terminal alkene, the
three vinylic hydrogens are
nonequivalent.

44

45

46

47

 Complex coupling in flexible molecules.

Coupling in molecules with unrestricted
bond rotation often gives only m + n + I
peaks.
That is, the number of peaks for a signal is
the number of adjacent hydrogens + 1, no
matter how many different sets of
equivalent H atoms that represents.
The explanation is that bond rotation
averages the coupling constants
throughout molecules with freely rotation
bonds and tends to make them similar; for
example in the 6- to 8-Hz range for H
atoms on freely rotating sp3 hybridized C
atoms.
48

49

13

C-NMR Spectroscopy

The study of carbon nuclei through nuclear magnetic

resonance (NMR) Spectroscopy is an important
technique for determining the structures of organic
molecules. Using it together with proton NMR and IR
spectroscopy, it is possible to determine the complete
structure of an unknown compound.
Two major problems were involved.
First, the 13C signal was very weak because the
natural abundance of 13C is low, only 1.1% of the
total carbon present in a sample. Also, the ratio g
and the sensitivity are low compared to that for 1H.
Second, the chemical shift range was up to 200 ppm

using TMS as a standard. This increased range precluded a

simple add on to the 1H NMR instruments already
commercially available.

In a proton-decoupled mode, a sample is irradiated with

two different radiofrequencies,
one to excite all 13C nuclei.
a second broad spectrum of frequencies to cause all
protons in the molecule to undergo rapid transitions
between their nuclear spin states.

50

 There are several advantages to obtaining 13C NMR spectra

for structural identification of organic molecules.
First, the wide range over which chemical shift occurs,
200 ppm for
carbon compared with only 10 ppm for protons, greatly
diminishes
overlap between carbons in different chemical
environments.
The spectra are less crowded and a peak is usually seen
for each
unique carbon nucleus.
Second, adjacent 12C atoms do not induce spinspin
splitting, and
the probability of two 13C atoms being adjacent to each
other is
very low. Therefore spinspin coupling between 13C
nuclei is not
seen
Coupling between 13C and 1H nuclei does occur, but there
51
are techniques

52

C-NMR Spectroscopy

13

On the time scale of a 13C-NMR spectrum,

each proton is in an average or effectively
constant nuclear spin state, with the result
that 1H-13C spin-spin interactions are not
observed; they are decoupled.
Each nonequivalent 13C gives a different
signal
A 13C signal is split by the 1H bonded to it
according to the (n + 1) rule.
Coupling constants of 100-250 Hz are
common, which means that there is often
significant overlap between signals, and
splitting patterns can be very difficult to
53
determine.

13

C-NMR Spectroscopy

54

55

Chemical Shift Type of

Carbon

Chemical
Shift ()

RCH3
RCH2R

10-40
15-55
20-60

R3CH
RCH2I
RCH2Br

0-40
25-65

RCH2Cl
R3COH

35-80

R3COR

40-80
65-85

RC CR
R2C=CR2

40-80

100-150

C-NMR

13

Type of
Carbon
C R

Chemical
Shift ()
110-160

O
RCOR

160 - 180

O
RCNR2

165 - 180

O
RCCOH

165 - 185

O
O
RCH, RCR

180 - 215
56

57

Interpreting NMR Spectra

Alkanes
1H-NMR signals appear in the range of
0.8-1.7.
13C-NMR signals appear in the considerably
wider range of 10-60.
Alkenes
1H-NMR signals appear in the range 4.65.7.
1H-NMR coupling constants are generally
larger for trans-vinylic hydrogens (J= 11-18
Hz) compared with cis-vinylic hydrogens (J=
5-10 Hz).
13C-NMR signals for sp2 hybridized carbons
appear in the range 100-160, which is to58

Interpreting NMR Spectra

59

Interpreting NMR Spectra

Alcohols
1H-NMR O-H chemical shift often appears in
the range 3.0-4.0, but may be as low as
0.5.
1H-NMR chemical shifts of hydrogens on
the carbon bearing the -OH group are
deshielded by the electron-withdrawing
inductive effect of the oxygen and appear
in the range 3.0-4.0.
Ethers
A distinctive feature in the 1H-NMR spectra
of ethers is the chemical shift, 3.3-4.0, of
hydrogens on the carbons bonded to the 60

Interpreting NMR Spectra

61

Interpreting NMR Spectra

Aldehydes and ketones
1H-NMR: aldehyde hydrogens appear
at 9.5-10.1.
1H-NMR: -hydrogens of aldehydes
and ketones appear at 2.2-2.6.
13C-NMR: carbonyl carbons appear at
180-215.
Amines
1H-NMR: amine hydrogens appear at
0.5-5.0 depending on conditions.
62

Interpreting NMR Spectra

63

Interpreting NMR Spectra

64

NMR spectra of cyclohexanol, C6H11OH

65

Introduction
Two-dimensional
2D
NMR is a set ofto
NMR
methods which give data
nuclear
plotted
in a magnetic resonance
spectroscopy
(2Dfrequency
NMR) axes rather than
space
defined by two
one.
Examples of types of 2D NMR
correlation spectroscopy (COSY),
exchange spectroscopy (EXSY), and
Nuclear Overhauser effect spectroscopy (NOESY).
Two-dimensional NMR spectra provide more
information about
a molecule than one-dimensional NMR spectra
and are
especially useful in determining the structure of a
molecule,
particularly for molecules that are too complicated
to work with
using one-dimensional NMR.
66

Introduction to 2D NMR
Each
experiment
consists ofexperiments
a sequence have
of radio
Almost
all two-dimensional
four
frequency
stages:
pulses with
delay
in between
them.
(RF)
preparation
period
- aperiods
magnetization
coherence
It isisthe
created
timing,
frequencies,
intensities of these
through
a set of RFand
pulses;
pulses
that period, a determined length of time
evolution
distinguish different NMR experiments from one
during
another
which no pulses are delivered and the nuclear
spins are
allowed to freely precess (rotate);
mixing period, where the coherence is
manipulated by
another series of pulses into a state which will
give an
observable signal; and
67
detection period, in which the free induction

Introduction to 2D NMR

The two dimensions of a 2D NMR experiment are

two frequency
axes representing a chemical shift.
Each frequency axis is associated with one of the
two time
variables, which are the length of the evolution
period (the
evolution time) and the time elapsed during the
detection
period (the detection time)
They are each converted from a time series to a
68
frequency series

Introduction to 2D NMR
Types of 2D NMR
1. Homonuclear through-bond
correlation
methods
In
these methods,
magnetization transfer occurs
between nuclei of the same type, through j-coupling of
nuclei connected by up to a few bonds.

Correlation spectroscopy (COSY)

-Used to identify spins which are coupled to each

other
- The two-dimensional spectrum that results
from the COSY experiment shows the
frequencies for a single isotope, most commonly
hydrogen (1H) along both axes.

69

orrelation spectroscopy (COSY)

1-D spectra are plots of intensity vs a frequency (chemical
shift). In 2-D
spectra the intensity is plotted as a function of two
frequencies, usually
represented as F1 and F2.
Each peak is specified by the two frequency co-ordinates (F1,
F2).
2-D NMR spectra are always arranged so that the F2 coordinates of the peaks
correspond to those found in the normal 1-D spectrum. (Often
1-D spectrum
is presented on the horizontal F2 axis).
F1 co-ordinates of the peaks also correspond to those of the
normal 1-D
spectrum (1-D spectrum plotted on the F1 axis) in H-H COSY

70

rrelation spectroscopy (COSY)

In a homonuclear COSY spectrum, the presence of a cross-peak multiplet

F1 = A, F2 =
X indicates that the two protons A and X at chemical shifts A and X
are scalar coupled.
If there had been no coupling, their magnetizations would not have given
rise to offdiagonal peaks.
COSY spectrum shows which pairs in a molecule are coupled
(through bond coupling,
71
hence connectivity).

Mass Spectroscopy
Mass Spectrometry is an analytical spectroscopic tool
primarily concerned with the separation of molecular
(and atomic) species according to their mass.
What information can be determined?
Molecular weight
Molecular formula (HRMS)
Structure (from fragmentation fingerprint)
Isotopic incorporation / distribution
Protein sequence (MS-MS)

72

Applications of Mass Spectrometry

Pharmaceutical analysis
Bioavailability studies
Drug metabolism studies,
pharmacokinetics
Characterization of potential drugs
Drug degradation product analysis
Screening of drug candidates
Identifying drug targets
Biomolecule characterization
Proteins and peptides
Oligonucleotides
Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Forensic analysis/clinical

73

Mass Spectroscopy
Mass spectrometry is the study of systems causing
the
formation of gaseous ions, with or without
fragmentation,
which are then characterized by their mass to
charge ratios
(m/z) and relative abundances.
In MS, compounds are ionized, ionized molecule
decomposes
into smaller ions/radicals/radical ions/ neutrals.
One way to ionize molecules is to extract electrons
from a
molecule.
The positively charged fragments produced are
74
separated,

Mass spectrometry as process

INPUT:
Sample
Separation to
Sample Preparation,
components Chromatography
(optional)
Creation of
ions
Ionisation
in gas phase
Separation
Mass spectrometer (analyser)
ions
Detection of
Detector
ions
OUTPU
T:
Spectru
m

75

Diagram of mass
spectrometer
instrument
Sample

Inlet
System

Ion
Source

Data
System

Mass
Analyzer

Ion
Detector

Vacuum
Pumps

High Vacuum Area

Data

76

Principle of Mass Spectrometry

Molecules are ionised
Ionisation produces several ions and
neutral fragments
Ions are separated and measured
according to their mass-to-charge ratios
(m/z)
Instrument used for the separation and
measurement is called mass
spectrometer (MS)

Ion Sources make ions from sample

Ionisation of a molecule produces typical
molecules

pattern of ions which can be used to

(Ions are easier to detect than neutral molecules.)

77

Ionisation methods and mass

analysis techniques

Mass spectrometry can combine different

ionisation methods and mass analysis techniques
Different ionisation methods are needed
as not all molecules ionise the same way
so that different aspects of the molecules are
seen
Different mass analysis techniques are needed
to get more information from ions
enable creation/detection of different ions
Electron ionisation (EI) is the most common
ionisation technique for systems hyphenated
with gas chromatographs (GC)
78

Electron Ionization
Electron Ionization (EI) is the most common
ionization technique
used for mass spectrometry.
EI works well for many gas phase molecules, but it
does have
some limitations.
Although the mass spectra are very reproducible
and are widely
used for spectral libraries, EI causes extensive
fragmentation so
that the molecular ion is not observed for many
compounds.
Electron
Ionization Source

79

Electron Ionization
The electrons used for ionization are produced by
passing a
current through a wire filament (see above Figure).
The amount of current controls the number of
electrons emitted
by the filament.
An electric field accelerates these electrons across
the source
region to produce a beam of high energy electrons.
When an analyte molecule passes through this
electron
a
Knockbeam,
an electron out of the molecule generates
valence shell electron can be removed
from the
the Molecular ion (M+.)
M+. fragments
molecule to
All charged species are accelerated through a
produce an ion.
magnetic field
based
on further
their mass-to-charge
Depending
on the
energy
fragmentation
ratio
(m/z)
1
eV
(per
molecule
may
take
place or atom) = 96.415 220 6
80
kJ/mole

Chemical Ionization
In Chemical Ionization the source is enclosed in a small cell with
openings for
the electron beam, the reagent gas and the sample.
The reagent gas is added to this cell at approximately 10 Pa
(0.1 torr) pressure.
This is higher than the 10-3 Pa (10-5 torr) pressure typical for a
mass
spectrometer source.
At 10-3 Pa the mean free path between collisions is
approximately 2 meters and
ion-molecule reactions are unlikely. In the CI source, however,
the mean free
path between collisions is only 10-4 meters and analyte
molecules undergo
many collisions with the reagent gas.
The reagent gas in the CI source is ionized with an electron
beam to produce a
+
CH5in
ion. cloud react and
cloud of ions. The reagent gas ions
this
81
produce adduct ions

Chemical Ionization
When analyte molecules (M) are introduced to a source
region with
this cloud of ions, the reagent gas ions donate a proton to
the analyte
molecule and produce MH+ ions.
The energetics of the proton transfer is controlled by using
different
reagent gases.
The most common reagent gases are methane, isobutane
and ammonia.
Methane is the strongest proton donor commonly used with
a proton
affinity (PA) of 5.7 eV. For softer ionization, isobutane (PA
8.5 eV) and
ammonia (PA 9.0 eV) are frequently used.
Fragmentation is minimized in CI by reducing the amount of
excess
energy produced by the reaction.
82
Because the adduct ions have little excess energy and are

A) EI of reagent gas to
form ions:
B) Reaction
CH4 + e of reagent
CH4++gas ions to form
adducts:
+2e
OR

C) Reaction of Reagent Gas Ions with

analyte molecules:

83

Fast Atom Bombardment and Secondary Ion Mass

Spectrometry
Fast Atom Bombardment (FAB) and Secondary Ion Mass
Spectrometry (SIMS)
both use high energy atoms to sputter and ionize the sample
in a single step.
In these techniques, a beam of rare gas neutrals (FAB) or ions
(SIMS) is focused
on the liquid or solid sample.
The impact of this high energy beam causes the analyte
molecules to sputter
into the gas phase and ionize in a single step (Figure 6). The
exact mechanism
of this process is not well understood, but these techniques
work well for
compounds with molecular weights up to a few thousand
dalton.
84

Since no heating is required, sputtering techniques (especially

Fast Atom Bombardment Source.

85

Atmospheric Pressure Ionization and

Electrospray Ionization

Atmospheric Pressure Ionization (API) sources ionize the sample

at
atmospheric pressure and then transfer the ions into the mass
spectrometer.
These techniques are used to ionize thermally labile samples
such as
peptides, proteins and polymers directly from the condensed
phase. The
sample is dissolved in an appropriate solvent and this solution is
introduced
into the mass spectrometer.
With conventional inlets the solvent increases the pressure in
the source
region of the mass spectrometer.
In addition, Joule-Thompson cooling of the liquid as it enters the
vacuum
causes the solvent droplets to freeze. The frozen clusters trap
analyte molecules
86
and reduce the sensitivity of the experiment.

Electrospray ionization:
Pressure = 1 atm
Inner tube diam. = 100 um

Partial
vacuu
m

Sample Inlet Nozzle

(Lower Voltage)

MH+

N2
Sample in solution
N2 gas

+
+ ++
++

++++
++
+
+ ++
++

+
++
+
++

+
++

+
++
+
++

+
++

+ +
+
+
+

+
+

MH2+

MH3+
High voltage applied
to metal sheath (~4 kV)

Charged droplets

87

Electrospray Ionization Source

88

Atmospheric Pressure Ionization and

Electrospray
Ionization
ElectroSpray Ionization (ESI) is the most common API
application.
Is frequently used for LC/MS of thermally labile and high
molecular weight
compounds.
The electrospray is created by applying a large potential
between the metal
inlet needle and the first skimmer in an API source (Figure
above).
The mechanism for the ionization process is not well
understood and there
are several different theories that explain this ionization
process.
One theory is that as the liquid leaves the nozzle, the electric
field induces a
net charge on the small droplets. As the solvent evaporates,
the droplet
shrinks and the charge density at the surface of the droplet
89
increases. The

Matrix Assisted Laser Desorption/Ionization

Matrix Assisted Laser Desorption/Ionization (MALDI) is used to
analyze
extremely large molecules. This technique directly ionizes and
vaporizes the
analyte from the condensed phase.
MALDI is often used for the analysis of synthetic and natural
polymers,
In
MALDI, and
bothpeptides.
desorption
and ionization
are induced
by a
proteins,
Analysis
of compounds
with molecular
single laser
weights
up topulse
(Figure
200,000below).
dalton is possible and this high mass limit is
The sample
is prepared by mixing the analyte and a matrix
continually
increasing
compound chosen
to absorb the laser wavelength. This is placed on a probe tip
and dried.
A vacuum lock is used to insert the probe into the source
region of the mass
spectrometer. A laser beam is then focused on this dried
mixture and the
energy from a laser pulse is absorbed by the matrix. This
90
energy ejects analyte

1. Sample is mixed with matrix (X)

and dried on plate.

MALDI Ionization

2. Laser flash ionizes matrix

molecules.
3. Sample molecules (M) are
ionized by proton transfer:
XH+ + M MH+ + X.
91

Summary
Ionisation methods
Electron Impact ionisation (EI)
Strong ionisation method
Useful for identification
Structural information due to excessive
fragmentation
Commercial libraries
Chemical Ionisation (CI)
Soft ionisation method
Useful for molecular weight
determination
Reactant gas required
Less structural information

92

Ionisation methods
Atmospheric Pressure Chemical Ionisation
(APCI)
Soft ionisation
For aqueous samples and smaller
molecules
No commercial libraries
Electrospray Ionisation (ESI)
Soft ionisation
For aqueous samples and large
molecules
Multiply charged ions large
molecules can be measured (MW
100,000)
No commercial libraries

93

Ionisation methods
Atmospheric Pressure Photoionisation
(APPI)
Soft ionisation
For non-polar molecules
No commercial libraries available
New technique (introduced in 2000)
Matrix-Assisted Laser Desorption
Ionisation (MALDI)
Sample mixed with suitable matrix
Sample is hit with energetic laser beam
ions are formed and desorbed from
the sample
Soft ionisation
Large molecules can be ionised

94

MASS ANALYZERS:
After ions are formed in the source region they are accelerated
into the mass
analyzer by an electric field. The mass analyzer separates
these ions according
to their m/z value.
The selection of a mass analyzer depends upon the resolution,
mass range,
scan rate and
detectiondescribed
limits required
for an
application.
Analyzers
are typically
as either
continuous
or pulsed.
Each
analyzer analyzers
has

Continuous
include quadrupole, filters and magnetic
very different operating characteristics and the selection of an
sectors.
instrument
These analyzers are similar to a filter or monochromator used for
involves
optical important tradeoffs.
spectroscopy. They transmit a single selected m/z to the
detector and the
mass spectrum is obtained by scanning the analyzer so that
different mass
to charge ratio ions are detected.
While a certain m/z is selected, any ions at other m/z ratios are
lost,
95
reducing the S/N for continuous analyzers. Single Ion

Quadrupole Mass Analyzer

Uses a combination of RF
and DC voltages to
operate as a mass filter.
Has four parallel
metal rods.
Lets one mass pass
through at a time.
Can scan through all
masses or sit at one
fixed mass.

96

Quadrupoles have variable ion transmission modes

m1

m2

m4

m3

m4

m3 m2

m1

mass scanning mode

m1

m2

m4

m3

m2

m2

m2

m2

single mass transmission mode

97

Time-of-flight (TOF) Mass Analyzer

Drift region (flight tube)

detector

Source

Ions are formed in pulses.

The drift region is field free.
Measures the time for ions to reach the
detector.
Small ions reach the detector before large
ones.

98

Magnetic Sectors
Magnetic Sectors are the traditional means of
separating ions of different
masses using the principle that when a beam of ions
passes through a
magnetic field they are deflected through an arc whose
radius is related to
the ratio of mass to charge.
By varying the field in a known manner, i.e. sweeping from
high to low field
with time, ions of different mass arrive at the detector
sequentially and
produce the mass spectrum.

99

Magnetic Sector
Magnetic Sector instruments have higher
resolution and
greater mass range than quadrupole instruments,
but they
require larger vacuum pumps and often scan more
slowly.
The typical mass range is to m/z 5000, but this may
be extended
to m/z 30,000.
Magnetic sector instruments are often used in
series with an
electric sector, described above, for high resolution
and tandem
mass spectrometry experiments.
Magnetic
Sector Mass
Spectrometer.
The low(Figure below)
Magnetic
sector
instruments
m/z ion (B+) is separated from the high m/z
100
separate
ions
in
a
ion (A+).

Magnetic Sector
Ions are accelerated from the source region into
the magnetic
sector by a 1 to 10 kV electric field. This
acceleration is
significantly greater than the 100 V acceleration
typical for a
quadrupole instrument.
Since the ions are charged, as they move through
the magnetic
sector, the magnetic field bends the ion beam in
an arc. This is
the same principal that causes electric motors to
turn.
The radius of this arc (r) depends upon the
101
momentum of the

Magnetic Sector
Ions with greater momentum will follow an arc
with a larger
radius. This separates ions according to their
momentum, so
magnetic sectors are often called momentum
analyzers.
The momentum of the ion is the product of the
mass (m)
and the velocity (v). The charge of the ion is
the product of
The
velocity
an ion is
by the
the
chargeofnumber
of determined
the ion (z) and
the
acceleration
charge of an
voltage
in (e).
the source
regionthese
(V) and
the mass
electron
Substituting
variables
intoto
charge
ratio
eq. 1 yields
(m/z) of the ion. Equation 2 rearranges to give
102
the m/z ion

Ion Trap Mass Analyzer

Top View

Cut away side

view
103

R in g E le c t r o d e

E n tra n c e
E ndcap
E le c t r o d e

E x it E n d c a p
E le c tr o d e
R in g E le c tr o d e

Ions fill the space between a ring electrode and a

pair of end-cap electrodes.
Mass analysis and fragmentation occur in the same
space.
Ions are formed within the ion trap or injected into
an ion trap from an external source; the ions are
dynamically trapped by the applied RF potentials
The trapped ions can be manipulated with RF
events to perform ejection, ion excitation and mass
selective ejection; MS/MS, MS/MS/MS... experiments
104

 Benefits
High sensitivity
Multi-stage mass spectrometry
Compact and cheap mass analyzer
Limitations
Poor quantitation
Poor dynamic range
Subject to space charge effects and ion molecule
reactions ([M+H]+ formation in EI); at least older
GC instruments

105

Ion Trap Types

- Linear Quadrupole
Ion Trap (LIT)
- -Also called 3D ion
trap

Quadrupole Ion Trap

(QIT)

Also called 3D ion

trap
106

- Linear Ion Trap (LIT)

107

 In full scan mode: Ions fill and are trapped in space

then masses are scanned out of the trap sequentially.
Ions are not lost, so full scan sensitivity is better, but
filling/closing cycles make them poorer at quantitation.

Mass resolution is controlled by the speed at which

masses are scanned out of the trap.
slower scanning = better mass resolution.

In MS/MS mode: Ions trapped. Fragmentation occurs

when the selected ion is excited by a so called
tickle voltage and collides with bath gas (He). This
process can occur recursively thus MS/MS/MS/MS.

Others

108

Summary
..\..\Files from old computer\
English+books
\Spectrometric Identification of Orga
nic
Compunds 7th ed - R[.pdf

109

Detectors
These are usually electron multipliers which
produce up to 106 electrons for every ion that hits
the detectors. This produces a current which can be
amplified and measured.

Electron multipliers

Most common means of detecting ions,

especially when positive and negative ions need
to be detected on the same instrument
Ions strike the initial amplification dynode
surface producing an emission of secondary
electrons which generates more secondary
electrons in a repeating process ultimately
resulting in a cascade of electrons
Typical amplification is of the order of one
million to one
110

Electron multipliers...

111
sources: http://www.chm.bris.ac.uk/ms/theory/detection.html &

Basic Mass Spectrum

Spectrum of air
Contains: H2O (MW 18), N2 (MW 28), O2
(MW
32), CO2 (MW 44)

112

Basic Mass Spectrum

Y-axis: Relative abundance: highest ion is
set to

100%

X-axis:mass/charge (m/z);
Note: not just mass of the ion

VERIFIN 113

Molecular ion

114

Molecular Ion
The molecular ion provides the molecular mass of the analyte
and is the first clue used to interpret a mass spectrum.
The mass of the molecular ion is based upon the mass of the
most abundant
isotope for each element in the molecule. This is not the atomic
weight from
the periodic table. Since many mass spectrometers have unit
mass
resolution, the atomic mass is rounded to the nearest whole
number, this is
called the nominal mass. For example the molecular ion for
CHBr3 is observed
at m/z 250, not at the formula weight of 253 (CHBr3 ; (12 + 1 +
379) = 250).
In many mass spectra, the molecular ion is easily identified as
the ion with the
highest mass to charge ratio. However, this assignment should
be made with
115
caution because the highest mass to charge ion be an impurity

Fragmentation
Although the molecular ion is useful for identification, it does
not provide any
structural information about an unknown.
The structural information is obtained from the fragmentation
patterns of the
mass spectrum. Identifying an unknown without analyzing
the fragmentation
patterns is like putting together a jigsaw puzzle without the
picture.
Fragmentation patterns are often complex, but they fit
together like pieces of
the puzzle to identify the structure of the molecule.

Fragmentation mechanisms
116

Fragmentation
After a molecule is ionized, the molecular ion retains the
excess ionization
energy. If this excess energy is greater than the energy
required to break a
chemical bond, the molecule can fragment.
The fragmentation processes are typically categorized as
direct cleavage or
Rearrangement Cleavage reactions are simply the breaking
of a bond to
produce two fragments. These reactions usually produce an
even electron
ion (AB+).
The even electron ion is detected at an odd m/z value
(assuming no
nitrogen) and a neutral odd electron radical.* Since the
radical is a neutral
fragment it is not observed in the mass spectrum.
Rearrangements are more complex reactions that involve
both making and
117
breaking bonds. These reactions are thermodynamically

Fragmentation
Odd electron ions are radical species with an unpaired
electron. They are
produced by removing a lone pair electron or a bonding
electron from a
molecule. For example, water is ionized by removing a nonbonding electron
from oxygen to produces H2O+, . This is an example of an odd
electron ion.
Odd electron ions have an even m/z value. The exception to
this is if the ion has
an odd number of nitrogen atoms. Calculate the m/z value for
some ions to
verify this statement.
Even electron ions have all paired electrons. Even electron ions
are usually
produced by cleavage reactions that result in loss of a neutral
radical. An
.
example of this is the cleavage of CH3 CH2 CH3+. to form CH
1183
+

Isotope Peaks
The peaks from the isotopes
The intensity ratios (relative intensities) in the
isotope patterns
are arising from the natural abundance of the
isotopes, thus are
valuable to ascertain the atomic composition of ions.
M+1 peaks are primarily due the presence of 13C in
the sample
M+2 peaks useful for N, O calculation (< seven C
cpds)
M+2, M+4, .. indicative of presence of Br, Cl, S;
(79Br : 81Br 1:1, 35Cl : 37Cl 3:1)

119

120

121

122

123

124

125

Index of Hydrogen
Deficiency
IHD = number of pairs of
hydrogen that must be
removed from the
corresponding saturated
hydrocarbon.
IHD = The SUM of the number
of rings, double bonds, or 2*the
number of triple bonds.

Formula
Used to determine an
approximate structure
once all of the atoms
have been identified.

The R+DB value can be

calculated as follows:

Where C, H, X, and N stand for the

number of carbons, hydrogen,
halogen, and nitrogen atoms in the
ion.
Si is treated as a carbon and P as
nitrogen.
Neither oxygen or sulfur are
considered in the calculation.

IHD = (2C + 2 H
X + N)/2 or
R + DB = C (H +
X)/2 + N/2 + 1 (round
down)

126

Structural Interpretation
Rings Plus Double Bonds Rule
Examples:
Ion Formula
CH3COCH2+
=1

Calculation
R+DB = 3 (5+0)/2 + 0/2 + 1 = 1.5

C5H4NCl+

R+DB = 5 (4-1)/2 + + 1 = 4

127

Structural Interpretation
Formation of Ions
Three factors determine which bond in a molecular ion will
break and which ions will form:
-stability of the ions formed

cleavage often occurs at branching points to form a stable secondary

or tertiary carbonium ion.

-relative bond strength

weak bonds such as those adjacent to hetero atoms are easily

broken. This is called alpha cleavage and is common in esters,
ketones, alcohols and amines.

-neutral fragment stability

cleavage to from a stable neutral molecule is preferred.

128

Structural Interpretation
Libraries
NIST/EPA/NIH library contains about 130,000
spectra with 10 million peaks.
Wiley library contains about 233,000 with 8
million peaks.
There are other smaller libraries that specialize in
certain kinds of compounds, i,e. drugs, poisons,
pesticides, flavors, and fragrances.
Of course manufacturers supply smaller
databases with their instruments.
129

Structural Interpretation
Libraries
Search routines often use a presearch to compare
8 most intense peaks in the unknown with 16
intense peaks in each library spectrum.
This reduces the number of candidates, usually to
about 100.
Ions are weighted for significance based on their
intensity and mass, higher mass ions are
considered more important.
130

Hyper link-introduction to spectroscopy

The mass spectra

of alkanes are
characterized by
strong molecular
ion peaks and a
regular series of
fragment ion peaks
separated by 14
amu

131

As the carbon skeleton becomes more highly

branched, the intensity of the molecular ion peak
decreases e.g. (pp406-407)
132

Cycloalkanes
Cycloalkanes generally form strong molecular ion
peaks. Fragmentation via the loss of a molecule of
ethene (M-28) is common
- Strong M+
- Fragment ions: M-28, a series of peaks, M-15, M-29, M43, M-47,
etc .
e.g. pp 409 410

Alkenes
-The mass spectra of most alkenes show distinct
molecular ion peaks.
- Apparently, electron bombardment removes one f the
electrons in the pi bond, leaving the carbon skeleton
relatively undisturbed.
- Fragmentation to form an allyl cation (m/e = 41) is
favored
133
- Strong M+

ynes
e mass spectra of alkynes are very similar to those of alkene
e molecular ion peaks tend to be rather intense, and
agmentation patterns parallel those of the alkenes
rong M+,
agment ions: M/e = 39, strong M-1 peak. E.g. p 413

ntinue from pp 413-onwards

134

Generally

135

136

Hyphenation Techniques
Techniques in which a separation device is
combined with a detector, such as gas
chromatography-Fourier transform infrared
spectroscopy (GC-FTIR and GC-mass
spectrometry (GC-MS), have already become well
accepted analytical tools
GCMS should be considered a relatively mature
technique. One does not see major changes in
the technique and practices of this method
appearing in the current literature.
LCMS is an almost mature technique. One
occasionally sees a new innovation concerning
how this experiment is done. By distinct contrast
LCNMR is a technique still transitioning between
infancy and adolescence
137

LC-MS

Thermospray Interface
Mass Spectrometric Detectors
Direct Introduction of Total
Fundamental Problem
Effluent
Large Solvent Volume of
Flow: up to 2 mL/min
HPLC
Liquid Vaporized in Heated
High Vacuum Requirement of
Stainless Steel Capillary
MS
Aerosol of Solvent &
Solutions
Analyte Produced
Column Split
Analyte Ionized by
Uses a Small Faction of
Ammonium Salt in Eluent
Eluent
Simple Spectra (Soft
Best with Microbore
Ionization)
Columns
Disadvantages:
Flow: 10 to 50 L/min
Only polar solvents
Moving Belt/Moving Wire
(for ammonium salt)
Only polar analytes
Effluent Dripped onto
Column Wire
Advantage - Excellent
Evaporator
Solvent Evaporated MSDesorptionChamber
LOD: 1 to 10 pg
Desorption-Ioniztion in MS
MovingWire

cartoonschematic

138

System Configuration

Liquid
Chromatography

Ionization

Very

important!
Many columns
Many solvent

ESI
APCI
APPI

Mass Analyzer

Detector/
Data
Collection

Triple Quadrapoles
Ion-Traps
Hybrids

systems
139

LC/MS system diagram

140

GCMS

A typical GC/MS system

diagram
141

Methods in GCMS
Sample
introduction

Split
Splitless
On-column
Thermodesorption
Air Samples
Dynamic

Ionisation

Electron impact
Chemical
Ionisation

Ammonia
Isobutane

Mass Analysis
Quadrupole
Triple
Quadrupole
Ion trap
Time-of-flight
Magnetic Sector

Methane
Other

head-space

SPME
Etc.

142

GCMS Data: Spectrum

Chromatogram

Mass Spectrum is a distribution of

ion m/z values at a certain retention time

Ion m/z

3-Dimensional Data

Ion Count

Retention time

143

GCMS Data: Total Ion

Chromatogram
Chromatogram

Total Ion Chromatogram (TIC) = Sum of all ions in data

Ion m/z

3-Dimensional Data

Ion Count

Retention time

144

GCMS Data: Extracted

Ion Chromatogram
TIC

EIC

Ion m/z

Extracted Ion Chromatogram (EIC) = Sum of selected m/z ions in data

Ion Count

Retention time

145