CHIM H407 Part B MolStructure 2016

Molecular Structural
Characterization and Analysis

Academic year 2015-2016
Master of Science in Chemical and Materials Engineering Commonn core 1
CHIM-H407 / 4016478ENR
Course structure
Introduction
Separation Sciences
Ia.
IIa.
IIIa.
IVa.
Va.
VIa.
VIIa.
VIIIa.
HPLC instrumentation
Types of LC
Optimization of HPLC separations
Band broadening in LC
Kinetic performance limits
Multi-dimensional separations
Chip Technology
Gas Chromatography
Molecular Structure Characterization

Ib.
IIb.
IIIb.
IVb.
Vb.
Spectroscopy: General considerations

Electronic spectroscopies
Vibrational spectroscopies
NMR spectroscopy
Mass spectrometry
Practicals and exercises illustrating both parts

of the course
Prof. S. Eeltink
seeltink@vub.ac.be
Prof. K. Bartik
kbartik@ulb.ac.be
Prof. G. Bruylants
gbruylan@ulb.ac.be
2
Molecular Structure
Characterization
Ib.
IIb.
IIIb.
IVb.
Vb.
Spectroscopy : General considerations

Electronic spectroscopies
Vibrational spectroscopies
NMR spectroscopy
Mass spectrometry
Ib. Spectroscopy : General

considerations
1. Spectroscopy
2. Spectroscopic transitions
I. Spectroscopy
1. Spectroscopy
Spectroscopy is the study of the interaction between matter and electro-magnetic radiation
which can be absorbed, emitted or scattered.
The mechanism by which this occurs involves the interaction between the oscillating
electric (or magnetic) field in the radiation with an electric (or magnetic) moment of the
molecule.
Electric transitions are more probable than magnetic transitions. Dipolar interactions are
more probable than those of a higher order.
I. Spectroscopy
2. Spectroscopic transitions
2.1. Energy levels
The electronic, vibrational and rotational energy levels of a molecule are quantized.
E1
Representation of the electronic, vibrational and

rotational energy levels of a diatomic molecule.
E0.
The nuclear and electronic spin levels are also quantized.

Transition between the energy levels are at the origin of the lines/bands observed in spectra.
6
I. Spectroscopy
Energy levels are determined by defining the Hamiltonian of the system and solving the Schrdinger
equation :
H = E
Selection rules and line intensities are obtained by solving the time-dependent Schrdinger equation :
H = i ! / t
A spectroscopic experiment registers the change of a molecule from one quantum state to another.
The transitions depend on the wavelength of the emitting source.
h = E = E
final
E
initial
7
I. Spectroscopy
X-ray spectroscopy (= 0,1-100 ):

Transitions between electronic energy levels of core electrons.
UV-Visible spectroscopy (UV : = 180-380 ; Vis : = 380-780 nm):
Transitions between electronic energy levels accompanied by rotational and
vibrational transitions (rovibronic spectroscopy).
IR spectroscopy IR (= 780 nm-300 m):
Transitions between vibrational energy levels accompanied by rotational
transitions (rovibrational spectroscopy).
Micro-wave spectroscopy (= 300 m-3,75 mm):
Transitions between rotational energy levels within the same vibrational state
(rotational spectrsocopy).
NMR spectroscopy (= 0,6 m-10 m):
Transitions between nuclear spin levels in a static magnetic field.
I. Spectroscopy
UV absorption and emission

spectra of rhodamine-B
0.1
absorption
NMR spectrum of isobutylamine
60
emission
50
0.08
(C 2H5)2N
N(C2H5)2
0.06
30
/ U. A.
40
mis
CO2H
0.04
20
0.02
0
500
10
550
600
650
0
700
Longueur d'onde / nm
IR spectrum of 2-methylbutanamide
CH3CH2CHC
CH3
NH2
I. Spectroscopy
2.2. Transition probabilities and Einstein coefficients

Radiative absorption and emission processes:
Excited state
Fundamental state
1. Induced absorption
F + h
2. Induced emission
E + h
F + 2 h
3. Spontaneous emission
F + h
Transition probability per unit of time

is proportional to the number of
photons.
is independent of the number of
photons.
10
I. Spectroscopy
The rate of induced absorption from a fundamental to an excited state :
TA =
dN F (t )
= BFE ( EF ) N F (t )
dt
= d/d : radiation density at a given frequency

BFE : Einstein induced absorption coefficient
with
BFE
N F (t ) = population of the fundamental state
2 1
=
3h 2 4 0
FE
FE = transition dipole moment

The rate of induced (stimulated) emission from an excited to a fundamental state :
TEst
dN (t )
= E = BEF ( EF ) N E (t )
dt
BEF : Einstein induced emission coefficient

1
The rate of spontaneous emission from an excited to a fundamental state :
TESp =
dN E (t )
= AEF N E (t )
dt
AEF : Einstein spontaneous emission coefficient
11
I. Spectroscopy
The net variation of a state is given by :
dN F (t) dN E (t)
=
= BFE ( FE ) N F (t) AEF N E (t) BEF ( FE ) N E (t)
dt
dt
At equilibrium :
dN F (t ) dN E (t )
=
=0
dt
dt
Considering the radiation density of a blackbody (Plancks law) :
8h
v EF 3
( EF ) = 3 h k T
c e EF B 1
And the Boltzmann distribution :
AEF
BFE eh EF
k BT
BEF = BFE
BEF
8h
v EF 3
= 3 h k T
c e EF B 1
AEF 8 hv EF
=
BEF
c3
12
IIb. Electronic spectroscopies

1. Absorption spectroscopy
2. Emission spectroscopy
3. Absorbing species
13
II. Electronic spectroscopies
1. Absorption spectroscopy
1.1. UV-Vis absorption spectra
Absorption spectrum of Na vapour :

few transitions spectral lines.
For a molecule, the number of transitions is

considerably higher (electronic, vibrational,
rotational): spectral bands with some fine
structure.
In the condensed phase, the fine structure is no

longer observed. Interaction with the solvent
broadens the lines.
14
1.2. Beer-Lambert Law

The empirical Beer-Lambert Law relates the absorption of light to the properties of the material
through which the light is travelling.
The law states that there is a logarithmic dependence between the transmissivity of light through a
substance and the product of the molar absorptivity of the substance, the concentration of
absorbing species and the distance the light travels through the material.
I A = I0 1 10 A
I0
C,
A = log T = b c
T=
I I0 I A
=
I0
I0
I0 = intensity of incident light
b = path length
IA = absorbed intensity
= molar absorptivity constant
T = transmissivity
c = concentration of the absorbing species
A = absorbance
is a measure of the absorption band intensity which is a measure of the transition probability.
The band intensity is directly related to the square of the dipolar transition moment.
15
1.3. Franck-Condon Principle

The transition probability (and consequently the band intensity) requires the calculation of the square of
the transition dipolar moment :
2
1
PA = BFE v (vFE ) =
3h 2 4 0
FE
v (vFE )
It can be shown that during an electronic transition, a change from one vibrational energy level to
another will be more likely to happen if the two vibrational wave functions overlap more significantly.
IA
vi v j d
The most intense transition is the one for which the overlap is the
greatest. The FC transition is rarely the 0-0 transition.
Classical Formulation : Vertical Transition

Classically, the Franck-Condon principle is the approximation
that an electronic transition occurs without changes in the
positions of the nuclei in the molecular entity and its
environment.
16
17
2. Emission spectroscopy
Molecules that are excited to high energy levels can decay to lower levels by
emitting radiation: photoluminescence.
Transition rates of spontaneous and stimulated emission
dN E
= TESp = AEF N E
dt
dN
E = TESt = BEF () N E
dt
Knowing the ratio between the Einstein coefficients and

in the presence of blackbody radiation
AEF
v3
= 8 h 3
BEF
c
TESp
TESt
( ) =
(eh
k BT
8h
v3
c3 e h k BT 1
1)
For the range of wavelengths used in electronic spectroscopy, spontaneous emission is the dominant
radiative relaxation mechanism.
No saturation in UV-Visible spectroscopy
18
2.1. Fluorescence and phosphorescence
S0
fundamental singlet
state
Fluorescence: spontaneous emission from

the excited singlet state (short lifetime: ns).
More probable than phosphorescence.
S1
T1
excited singlet
state
excited triplet
state
Phosphorescence: spontaneous emission from

the excited triplet state (lifetime: ms to hours)
(The Franck-Condon principle also applies in emission.)
19
2.2. Jablonski diagramme

Molecules that are excited from a singlet sate (S0) to a higher energy levels (S1) will relax to the
lowest vibrational state of S1 by radiationless decay. This relaxation involves dissipation of energy
from the molecule to its surroundings, and thus cannot occur for isolated molecules.
The molecules can deactivate themselves from the S1 state via the
following processes :
Fluorescence (kR) (radiative process)
Radiationless relaxation (kNR) (the most efficient process)
Intersystem conversion (ISC) to T1 state (kISC)
(radiationless transition to a state with a different spin multiplicity
and occurs in molecules with large spin-orbit coupling)
The molecules can deactivate themselves from the T1 state via the
following processes:
Phosphorescence (radiative process)
Radiationless relaxation
kISC
S1
IA
T1
kR kNR
S0
20
2.3. Emission intensity

Emission occurs in all directions and it is consequently difficult to measure emission intensity.
I0
IT
IE
A fraction of the intensity is measured (detector placed at a specific orientation relative to the source
generally orthogonally). This fraction of fluorescence (or phosphorescence) is characterized by the
quantum luminescent yield .
number events
I
= E
photons absorbed I A
The quantum yield can also be defined relative to the rate constants :
kR
k R + k NR + k ISC
21
2.4. Stokes shift

The absorption and emission spectra of a molecule should have the 0-0 transition in common.
This overlap is however rare :
DG
the spectra rarely exhibit any fine structure;

the 0-0 transition is not the most intense transition;
the solvent induces a shift called the Stokes shift.
The Stokes shift :

When a photon is absorbed, the dipolar moment of the
molecule is modified. The solvent does not have the time
to rearrange itself to a minimum energy state during the
time that it takes for the transition to occur.
When emission occurs and the molecule returns to its
ground state the solvent once again reorients itself more
slowly than the transition.
22
3. Absorbing species
Organic species possess n, and molecular orbitals. Four types of
electronic transitions can occur in the UV-Visible range.
antibonding
- *
< 200nm
antibonding
n - *
< 250nm
: 10 103 l.cm-1.mol-1
- *
< 400nm
: 103 - 104 l.cm-1.mol-1
n - *
200 < < 700nm
: 10 - 100 l.cm-1.mol-1
nonbonding
bonding
bonding
Allowed Transitions : > 103 l.cm-1.mol-1

Less strongly forbidden/Not allowed : < 100 l.cm-1.mol-1
Forbidden Transitions : (-* and -*)
* are difficult to exploit in solution.

23
3.1. Definitions
Chromophore
Section of molecule which can undergo electronic transition

and responsible for absorption ( > 190 nm)
Bathochrome shift
Shift to a longer wavelength due to substitution or solvent

effect (red shift)
Hypsochrome shift
Shift to a shorter wavelength due to substitution or solvent

effect (blue shift)
HYPERCHROME effect
Increase in absorption intensity
Hypochrome effect
Decrease in absorption intensity
Absorbance
HYPERCHROME
bathochrome
hypsochrome
hypochrome
24
3.2. Chromophores
Sections of molecules which can undergo detectable electron transitions are referred to as
chromophores and absorb electromagnetic radiation (which may be perceived as color
somewhere in the electromagnetic spectrum).
Chromophore
Example
max / nm
max / M-1cm-1
Transition
Solvent
Ketone
Acetone
186
280
1000
16
*
n *
n-hexane
Aldehyde
Acetaldehyde
290
16
n *
n-hexane
Carboxylic acid
Acetic acid
204
41
n *
ethanol
Amide
Acetamide
214
60
n *
water
Azo
Azomethane
339
n *
ethanol
Nitro
Nitromethane
280
22
n *
isooctane
max and max only constitute an approximate guide for structure determination as:
they are solvent dependant

can be difficult to determine
are dependant on neighbouring effects (chromophores are not not always isolated)
25
Polyenes: * transition (K band)
Compound
max / nm
max / M-1cm-1
Solvent
vapour
Ethene
171
15500
Penta-1,4-diene
178
Buta-1,3-diene
217
21000
hexane
2, 3-dimethylbuta-1,3-diene
226
21400
cyclohexane
2,5-dimthylhexa-2,4-diene
241
13100
Cyclohexa-1,3-diene
256
8000
497
133000
hexane
(-carotene)
Observation: Conjugation and hyper-conjugation stabilizes the system (increase in max)
26
Bathochromic shift due to p orbital overlap
4*
*
165 nm
217 nm
3*
H2C
CH2
CH CH2
CH2 CH
27
Carbonyle group : n * transition (R band)

Compound
max / nm
max / M-1cm-1
Solvant
Acetone
279
13
Isooctane
Diisobutyle ketone
288
24
Isooctane
Acetaldehyde
290
17
Isooctane
Propionaldehyde
292
21
Isooctane
Acetic acid
204
41
Ethanol
Acetamide
214
60
Water
Hexa-1-ene-5-one
278
30
Buta-1-ene-3-one
324 (R)
219 (K)
24
3600
Observations:
Batochrome shift (stabilization; red shift) of the R band of ketones (not aldehydes) when larger alkyl
groupes are introduced. (1-2 vs 3-4)
Hypsochrome shift of the R band (blue shift) for nonbonding electron containing groups (carboxylic
acids, amides). (5 and 6)
Bathochrome shift of the R band (red shift) if the carbonyle group is conjugated (enones); the K
band of the ethylenic chromophore is also observed. (7 vs 8)
28
Benzene and aromatic compounds

Benzene has three caracteristic * transitions bands:
E1 (max = 184 nm, = 6.104 M1cm-1)
E2 (max = 204 nm, = 7900 M-1cm-1 ; also called K band)
B (max = 255 nm, = 200 M1cm1)
Substitution on the aromatic ring has a strong effect on the absorption spectrum of the compound.
CH3
max
255nm
261nm
OH
270 nm
O-
287 nm
NH2
280 nm
NH3+
254 nm
(B band)
29
UV absorption spectra of different aromatic compounds :
30
The Color Wheel
31
IIIb. Vibrational spectroscopies
1. Molecular vibrations
2. Infrared spectroscopy
32
III. Vibrational spectroscopies
1. Molecular vibrations
1.1. Definitions
Vibration : Periodic and concerted motion of all the nuclei of a molecule that does not lead to change
in the position of the centre of masse (no translation).
All atoms within a molecule are in continuous motion, vibrating at defined frequencies called normal
modes of vibration.
The number of independent vibrational degrees of liberty (normal modes) of a molecule composed
of N atoms:
- non linear molecule : # = 3 N - 6
- linear molecule : # = 3 N - 5
Normal (or vibrational) coordinates are used to describe the vibrations of a molecule (use of a
molecular frame instead the laboratory frame). These coordinates (q) are a linear combination of
the changes in the positions of atoms in the molecule relative to their equilibrium position.
Specific normal coordinates are associated to each normal mode of vibration of a molecule.
33
1.2. Diatomic molecule

For a diatomic molecule, the stretching of the bond between the two atoms is the only mode of vibration
(centre of masse is represented by the cross).
r
r2
m1
m2
Vibrational coordinate : q = r re
r1
r and re are respectively the instantaneous and equilibrium bond lengths
The potential energy associated to the change in bond length is a function of the
vibrational coordinate : U(q).
At short distances, the potential energy of the molecule increases rapidly due to the strong repulsion
between particles in close proximity.
At longer distances, the bond is stretched (increase in potential energy) and can break (asymptotic
behaviour).
The distance at which the bond is most stable is the minimum in the potential.
The mathematical expression for this potential energy curve is not known but simple
models can be used, in the case of diatomic molecules, to approximate it.
34
Morse potential : U (q) = De 1 e
q 2
De : dissociation energy
1 d 2U 2 1 2
Harmonic potential : U (q) =
q = kq
2 dq 2
2
k : force constant characteristic of the bond (Hookes law)
Potentla energy U(q)
: constant characteristic of the bond
De
Morse potential
Harmonic potential
Vibrational coordinate , q
In the harmonic approximation, the potential energy is a quadratic function of the normal coordinate.
35
Quantum harmonic model

m1
The energy of the different levels for a harmonic oscillator :
E = n + h
2
with
1
2
k (m1 + m2 ) 1
=
m1m2
2
m2
m1m2
m1 + m2
Reduced mass
n is the vibrational quantum number (0, 1, 2 ...) ; m1 and m2 are the atomic masses of the two nuclei at
the extremities of the bond ; k is the force constant characteristic of the bond
The energy of the fundamental state is non-zero and the difference between consecutive energy levels
is constant :
h k
E = h =
2
For a harmonic oscillator transitions are allowed only

when n = 1 (selection rule).
This does not apply to an anharmonic oscillator; the
observation of overtones is possible (n = 2,3).
Comparison between harmonic and anharmonic oscillators

good energy approximation for the first energy levels.
36
1.3. Polyatomic molecules

All the atoms of a molecule vibrate at the same frequency: normal modes of vibration.
Two classes of molecular motions exist :
Stretching (symmetric and asymmetric) : mouvement of the atoms along a bond axis.
Bending (scissoring, rocking, wagging and twisting) : deformation of the angle between bonds.
H 2O
Elongation symtrique
Symmetric
stretching
Asymmetric
stretching
Elongation
antisymtrique
Bending
Cisaillement
CO2
Elongation symtrique
Symmetric
stretching
Asymmetric
stretching
Elongation antisymtrique
Bending
Cisaillement 1
Scissoring
Bending
Cisaillement 2
Scissoring
The bending modes are

degenerate (same frequency).
37
Vibrations of a methylene group in a molecule :
Symmetrical stretching
scissoring
wagging
antisymmetrical stretching
rocking
twisting
38
2. Infrared spectroscopy
2.1. IR transitions
The vibrational states of a molecule can be probed via infrared spectroscopy, as vibrational
transitions typically require an amount of energy that corresponds to the infrared region of the
spectrum.
Near infrared NIR (: 780 nm 2.5 m)
( ~ : 12800 cm-1 4000 cm-1)
Mid Infrared MIR ( : 2.5 m 50 m)
( ~ : 4000 cm-1 200 cm-1)
Far Infrared FIR ( : 50 m 1 mm )
( ~ : 200 cm-1 10 cm-1)
E = h = h = hc
The wave number (expressed in cm-1) is the inverse of the wavelength.
Selection rule : in order for a vibrational mode in a molecule to be IR active ,

it must be associated with changes in the permanent dipole of the molecule.
39
Example : carbon dioxide has 2 active modes
Symmetric
Elongation stretching
symtrique
Asymmetric
stretching
Elongation antisymtrique
= 0
=0
Bending
Cisaillement
! 0
Example : water has 3 active modes
Symmetric
Elongation stretching
symtrique
!
0
Asymmetric
stretching
Elongation
antisymtrique
!
0
Bending
Cisaillement
!
0
40
Not all the normal modes of vibration are observed in the IR spectrum of a molecule.
- Some fall outside the zone experimentaly exploited between 4000 et 400 cm-1.
- Some are of weak intensity (small perturbation of the dipolar electric moment).
- Some modes are degenerate.
- Line overlap can occur.
Additional lines can be observed due to the limits of the harmonic potential approximation.
- Overtones
- Combination tones
Coupling between normal modes of vibration is frequent:

(coupling effects can yield information on the relative position of functional groups in a molecule)
- Stretching modes with a common atom
- Bending modes with a common atom,
Beer-Lambert law is rarely valid in solution: few quantitative applications of IR spectrscopy.

41
Toluene
CH3
4000 1400 cm-1 =
vibrations characteristic of functional groups

(help for structure determination)
1400 400 cm-1 =
fingerprint region
(help for compound identification)
42
2.2. Identification of organic compounds

The characteristic frequencies of different functional groups have been tabulated:
Bond
C-H
Type of compound
Energy / cm-1
Intensity
2850-2970
Strong
1340-1470
Strong
3010-3095
Medium
675-995
Strong
3300
Strong
3010-3100
Medium
690-900
Strong
Alcohol and phenol
3590-3650
Variable
with H bond
3200-3600
Variable
Carboxylic acid
2500-3300
Broad
Alcane
Alcene
Alcyne
Aromatic
O-H
43
Bond
Type of
compound
Energy / cm-1
Intensity
N-H
Amine / amide
3300-3500
Medium
C=C
Alkene
1610-1680
Variable
Aromatic
1500-1600
Variable
CC
Alkyne
2100-2260
Variable
C-N
Amine / amide
1180-1360
Strong
CN
Nitrile
2210-2280
Strong
C-O
Alcohol, ether,
ester, acid and
anhydrides
1300-1050
Strong
C=O
Aldehyde,
ketone, acid and
ester
1540-1870
Strong
Approximate values due to the interaction between the different vibration modes
of a molecule.
44
The position of the C=O stretching band is function of the electronic and mass effects of the
neighbouring substituents as well as conjugation and possible hydrogen bonding.
A saturated aliphatic ketone has its stretching band at 1715 cm-1.
G+
C
O-
An inductively electron withdrawing group

reduces the bond length and increases
the frequency of absorbance.
A resonance donor group increases

the bond length and reduces the
frequency of absorbance.
( ~ > 1700 cm-1)
( ~ < 1700 cm-1)
G = Cl
F
Br
OH
OR
1815 cm-1
1870 cm-1
1812 cm-1
1760 cm-1
1750-1730 cm-1
G = NH2
SR
1695-1650 cm-1
1720- 1690 cm-1
45
46
2.3. Characteristic spectra

dodecane
CH2
CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
CH2
A: Stretching: symmetric (all the bonds contract at the same time) and asymmetric of CH2 and CH3.
B: Bending: CH3 symmetric (1375 cm-1 ; closing petals of a flower) ;
CH3 asymetric (1450 cm-1; one petal opens and two close)
CH2 asymmetric (1465 cm-1; in plane scissoring)
C: Bending: CH2 in plane rocking
47
dec-1-ene
CH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
CH2
A: alkane C-H stretching

B: alkene =C-H stretching
C: C=C stretching (between 1670 and 1600 cm-1)
frequency depends on substitution ; cis/trans nature and conjugation.
D: out-of-plane =C-H bending (strong bands between 1000 and 650 cm-1 ; depends on substitution)
E: CH2 rocking
48

CH3
CH3
o-xylene
A: aromatic C-H stretching between 3100 and 3000 cm-1

B: aliphatic C-H stretching
C: weak overtones or combinations of the aromatic C-H streching (2000 -1650 cm-1 region)
D: C=C ring stretching (1600 to 1585 cm-1 and 1500 to 1400 cm-1 )
E: in-plane =C-H bending
F: out-of-plane =C-H bending
G: out-of-plane ring C=C bending
49
2,2,4-trimethylpentan-1-ol
H3C
H3C
CH
H3C
CH3
OH
C
C
H2
C
H2
A: broad O-H stretching above 3200 cm-1 (broadening due to H bonding)

B: alkane C-H stretching
C: alkane symetric and asymetric C-H bending
D: C-O stretching between 1260 and 1000 cm-1
50

O
pentan-2-one
CH2
CH3
C
CH2
CH3
acetophenone
C=O stretching (between 1650 and 1850 cm-1 depending on substitution)
51

O
2-methylbutanamide
NH2
A: N-H2 asymmetric and symmetric stretching around 3520-3400 cm-1

B: C-H stretching
C: C=O stretching and N-H bending (amide I and II bands)
D: C-N stretching
52
heptanoic acid
OH
A: O-H stretching with H bonding

B: C-H stretching
C: C=O stretching
D: C-O-H in-plane bend
E: C-O stretching
F: O-H out-of-plane bend
53
IVb. NMR spectroscopy
1.
2.
3.
4.
5.
6.
Introduction
Basic principles of NMR
The chemical shift
Spin-Spin coupling
Fourier transform NMR
Experimental and instrumental aspects of pulsed Fourier
transform NMR
7. General considerations about different nuclei
8. 1D and 2D pulse sequences
54
IV. NMR spectroscopy
1. Introduction
Absorption in NMR is the result of the interaction between a time dependent magnetic field
and the dipolar magnetic moment of nuclear spin placed in a static magnetic field.
Low energy absorption spectroscopy :
-
radio-frequencies = 107 to 109 Hz (UV-Vis : = 1013 1015 Hz)
E : 10-4 to 10-2 J/mole

Molecules are in their electronic and vibrational ground state
Spectroscopy of low sensitivity
The intensity, frequency and shape of the signals yield the same type of information as in other
spectroscopies: quantitative and structural.
NMR spectroscopy probes the nucleus of the atom and yields information on the
atom environment and on its connectivity.
Fields of application :
- Structural analysis in the gas, liquid or solid state

- Study of molecular motion:
dynamics of systems at equilibrium
diffusion in the liquid phase
- Study of in vivo metabolisms
- Imaging
55
Important dates
1944
Isidor Isaac Rabi (Columbia, NY): Nobel prize in physics "for his resonance method for
recording the magnetic properties of atomic nuclei".
1946
Edward Purcell (Harvard) and Felix Bloch (Stanford) perform the first NMR experiment on
condensed matter.
1952
Edward Purcell and Felix Bloch: Nobel prize in physics "for their development of new
methods for nuclear magnetic precision measurements and discoveries in connection
therewith".
1950s First spectra recorded to elucidate molecular structure; permanent and electro-magnets.
1960s Superconducting magnets are introduced; implementation of Fourier Transform NMR.
1970s First applications of NMR to biology and medicine.
I. Rabi
E. Purcell
F. Bloch
56
1991
Richard Ernst (ETH Zurich): Nobel prize in chemistry "for his contributions to the
development of the methodology of high resolution nuclear magnetic resonance (NMR)
spectroscopy".
2002
Kurt Wthrich (ETH Zurich): Nobel prize in chemistry "for his development of nuclear
magnetic resonance spectroscopy for determining the three-dimensional structure of
biological macromolecules in solution".
2003
Paul Lauterbur (U. Illinois) and Peter Mansfield (U. Nottingham): Nobel prize in
physiology or medicine "for their discoveries concerning magnetic resonance imaging".
R. Ernst
K. Wthrich
P. Lauterbur
P. Mansfield
57
2. Basic principles of NMR

2.1. Nuclear spin
Nucleus :
- Composed of protons + neutrons (fermions).
- The combination of the individual momentums of the protons and neutrons
gives the total kinetic (angular) momentum : I
- The total angular momentum is quantized as a function of the nuclear
quantum (spin) number : I
- Nuclei with I 0 are paramagnetic and possess a dipolar magnetic moment.
Paramagnetic nuclei :
Non paramagnetic nuclei :
1
1H
2
1H
12
6C
16
8O
13
19
6C 9 F
129
54 Xe
131
54 Xe
18
8O
58
Nuclear quantum number : I

If A (mass number) is odd
If A is even and Z (atomic number) is odd

If A and Z are even
I = half-integer
1H 13C 15N 19F
1/2 :
23Na 35Cl
3/2 :
17O
5/2 :
I = integer 2H 14N (I =1)
12C 16O
I=0
(I can take all half-integer and integer values between 0 and 7)

Nuclear total angular momentum operator (spin operator) :
- Eigenvalues of
: ( h ) I(I+1) = ! I(I+1)
2
- Eigenvalues of
: !m
Nuclear magnetic moment operator :
I
h: Planck constant
m = -I, -I + 1, ..., I -1, I (2I+1 possible values)

m : magnetic quantum number
= I
- = magnetogyric ratio (positive or negative scalar); characteristic of a specific isotope

- Eigenvalues of
: (
- Eigenvalues of
h
)
2
I (I +1) = !
h
m = !m
2
I (I +1)
59
2.2. Zeeman energy levels

In the absence of a static magnetic field
B0
The 2I + 1 spin nuclear states have the same energy: degenerate energy levels.
In the presence of a static magnetic field
B 0 ( B0 1z )
2I + 1 non-degenerate levels (corresponding to the 2I+ 1 values of m)

ZEEMAN spectroscopy
(Hamiltonian describing the coupling of the spin with the applied static magnetic field)
Energy of the eigenstates
60
2.3. Larmor frequency: fundamental equation of NMR

Selection rule for a transition : m = 1
Energy difference between consecutive Zeeman energy levels :
0=
B0 s-1
2
0 = B0
rad s-1
Larmor frequency and angular velocity

For 1H ( >0):
I = and m = + or -. The eigenfunctions are labeled and
: 1/ 2, +1/ 2
: 1/ 2, 1/ 2
(eigenfunctions or wavefunctions describing the possible states of the system)
1
2
1
2
(m = -1/2)
E1/2 = E = E = + ! B0 = +h 0
(m = + 1/2)
E1/2 = E+ = E = ! B0 = h 0
1
2
1
2
61
2H
( >0) I = 1; m = -1, 0, 1
3 energy levels
(m = -1)
(m = 0)
(m = + 1)
NMR: Absorption spectroscopy

Transitions occur between the nuclear spin energy levels when the sample is submitted to an
electromagnetic radiation with a frequency = Larmor frequency
Use of a time-dependent magnetic field B1 to induce the transitions between the energy levels.
62
E (and 0 )
B0
1H
19F
w0
E
Different frequency for each type of isotope (different )
13C
Frequency is proportional to the static field strength.
B0
B0 (T)
0 (1H ) (MHz)
permanent
magnets
0.47
1.4
4.7
9.4
14.1
18.8
22.3
20
60
200
400
600
800
950
electromagnets
superconducting magnets
63
2.4. Macroscopic equilibrium nuclear magnetization : M 0

Probability that the spin is in the eigenststate characterized by m (Boltzman distribution)
pm =
pm =
exp(
Em
)
kT
Em
exp(
)
kT
m
1
=
Em
kT
( Em << kT )
Em
1
kT
m
(m : from I to + I ; 2I + 1 energy levels)
1 Em kT
1 + h m B0 kT
=
2I + 1
2I + 1
Macroscopic magnetization
M o = N Zm pm
(N = total number of spins)
Zm = !m
( !) 2
Mo = N
I I +1 Bo
3kT
M0 is a macroscopic entity to which the laws of classical mechanics can be applied.

Difference in population between two consecutive energy levels
! B0
h0
N = N ( pm pm1 ) = N
=N
kT (2I +1)
kT (2I +1)
For 1H ( = 2.675 108 rad s1 T 1)

300K; 9.4T magnet :
N = N 3.2105
64
2.5. Radiative and nonradiative processes

2.5.1. Radiative processes :
Excited state
Fundamental state
4
5
1. Induced absorption
F + h
2. Induced emission
radiatifs
E + h
F + 2 h
3. Spontaneous emission
F + h
Transition probability per unit of

time
is proportional to the number
non
radiatifs
of photons (B).
is independent of the number of photons (A).
3
In the case of nuclei of spin :
2 2 ! $
A = ! # 2 & <<< 1
3
" c%
Spontaneous emission is negligible in NMR spectroscopy.

(A quantum mechanical description of the radiation field is not required.)
65
The radiofrequencies used in NMR are produced by means of electronic generators : the frequencies
are well defined and can be generated with considerable precision (narrow frequency range).
As the photons associated to radiofrequencies are of low energy, very low power suffices to generate
a large number of photons per unit frequency.
The photons induce transitions between consecutive energy levels (from F to E and from E to F).
As the population of the fundamental energy level is greater than that of the excited level ([F] > [E]),
the net balance is an absorption of energy.
In the absence of other processes inducing transitions : saturation ([F] = [E]).
66
2.5.2. Non-radiative processes :

E
F
1
radiative
radiatifs
non
radiatifs
nonradiative
4. F + Q
transition probability : WFE
5. E
F+Q
transition probability : WEF
At thermodynamic equilibrium : [F] WFE = [E] WEF
WEF [F ]
=
>1
WFE [E ]
Energy receptor : external lattice
(the calorific capacity of the lattice is considered to be infinite.)
Due to the non-radiative processes, saturation can be avoided.
Non-radiative processes are at the origin of relaxation (spin-lattice and spin-spin; see later).
67
2.6. Vector model of NMR (classical model)

2.6.1. Larmor frequency
The nucleus is considered as a charged particle spinning around an axis.
Angular momentum I
Magnetic moment
!
B0
When placed in a static magnetic field,

, the individual moments align themselves relative
to the field in a discrete number of orientations (2I +1 orientations)
The static magnetic field will impose a torque on the moments :
Solution of the equation of movement :
0 = B0
d
= B0
dt
Larmor precession
Bo
y
z (t ) = z (0) = constant
x (t ) = x (0) cos ot + y (0) sin ot
y (t ) = x (0)( sin ot ) + y (0) cos ot
68
2.6.2. Macroscopic magnetization

Allowed orientations of the nuclear magnetic moments
( angle between magnetic moment and Bo direction) :
cos =
m
I (I +1)
For 1H ( I=1/2 and >0) :

N spins parallel to
N = Np =
h
N
(1 + 0 )
2
2kT
!
N spins anti-parallel to B0
N = Np =
!
B0
z
M
Bo
y
x
y
x
h
N
(1 0 )
2
2kT
M = = Macroscopic magnetization
Equilibrium situation : isotropic distribution and M = (0,0, M 0 )
Transitions are induced between the orientations, thereby changing M , by using a time-dependent
magnetic field B1 which has a frequency which matches that of the nuclear precession: resonance.
(see later)
69
3. The chemical shift

3.1. Introduction
Larmor angular velocity : 0 = B0
The magnetogyric ratio is a constant characteristic of an isotope.
1H
w0
19F
13C
B0
Is the resonance frequency the same for all atoms of a same isotope
in a molecule?
70
1H
spectrum of CH3COCH2OH (hydroxyacetone) :
The resonance frequency is different for the different 1H.
= Blocal
The chemical environment of each nucleus is at the origin of this effect:

chemical shift.
71
3.2. Shielding constant shielding tensor

3.2.1. Isolated spherical atom: unperturbed spherical electron distribution
- The external static magnetic field induces circulation in the electronic cloud.
- Circulation induces a secondary field which opposes the primary field: diamagnetic effect.
Blocal = B0 + Binduced = B0 (1 )
Series limited to the 1st term ; = screening or shielding constant
Lamb formula :
oe 2
3m
re dr
0
Bo
Binduced
72
E = !B0
E = !B0 (1 )
B0
B0
noyau
dans
noyau
nu
naked
nucleus
in
un
atome
isol
an isolated atom
nucleus
Z=1
= 17.8 ppm
(H)
Z=6
= 260.7 ppm
(C)
Z=54
= 5642.3 ppm
(Xe)
73
3.2.2. Atom in a molecule
Non spherical electron distribution
BB
0 o
Blocal = B0 + Binduced = B0 (1 )
Bind
xx xy xz
= yx yy yz
zx zy zz
Second rank Cartesian tensor
Diagonalized tensor : 3 principal components
(use of a principal axis frame)
11
= 0
0
22
0
0
33
ij = positive or negative scalar !!!

(Several different models exist to calculate these components.)
74
3.3. Chemical shift in an isotropic environment

3.3.1. Definition of an isotropic environment in NMR
Molecule in an isotropic environment :
The environment of the molecule is the same in all directions. There is no
privileged direction.
In the liquid and gas phase : the environment is on average isotropic because:
- Molecular reorientation (molecular tumbling) is fast compared to the
time scale of the interactions affecting the nuclear spins.
- The molecule can adopt all orientations with the same probability.
NMR parameters are averaged over all possible orientations.
(time averaging)
= 11 + 22 + 33 trace of the tensor

3
NMR signals are narrow and symmetrical (Lorentzian lineshape).
For a nucleus A :
A = B0 (1 A )
75
3.3.2. Chemical shift reference

Experimentally, one does not know where the origin of the scale is situated.
Absolute shifts are rarely needed and it is common practice to use a reference
and to measure differences in frequencies.
= ref = 0 ref
The chemical shift is introduced in order to have a property independent of the B0 magnetic field.
ref 6
10
0
(ppm)
d,n
s , B0
dblind
shielded
blind
Conventional references:
19F
1H
CFCl3
TMS
13C
(tetramethylsilane)
TMS
76
3.3.3. Chemical shift tables

Based on chemical shifts measured in homologous molecules :
-
Chemical shifts of common types of 1H et 13C found in organic molecules.
Chemical shifts can also be calculated using empirical increments.
Example of tables (see practicals):

13C
77
Cyclohexane :
Benzene :
1H = 1.6 ppm
1H = 7.2 ppm
Internal protons : -1.8 ppm

External protons : 8.9 ppm
78
4. Spin-Spin coupling
4.1. Dipolar coupling (or direct coupling)
Direct magnetic interaction of nuclear magnetic moments through space.
mX
mA
B0
Bo
q
Modification of the local field around A due to the dipole of spin X : B =
x (3 cos2 1) / r 3
For a molecule in solution or in the gas phase the angle varies due to the rapid molecular
tumbling and the effect is averaged to zero:
2
(3cos 1)sin d = 0
79
4.2. Scalar coupling (or indirect coupling)

1H
spectrum of ethanol in solution: a fine structure is observed in the signals (multiplicity).
Manifestation of the magnetic interaction between spins transmitted through chemical bonds
Scalar coupling characterized by a constant J (Hz)
80
Origin of scalar coupling:

The magnetic moment of spin A causes a weak magnetic polarization of the bonding electrons
(Fermi contact interaction between an electron and an atomic nucleus when the electron is inside that
nucleus ) that is transmitted by way of the overlapping orbitals to nucleus X (following Hunds rule and
the Pauli exclusion principle).
The Dirac model shows that for vicinal nuclei (J3) with > 0, the situation which is most favoured
energetically is the one where the individual nuclear moments are antiparallel. The situation where
the two individual nuclear moments are parallel is energetically less favoured.
>0
electron spin
nuclear spin
A (H)
C12
C12
X (H)
Favoured configuration : example for vicinal coupling.
81
4.2.1. System composed of two coupled spins 1/2 (J >0) and /J > 10 (1st order)
For two spin spins A et X, the Hamiltonian is :
H = ! A B0 (1 A ) IZA ! X B0 (1 X ) IZX + hJIA IX

H = h A IZA h X IZX + hJIA IX
If
J: coupling constant
H = h A IZA h X IZX + hJIZA IZX
A X >>> J (first order spectrum) :
The eigenfunctions of the system are :

The energy of the two spin system is :
J
2
2
4
J
E2 = +h A h X h
2
2
4
J
E3 = h A + h X h
2
2
4
J
E4 = +h A + h X + h
2
2
4
E1 = h
+h
E = h AmA hX mX + hJmAmX
for mA = and mX =
for mA = - and mX =
for mA = and mX = -
for mA = - and mX = -
(>0)
82
E = h AmA hX mX + hJmAmX
h A
h X
h X + h J 2
h A + h J 2
h A
h X
h X h J 2
h A h J 2
Energy levels in the

absence of coupling
Energy levels
with coupling
Spectrum
Spectrum
A
A
M X M X
J
M A M A
J
83
4.2.2. First order coupling with several spins with different J

If a particular spin couples to several spins : doubling rule
JA
J A> J B
J A> J B > J C
84
4.2.3. First order coupling with the same J with several spins
If a spin couples to several spins with the same J there will be 2nI+1 lines for this spin, where n is
the number of neighbouring nuclei and I the spin of these neighbouring nuclei (overlap of lines).
Example: spin A coupling to 3 X (AX3 spin system) ; ; IX =
JAX
JAX
JAX
JAX
JAX
JAX
Multiplet with intensities 1:3:3:1
85
Energy levels of the AX3 spin system with IX = ( m = or )
spin A
X1
X2
X3
configuration with
highest energy
Multiplet with intensities 1:3:3:1
3 configurations with
same energy
3 configurations with
same energy
configuration with
lowest energy
86
Generalisation for I = : there will be n+1 lines and the intensities will follow Pascals triangle:
spin next to a CH2 group
spin next to a CH3 group

87
4.2.4. Strong coupling and second order spectra (JAX < 10 AX)
The eigenfunctions of the Hamiltonian are linear combination of the first order eigenfunctions :
The frequency, number and intensity of the lines do not follow a simple rule.
JAX
n1
JAX
n2
n3
DnAX
Example : AB system
J AB = 1 2 = 3 4 = J AX
n4
= ( 1 4 )( 2 3 )
I3
I4
I2
I1
DnAB
= (1 4 ) /( 2 3 )
JAB
Nomenclature AX or AB : function of the /J ratio
JAB
DnAB
88
89
4.2.5 Example : Ibuprofen; full 1H NMR spectrum
Broad singlet
Labile proton (exchange)
2&2
Relative intensities of the resonance lines
6
90
Example : Ibuprofen; full 1H NMR spectrum
Three
1H
Doublet
coupling to one 1H
Six
One
1H
Quartet
coupling to three 1H
Two
1H
1H
Doublet
coupling to one 1H
Doublet
coupling to one 1H
One
Aromatic protons
Second order coupling, magnetically non-equivalent
(see further)
1H
Nonet
coupling to 8 1H with same J
91
4.3. Factors affecting the value of J

Number of bonds :
saturated compounds : coupling is rarely observed if the nuclei are separated by more than 3 bonds.
unsaturated compounds : long distance coupling is observed in conjugated systems ( J4 J5).
Hybridization of the atom :
Ethane
sp3 hybridization
JCH = 125 Hz
Ethylene
sp2 hybridization
JCH = 156 Hz
Acetylene
sp hybridization
JCH = 248 Hz
Structure : Karplus equations (ex. : J3 vicinal coupling )
J = Acos2 + Bcos + C
The nature of the nuclei :

Comparison which is possible when isotopic substitution is performed (the electronic eigenfunctions are
JH H
not affected)
i j
= H
JD H
D
i
All factors which influence the electronic distribution and electronic density !!!
92
4.4. Chemically and Magnetically Equivalent Nuclei

4.4.1. Chemically equivalent nuclei :
Nuclei of same characterized by a same screening constant and consequently by the same
resonance frequency.
This can be due to :
- chance .
- symmetry
Cl
C C
OH
H
Br
Cl
H
H
C2
S1 (plane of the page)
Homotopic protons
Enantiotopic protons
- a fast interconversion process ( < k)

Hy
k
Hy
Hx
x = y
Hx
93
4.4.2. Nuclei which are not chemically equivalent

This is the case for :
- nuclei with different :
13C
et 1H
- nuclei with different chemical environments:
H3C CH2 Cl
System A3X2
- enantiotopic nuclei in a chiral environment ie: chiral solvent

- diastereotopic nuclei : nuclei next to a chiral centre
HA Cl
Cl
C C * Br
cysteine
HB F
A B
Nomenclature used : AX, AX2, ...., A3B2, AB2

(the letters used depend on the value of the /J ratio)
94
4.4.3. Nuclei which are not magnetically equivalent

Chemically equivalent nuclei that are not coupled in the same way to
all the other nuclei in the molecule.
X
H1
H3
H2
H4
Y
H1 and H3 chemically equivalent

H2 and H4 chemically equivalent
J12 = J34 but J12 J14 et J34 J32
The nuclei are not magnetically equivalent
Complicated spectra
Nomenclature AA'XX' or AA'BB
( depending on the value of the /J ratio)
95
Example
H3 H 4
H5 H 6
AAXX
AABB
96
Example
97
5. Fourier transform NMR

5.1. Principle of pulse spectroscopy
z
z
Acquisition
M
y
x
Macroscopic
magnetization at
equilibrium
Pulse of a timedependent
magnetic field
(B1)
y
x
System is no longer at
equilibrium
Return to equilibrium
The macroscopic
magnetization is
monitored in the xy plane
as a function of time
Perturbation of the population

and phase distribution of the
individual spins
98
For an ensemble of spins , with > 0

z
M
represents
y
Equilibrium population
& isotropic distribution of
individual spins
x
z
represents
y
x
Out of equilibrium
population
& phase coherence of
individual spins
99
5.2. The time-dependent B1 field

Oscillates in the transverse plane
B1
Oscillation frequency =
B1 << B0
M
Bo
Generated by a coil in the transverse plane perpendicular to the static field.
B1 = 2B1 cos(1 t)1x
100
!
B1
is considered to be composed of two counter-rotating radiofrequency (rf) fields
B1 = 2B1 cos(1 t)1x
-1
+1
Sum of the
two vectors
101
Introduction of the rotating frame of reference; rotating at angular velocity =
+1
yrot
yrot
xrot
+1
yrot
xrot
xrot
xrot
xrot
yrot
yrot
102
5.3. Pulse spectroscopy

B1 is applied for a very short time: rf pulse of length = tp
The effect of the pulse on a collection of nuclear spins at equilibrium is to
rotate the macroscopic magnetization away from the Z axis.
A pulse leads to a change in MZ, MX and MY :
- the change in MZ is the consequence of a change in the population of the Zeeman states.
- the presence of the transverse components MX and MY is the consequence of a phase coherence
in the precession of the individual magnetic moments.
z
z
pulse
M0
Mz
My
y
x
Mx
M A = (0,0, M 0 )
M A = (M x , M y , M z )
103
5.3.1. Equation of motion

Equation of motion for the macroscopic magnetization
dM A
= M A ( B0 (1 A ) + B1 )
dt
A = B0 (1 A )
dM A
= M A A + B1
dt

In the rotating frame :
z
B0
dM A
dM A
=
- 1 M A
dt rot
dt lab
dM A
A
+
B
1 ( 1 M A )
A
dt rot

= M A A + B1 + 1

Beff
B1
1 /
xrot
effective field
Beff = B1 +
tan =
1 A
A 1
B1
effective field
104
5.3.2. The Free Induction Decay - FID

When A = 1
Beff = B1
dM A
= M A B1
dt rot
During the pulse, a torque is applied on M A which consequently rotates from the z-axis towards
the transverse plane with an angular velocity = B1 .
Angle of rotation: = B1 tp
For a 90X pulse the magnetization ends in the transverse plane on the Y-axis.
After the pulse, M A will rotate in the laboratory frame around the z-axis with an angular velocity
A = B0 (1 A ) = BA which is the angular velocity of the rotating frame.
M A returns to its equilibrium position
M A = (M x , M y , M z )
M A = (0,0, M 0 )
The amplitude of M A is governed by the transverse relaxation (T2) and longitudinal relaxation (T1) times.
105
Evolution of the magnetization after a 90x pulse
time (t)
yrot
yrot
xrot
xrot
Myrot
M0
Mz
dM y
dt
M y
M0
dM z M 0 M z
=
dt
T1
T2
M yrot = M 0 exp(
t
)
T2
t/T
M Z = M 0 "# 1 e 1 $%
T1 : longitudinal or spin-lattice relaxation time (1st order reaction time constant)

T2 : transverse or spin-spin relaxation time (1st order reaction time constant)
(values depend on each specific nuclear spin; see later)
The signal in the transverse plane is recorded using a coil surrounding the sample (electric signal)
106
FREE INDUCTION DECAY (FID)
When A 1
dM A
= M A Beff
dt rot
Beff B1
During the pulse M rotates from the z-axis towards the transverse plane with an angular velocity :
A
= Beff
Angle of rotation: = Beff tp .

For a 90X pulse the magnetization ends in the transverse plane but NOT on the Y-axis: offset.
It is important to experimentally keep small in order to stay close to the Y-axis.
(A 1) << B1
After the pulse, M A will rotate in the laboratory frame around the z-axis with an angular velocity
= B (1 ) = B which is NOT the angular velocity of the rotating frame.
A
M A returns to its equilibrium position
M A = (M x , M y , M z )
M A = (0,0, M 0 )
The amplitude of M is governed by the transverse relaxation (T2) and longitudinal relaxation (T1) times
A
and a modulated exponential decrease is observed in the transverse plane.
107
Evolution of the magnetization after a 90x pulse
yrot
xrot
time (t)
A - 1
MAyrot
= (A - 1) t
yrot
xrot
MAxrot
t/s
damped cosine wave
M Ayrot = M 0 exp(
t
)cos( A 1 )t
T2 A
Free Induction Decay: FID
damped sine wave
M Axrot = M 0 exp(
f A (t) = M A (0)exp(
t
)sin( A 1 )t
T2 A
t
)exp(i At)
T2 A
108
5.3.3. Fourier Transform

The signals Myrot and Mxrot are recorded in the time domain.
Fourier Transformation (FT) will yield the frequency signal.
f j (t) = M j (0)exp(
t
)exp(i j t)
T2 j
+
F j () =
f j (t )eit dt
F j () = M j (0) A j () iD j ()
A j ( ) =
T2 j
1 + T22j ( j )
Absorption signal
D j () =
T2 j 2 ( j )
1 + T22j ( j )
Dispersion signal
109
The Fourier transformation of an exponential signal in the time domain

will give a Lorentzian absorption signal in the frequency domain.
For a modulated exponential signal ( A 1 ) Fourier transformation gives
a Lorentzian absorption signal shifted to a.
If several different nuclei are presnt in the sample, the FID will be the sum of the
modulated exponential of each nuclei
1 type of nucleus
Spectra after FT
2 types of nuclei
Several nuclei with

different resonance
frequencies
110
5.4. Relaxation
Relaxation is the return of a spin system to its thermodynamic equilibrium state after it has been
brought out of equilibrium by a time dependent RF perturbation.
!
M = ( M x ,M y ,M z )
Relaxation
z
Mz
dM x M x
=
dt
T2
dM y
out of
equilibrium
M0
dM z M 0 M z
=
dt
T1
M
My
Mx
!
Meq = (0,0,M0 )
dt
M y
T2
at
equilibrium
111
Longitudinal relaxation (for spin )
Nh2 2B0
M0 =
4kT
!
n0
2
!
Mz = n
2
M0 =
n0 = population difference at equilibrium

n = population difference out of equilibrium
The spin system absorbs when it is brought out of equilibrium and the excited state is more populated
than at equilibrium.
n < n0
Mz < M 0
Longitudinal relaxation implies return of the population difference n towards n0.
t/T
M Z = M 0 "# 1 e 1 $%
(after a 90 pulse)
Energy must necessarily be emitted from the spin system towards its molecular environment (the lattice).
Longitudinal relaxation is a phenomenon of enthalpic nature. The energy difference between the spin
states is extremely small and the temperature increase in the lattice is immeasurably small (B0 <<< kT).
112
Transverse relaxation
Transvers relaxation is the loss of the phase coherence of individual magnetic moments which
exists after excitation of the spin system.
z
z
relaxation
Leading to
loss of phase coherence
( with n
n0)
y
x
M y , x = M 0 exp(
y
x
t
)
T2
(after 90 pulse)
y
projections in transverse plane xy

x
out of equilibrium
immediately after application of a pulse
at equilibrium
Transversal relaxation is a phenomenon of entropic nature.
113
Molecular basis of relaxation
Spontaneous emission is completely negligible in NMR and only non-radiative relaxation is operative.
Relaxation in NMR requires the presence of local randomly time dependent magnetic fields B f (t )
(local randomly time dependent electric field gradients are also important for spins I 1 :
quadrupolar relaxation).
Only certain frequencies of the time-dependent fluctuations with lead to relaxation : those close to the
Larmor frequency of the nuclear spin.
Molecular motions are at the origin of the time-dependent fluctuations.
114
6. Experimental and instrumental aspects of

pulsed Fourier transform NMR
6.1. Basic configuration a NMR spectrometer
COMPUTER
ADC
SUPERCONDUCTING
MAGNET
AF
PLOTTER
QPD
PP
PA
T&A
SIGNAL (FID)
FROM SAMPLE
Sample
PA = pre-amplifier
QPD = Quadrature Phase Detector
AF = audio-filters
ADC = Analog-to-Digital Converter
RF EXCITATION TOWARD SAMPLE

PP = Pulse Programmer
T&A = radio-frequency transmitter and amplifier
The magnetic induction field, B0 , is generated by a superconducting magnet.

Radio-frequency pulses are generated by the pulse progarmmer.
The signal (FID) transits via a quadrature phase detector and analog-to-digital converter
before being processed (apodisation, FT, phasing, )
115
6.2. The NMR pulse

The time dependent magnetic field B1 ( 1) is applied as a pulse of duration tp (pulse length or pulse width).
The width of the excitation window is inversely proportional to the pulse length :
1 1/ tp rad.s-1 or 1 1/(2tp) Hz
tp
B1
FT
FT
B1tp
2/tp
1
116
6.2.1. Hard pulses

Short pulses used for the complete and homogeneous excitation of the whole NMR spectrum.
Examples:
1H
13C
nuclei: excitation window ~ 10 ppm

@ 500 MHz this corresponds to ~ 5000 Hz
tp < 400 s
nuclei : excitation window ~ 200 ppm

@ 125 MHz this corresponds to ~ 25000 Hz
tp < 80 s
In practice:
- to cover the full spectrum in a homogeneous manner shorter pulse times are used : tp ~10 40 s
- areas outside the spectrum are filtered away digitally (remove noise).
5 -25 kHz : typical spectral width
~ 200 kHz : spectral width covered by a tp ~ 10s

(problems for nuclei with wide chemical shift range such as Xe-129)
117
6.2.2. Soft pulses

Long pulses used for the selective excitation of very narrow frequency ranges.
The purpose of selective excitation : saturation of a well-defined individual resonance ( ~10 Hz) leading
to the suppression of the NMR resonance (equalization of the populations of the spin energy levels).
Typical application : suppression of an intense solvent signal or for selective decoupling.
Long saturation pulses (of the order of the second) are applied from a B2-excitation channel, which is
different from the B1-excitation channel used for the hard excitation of the full spectrum.
Soft pulse to excite 1 to 10 Hz (linewidth of a resonance)

Measuring B1channel
B1-pulse
FID
acquisition time
Hard pulse to excite 5 25 kHz (full spectrum)
Decoupling B2 channel
B2- soft pulse
118
Example of solvent pre-saturation

residual water signal
Spectrum of a sample recorded in H2O with
selective presaturation of water resonance
full water signal

Spectrum of a sample recorded in H2O without
selective presaturation of water resonance
Relevant resonances with poor amplitude

resolution because of dynamic range problem
119
Example of selective decoupling
CH3
CH3
CH2
triplet
quartet
Saturation of the triplet of the CH3 group

leads to decoupling, during FID acquisition,
of the coupled CH2 protons of which the
quartet becomes a singlet.
Saturation of the quartet of the CH2 group

leads to decoupling, during FID acquisition,
of the coupled CH3 protons of which the
triplet becomes a singlet.
ppm
4.0
3.5
3.0
2.5
2.0
1.5
1.0
120
6.2.3. Broad-Band Decoupling (BBD)

BBD of 1H is commonly used when recording in 13C or 15N spectra NMR. This leads to the suppression
of all fine structure in the signals due to C-H or N-H coupling (1J and nJ).
nJ(13C-1H)
(n 2) < 20 Hz
1J(13C-1H)
~ 130 to 230 Hz
The 13C signals are reduced to singlets

with broad band heteronuclear proton
decoupling.
60
50
40
30
20
10
ppm
As long pulses excite in principle only narrow frequency bands, the full proton band width can only be
decoupled when strong B2 powers are applied which is harmful for the spectrometer transmitter hardware.
In practice trains of short hard pulses are used (complex issue).
121
6.2.4. Pulse angle and phase

Pulse angle : = B1tp
The pulse angle can be set up by adjusting either the pulse length or the intensity of the B1-field
!
M0
r
r
M z = M 0 cos 1z
y (or yrot)
r
r
M y = M 0 sin 1y
!
B1
x (or xrot)
Pulse phase : B1 can be applied along the x, -x, y or -y axis
!
B1
!
M
!
M
!
B1
y
x
!
M
!
B1
!
B1
x
!
M
Position of the magnetization after a 90 pulse, as a function of its phase

122
6.3. Signal acquisition Signal intensity

NMR is not a sensitive technique compared to other spectroscopies.
M to mM concentrations are needed in order to obtain a signal which detaches itself from the noise.
Noise :
- comes from random electromagnetic sources in the environment
- mean noise intensity = maximum noise intensity /2,5
signal intensity
signal S
= =
noise N mean noise intensity
To increase signal intensity
- Increase population difference : N = N0hBo/4kT
increase sample concentration

increase Bo
decrease T
- Increase isotopic abundance in sample

- Signal accumulation
123
Signal accumulation
At: acquisition
time
Signal increases
n and noise increases n

S
n
N
Pulse train : n accumulations
124
To obtain quantitative signals
M Must return to its equilibrium value (0,0,M0) before each pulse.

If T2 < T1 , the signal is no longer detected but longitudinal magnetization is not at equilibrium.
Myrot
T2
M0
Mz
T1
Compromise between and Tr
90
80
45
30
My
Mo
0,985 Mo
0,707 Mo
0,5 Mo
Mz
0,174 Mo
0,707 Mo
0,866 Mo
Introduce a relaxation delay between pulses: Tr = (At + Rd) = 5 T1 and sum several FID
> 5 T1
90x
Rd
90x
90x
At
Rd
At
Rd
At
125
6.4. FID processing - Apodization

Apodization : mathematical treatments of the FID aim at optimizing the spectrum patterns and
outlook, in order to improve signal-to-noise ratio or to enhance spectral resolution (these two
requirements are usually contradictory.
6.4.1 Improvement of the signal-to-noise ratio : exponential multiplication
e-at
Fourier
Transform
Fourier
Transform
FID decay is made mathematically faster and the noise which is mainly in the tail of the FID is
smoothed away improving the signal-to-noise ratio but broadening the lines (by 2a Hz !).
126
Exponential multiplication is useful when the FID is truncated (detector switched off too early).
Truncated FIDs generate line distortions in the frequency domain.
Exponential multiplication cleans away sinc function distortions at the expense of line broadening
FT
sinc function
FT
FT
127
6.4.2 Improvement of spectral resolution : Gaussian multiplication
+at -bt 2
a:
1
T*
Fourier
Transform
Fourier
Transform
The FID is mathematically blown up at the longer times which enhances resolution.
Gaussian multiplication also emphasizes the noise and decreases the initial FID amplitude: there is a
lowering of the signal to noise ratio !
128
Overenhancement will lead to distortions in the spectrum
Standard Gaussian
Fourier
Transform
broader resonances are

attenuated more strongly
minor resonances disappear

into the noise
Overenhanced
Gaussian
Fourier
Transform
spurious spikes appear

129
7. General considerations about different nuclei

7.1. 1H and 2D
1H
I = 1/2
natural abundance 99.9%
2D
I=1

2D:
Importance for the monitoring of B0
= 26,75 107 rad s-1 T-1

H
= H 6, 5
Usual chemical shift range : 14 -4 ppm

Reference: TMS (12 equivalent protons, shielded signal, low reactivity, easily evaporated)
Deuterated solvents are used to avoid solvent signals.
130
7.2. 13C
12C
13C
I=0
I = 1/2

H 4
13
C
13C 1H
4
Receptivity 3N x I ( I + 1)
Taking natural abundance into consideration its

Receptivity relative to 1H : 1.7 10-4
Usual chemical shift range: 200 0 ppm

Reference: TMS
The probability that two 13C isotopes are in the same molecule is low.
The spectrum corresponds to the sum of spectra of molecules each containing one 13C nucleus.
131
As the probability that two 13C isotopes are in the same molecule is low, no 13C - 13C coupling is observed.
13C
- 1H coupling is observed
complex spectra
J1(1H-13C)
C sp3 ~ 125 Hz
C sp2 ~ 155 Hz
C sp ~ 250 Hz
Jn(1H-13C)
10-20 Hz for n = 2 and 3 ; < 3 Hz for n=4
60
50
40
30
20
10
ppm
Relaxation times vary considerably from carbon to carbon; quaternary carbons have very long T1 and T2
it is difficult to obtain quantitative 13C spectra.
132

13C
Satellites in the 1H spectrum : result from J1(1H-13C) coupling
Part of 1H Spectrum
(CH3)3C CO - CH3
CH3
(98.9 % of signal intensity)

12CH
3
Increase in
Y scaling (zoom)
J1(1H-13C)
(1.1 % of signal intensity)

13CH
3
C sp3 ~ 125 Hz
C sp2 ~ 155 Hz
C sp ~ 250 Hz
133

1H
decoupling of 13C spectra : remove fine structure due to coupling
60
ADVANTAGES :
INCONVENIENCE :
50
40
30
20
10
ppm
simplified 13C spectra

less signal dispersion and overlapping
no resonance broadening due to unresolved nJ(13C-1H) couplings
significantly improved signal-to-noise ratio (full resonance in one line and NOE).
loss of nJ(13C-1H) couplings, and hence, of connectivity information.
134
B1-pulse
Measuring B1channel for 13C
FID-acquisition
Decoupling B2 channel
for broadband 1H decoupling
acquisition time
B2-irradiation occurs during FID-acquisition for the effective decoupling of the 1H nuclei.
B2-irradiation occurs also prior to FID-acquisition for the effective development of the NOE-effect;
this is signal enhancement of 13C resonances due to the B2-irradiation of 1H nuclei close in space to the
13C nuclei.
(NB: other decoupling schemes are sometimes implemented, for example to obtain quantitative spectra)
135
7.3. 15N
14N
15N
I=1
I = 1/2
1H
10
15
C
Receptivity 3N x I ( I + 1)

15 1 1
N
10 H
Taking natural abundance into consideration its

receptivity relative to 1H : 3.8 10-6
Extremely low enriched samples are needed
Usual chemical shift range: 900 0 ppm

Reference: 90% CH3NO2 in CDCl3 or NH3 liquid
The probability that two 15N isotopes are in the same molecule is low.
136
8. 1D and 2D pulse sequences

8.1. Representation of pulse sequences
z
Relaxation (Rd)
90x pulse
Acquisition (At)
M
y
x
y
x
Pulse sequence with direct observation

90x
Rd
At
137
Representation of 1D pulse sequence with heteronuclear decoupling

x
The decoupling period can cover:

- the entire pulse sequence
- only the acquisition time (At): inverse-gated decoupling
decoupled spectrum with no NOE
- only the relaxation delay (Rd): gated decoupling
coupled spectrum with NOE
At
Rd
13C
1H
Direct
observation
observation directe
Noscans,
decoupling
128
30 degre
Decoupled spectrum
inverse-gated decoupling
16 scans, 30 degre
AQ=0.5s
D1=4.5s
AQ=0.5s
D1=4.5s
70
60
50
40
30
ppm
70
60
50
40
30
ppm
138
8.2. Inversion-Recovery : measurement of T1

180x (y)
Pulse sequence
90x
180 pulse:
z
M0
180x (y)
y
y
x
Mz= - M0
139
90 observation pulse after delay :

Longitudinal relaxation occurs during the delay
z
90x
=0
FT
y
x
z
90x
< 0.7 T1
FT
y
x
z
90x
> 0.7 T1
FT
y
x
T1 relaxation
140
Intensity of the signal I( ) as a function of delay :
I ( )
1
0.5
0
-0.5
-1
T1
I : integrated intensity of equilibrium magnetization ( M 0)

dM z 1
= [M 0 M z ]
dt
T1
t
M z (t ) = M 0 1 (1 cos ) e T1
M z (t ) = M 0 1 2 e T1
for =
I ( ) = I 1 2 e T1
T1 obtained from parametric

adjustment of this equation to
the experimental data.
141
Ref.: Claridge, 2000
142
8.3. Spin echo

8.3.1 Pulse sequence
180x (y)
90x
z
After /2 pulse :
y
x
x
143
During the first delay spins A (A) and X (x) precess (ie. A > X )
Refocusing takes place during the second delay .
180x
(Sign inversion)
x
180y
x
A
x
A
(Sign retention)
Chemical shift evolution is refocused by the spin-echo sequence.

144
8.3.2. Measurement of T2
Signal line-width is theoretically only function of T2 : 1 =
2
1
T2
Due to field inhomogeneities, the signal line-width is increased: 1 =

2
B0
1
1
=
+
T2* T2
During a spin echo pulse sequence:

Dephasing will occur during the first delay due to field inhomogeneity.
Refocusing will be achieved during the second delay .
y
x
180x
dephasing
refocusing
y
x
T2 relaxation takes place during both delays and at t = 2 , when acquisition

starts, the intensity of the magnetization along the detection axis is:
M y (2 ) = M y (0) e
T2
with M y (0) = M 0
145
To measure T2 , spectra are recorded using this modified spin-echo pulse sequence for different values of n:
90x
180x (y)
n
With the Carr-Purcell-Meiboom-Gill pulse sequence (CPMG), n spin-echoes with delay are performed
with n = . This enables to avoid diffusion problems.
I ( ): intensity of signal recorded with spin echo and different

Srie1
Srie2
Srie3
t or
My(t) (FID): T2* exponential decrease
T2 is obtained via the parametric adjustment of this equation to the experimental data :
I ( ) = I e
T2
I : integrated intensity of equilibrium magnetization ( M 0 )

146
147
8.4. Distortionless Enhancement by Polarization Transfer (DEPT)

90x
180x
1/2J
X
90x
180
1/2J
Acq(+/-)
y
1/2J
The theory is complex

and cannot be explained using
vector model.
Decoupling
1H
selection angle
The intensity of CH, CH2 et CH3 signals varies as a function of selection angle .
C
H
C
H3
C
H2
ex: DEPT135: CH3 and CH signals are antiphases relative to CH2 signals
All information due to 1H-13C coupling is lost in BBD 13C spectra (which have better S/N ratios and are simplified).
The appropriate choice of DEPT spectra enables ones to retrieve information on the nature of the carbons
(primary, secondary, tertiary or quaternary).
148
149
8.5. Multidimensional NMR

8.5.1. Basic principle
1D pulse sequence:
FT
FID = S(t)
C()
90x
90x
At
Rd
pulses acquisition (t)
150
2D pulse sequence : FID = S(t1, t2) FT twice
C(1, 2)
t2 = acquisition time domain; direct detection

t1 = evolution time domain; indirect detection
The principle is to record a series of 1D spectra, each one with a different t1 which is incremented
by a delay called the dwell-time.
preparation
period
evolution
time: t1
mixing
period
acquisition
time: t2
The preparation and mixing periods are composed of pulses and/or fixed time periods.
(For 3D, 4D spectra, additional evolution times are introduced.)
Utility of multidimensional spectra :

Spread the information out over several frequencies and map out interactions
within or between molecules of interest.
The interactions that can be probed can be divided according to the following physical phenomena :
- through bond coupling (homo- or hetero-nuclear);
- through space coupling (nOe);
- chemical exchange.
151
8.5.2. A simple 2D experiment

90x
90-y
t1
t2
The preparation and mixing periods are both composed of a simple 90 pulse.
The t1 delay is incremented by the dwell-time t
t2
t1 = 0
t2
t1 = t
t2
t1 = 2 t
etc.
152
Evolution for a single uncoupled spin
FT(t2)
90-y
t1 = 0
yrot
yrot
xrot
xrot
t1 = t
yrot
= 2t1
yrot
xrot
xrot
t1 = 2t
yrot
yrot
xrot
xrot
t1 = 3t
yrot
yrot
xrot
xrot
153
The amplitude modulation of the singlet resonance is periodic as a function of t1: interferogram.
The amplitude is diminished by spin relaxation (T2).
t1
2
t1
I = I 0 cos(2t1 ) et1 T2
After t1 FT : 2D spectrum
2D spectrum for a sample containing a single uncoupled spin :

one peak with a shift of Hz in both dimensions
154
2D spectrum for a sample containing two

uncoupled spins A and X. Each produces a
peak at its corresponding frequency
in both dimensions.
The equivalent contour plot representation
with the 1D spectra along the frequency axis.
Ref: Claridge, 2000
155
8.5.3. Examples of 2D pulse sequences and spectra

COSY (COrrelated SpectroscopY; homonuclear shift correlation)
90x
90x
t1
t2
Diagonal : 1D spectrum
Crosspeaks : provide evidence of J coupling between the correlated spins
Coupling between 3 and 4
Coupling between 2 and 3

500 MHz (H,H) COSY Spectrum of
Glutamic acid in D2O.
1-D spectra left and top
156
HSQC (Heteronuclear Single Quantum Correlation)
90
180
/2
90
180
90
/2
180
/2
/2
+/t2
1H
180
90+
-
90
t
180
13C
15 N
+-Decoupling
Correlates coupled heteronuclear spins across a single bond;

Identifies directly connected nuclei, most often 1H-13C and 1H-15N;
Employs inverse-detection: detection via the high-sensitive nuclei (1H generally).
157
HMQC (Heteronuclear Multiple Quantum Correlation)

90
180
D
D
+-
1H
t2
90+-
90
t1
13C
+Dcouplage
Decoupling
Variant of HSQC : experimentally more robust sequence

but provides poorer resolution and is less sensitive.
158
1H
13
159
750MHz 1H-15N HSQC spectrum of human alpha-lactalbumin
160
NOESY (NOE SpectroscopY)

90x
90x
90x
tm
t1
t2
Diagonal : 1D spectrum
Correlation peaks: indicate NOE interaction between the correlated peaks which arise from
transfer of magnetisation between the spins during the mixing time tm.
A
B
B
Schematic representation of the NOESY spectrum of a molecule
whose 3D conformation brings A in close proximity to B.
161
162
Vb. Mass spectrometry

1. Introduction
2. Data interpretation
(Instrumentation see LC-MS)
163
V. Mass spectrometry
1. Introduction
With mass spectrometry it is possible to determine, with a very small amount of matter, the molecular
weight of molecules (strictly speaking the mass/charge ratio of ionized molecules in the gas phase),
the molecular formula of unknown compounds and also obtain information on the connectivity within
the molecule.
1.1. Principle
Seminal experiment was made by Wien in 1898.
Sample is brought into the gas phase and bombarded with high energy electrons, leading to the
ejection of one or more electrons from the substrate and potentially to its fragmentation.
e
+
target molecule
radical cation
The cations and radical cations formed are accelerated by a difference in potential (accelerating
voltage, V ) and deflected by a magnetic field, H, according to their mass/charge ration (m/z).
164
The molecular ion is the charged unfragmented molecule.

The m/z ratio directly yields de molecular weight of the compound.
e-
2 e-
molecular ion
The energy of the incident electrons is relatively high (~70 eV) and causes further
fragmentation of the molecular ion. These fragments can be more abundant than the
molecular ion.
The relative intensities in a mass spectrum are calculated relative to the base peak, which
is the most intense peak to which a normalized intensity of 100% is assigned.
Relative
intensity
(%) 100
fragments
base
peak
molecular ion
m/z
165
1.2. The spectrometer

Mass spectrometry is the ensemble of methods where analytical information is
obtained from the m/z ratio of the different species present.
The ions must be in the gas phase and under a strong vacuum (~10-6 torr) in order to
avoid collisions between molecules/fragments.
All spectrometers present the following features :
166
The source : Can be classified into two groups: high-energy and low-energy .
Characteristics of high energy sources
- Ions formed are in an excited state relaxation fragmentation.
- Generally nebulisation occurs prior to ionization
Convenient for molecules with low boiling temperature (< 500 C)
Convenient for molecules of low molecular weight (< 1000 Da)
Electron impact ionization (EI)
Characteristics of soft sources

- Very little fragmentation
-Can be used to characterize non-covalent complexes
-When desorption methods are used (direct conversion of the sample into gas phase ions)
not limited to molecules of low molecular weight (can go to MW of 105 Da )
Chemical ionization (CI), Fast Atom Bombardment ionization (FAB), Matrix-Assisted Laser
Desorption Ionization (MALDI), Electrospray Mass Spectrometry (ESMS),
167
Comparison of the spectra of decanol obtained using 2 different sources
High energy ionisation
Soft ionisation
Information obtained is complementary for the determination of molecular structures.
168
The mass analyzer : Separates the mixture of ions generated during the ionization step
according to their m/z ratio.
The resolution of the analyzer gives the ability to distinguish in the spectrum two peaks of slightly
different mass-to-charge ratios M
R=
M
M
Examples :
R = 2000
Instruments capable of distinguishing/separating 1999 and 2000
R = 10000
Instruments capable distinguishing/separating 500,00 et 499,95
High resolution requires the use of very small slit widths : loss of sensitivity.
169
Low resolution spectrum of compound P

(R= 600)
High resolution spectrum of compound P

(R= 3400)
Figure 2. MALDI mass spectra of substance P in CHCA comparing mass resolution obtained in the linear
(upper) and reflecting (lower) modes utilizing continuous ion extraction at threshold laser irradiance. 25 kV
accelerating potential, 50 laser pulses averaged.
http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html
170
2. Data interpretation
2.1. Fragmention
When ions formed are in an vibrational and rotational excited state, fragmentation can occur.
.
Fragmentation of the radical cation molecular ion ( M+ ) can be homolytic or heterolytic.
H3C
CH2
CH3CH2CH2
CH3
Br
CH2
CH3CH2CH2+
Br
The resulting cations can undergo further fragment heterolytic fragmentation (homolitic
fragmentation is not thermodynamically favoured as it leads to the formation of a radical and
a radical cation).
H3C
CH2
CH2
CH3
H2C
CH2
171
Predicting the different possible fragments is difficult but certain rules can be
proposed, based on physico-chemical considerations, to predict prominent
peaks in EI spectra.
1 Cleavage is favoured at alkyl substituted carbons: the more substituted the more likely the cleavage.
This is a consequence of the increased stability of substituted carbocations.
+
+ C
Cation stability order :

CH3+ <
RCH2+ < R2CH+ < R3C+
Generally the largest substituent is eliminated most readily as a radical (can achieve some stability
by delocalisation of the lone electron).
Saturated cycles have a tendency to loose their side-chains.
172
2 Double bonds favor allylic cleavage and give the resonance-stabilized allylic carbocation.
+
-R
H2C
CH
CH2
H2C
CH
H2C
CH
CH2
CH2
3 Unsaturated rings can undergo a retro-Diels Alder reaction.
+
C
+
C
4 Cleavage is often associated with the elimination of small, stable neutral molecules such as :
CO, H2O, NH3, HCN, CH2CH2 .
173
5 In alkyl substituted aromatic compounds, cleavage is very probable at the bond to the ring, giving
the resonance stabilized benzyl ion, or more likely, the tropylium ion.
+
CH2
CH2+
CH2
R
-R
+
+
6 The C - C bonds next to a heteroatom are frequently cleaved, leaving the charge on the fragment
containing the heteroatom whose non-bonding electrons provide resonance stabilization.
Y = O, NH ou S
- CH3
H 3C
CH2
H 2C
H 2C
174
Examples of spectra obtained by Electron Impact Ionization
pentan-2-ol
pentan-1-ol
31
CH2O+H
42
M (H2O + CH2=CH2)
55
M (H2O + CH3)
70
M - H 2O
55
M (H2O + CH3)
70
M - H 2O
Different fragmentation patterns for these two alcohols which are isomers !
73
M CH3
175
2.2. Rearrangements
Rearrangement ions are fragments whose origin cannot be described by simple cleavage of bonds in
the molecular ion.
Rearrangements involving migration of hydrogen atoms in molecules that contain a heteroatom are
especially common.
The most important example is the McLafferty rearrangement.
CR2
CH2
- R2C
C
H2
CH2
Y
.O
CH2
CH2
+C
CH2
The McLafferty rearrangement requires the presence of an appropriately located heteroatom, a system
with an abstractable hydrogen atom in the position.
+
Random rearrangement of
hydrocarbons can also be observed :
CH3
+
H3C
CH3
C2H5
CH3
176
Example : methyl octanoate

McLafferty rearrangement product
177
2.3. Isotope peaks

Molecular species exist that contain the less abundant isotopes of the constituting atoms giving rise
to isotope peaks at M+1, M+2 etc.
p-chlorobenzophenone
Cl
Isotope peaks
178
Elements
Isotopes
Ab. Rel.
Isotope
Ab. Rel.
Isotope
Ab. Rel.
Carbon
12C
100
13C
1.11
Hydrogen
1H
100
2H
0.016
Nitrogen
14N
100
15N
0.38
Oxygen
16O
100
17O
0.04
18O
0.20
Fluorine
19F
100
Silicium
28Si
100
29Si
5.10
30Si
3.35
Phosphor
31P
100
Sulphur
32S
100
33S
0.78
34S
4.40
Chlorine
35Cl
100
37Cl
32.5
Bromine
79Br
100
81Br
98.0
Iodine
127I
100
Way to highlight the presence of heteroatoms
179
Relative intensity of isotopic peaks for different combinations of Br and Cl atoms

present in the molecule (relative to the intensity of the molecular ion).
Combinaison
M+2
M+4
M+6
M+8
M + 10
Br
97.9
Br2
195.0
95.5
Br3
293.0
286.0
Cl
32.6
Cl2
65.3
10.6
Cl3
97.8
31.9
3.47
Cl4
131.0
63.9
14.0
1.15
Cl5
163.0
106.0
34.7
5.66
0.37
Cl6
196.0
161.0
69.4
17.0
2.23
BrCl
130.0
31.9
Br2Cl
228.0
159.0
31.2
Cl2Br
163.0
74.4
10.4
M + 12
93.4
0.11
180
Graphical representation
181
Nominal mass, monoisotopic mass and average mass
Isotopic envelope of the molecular peak of the peptide HLKTEAEMK

Nominal mass 1086
The nominal mass is the mass calculated using the integer mass of the predominant (most abundant)
isotope of each element.
The monoisotopic mass is obtained by summing the exact masses of the predominant isotope for each
element (for small molecules it corresponds to the first peak in the envelope which is the most intense).
The average mass of a molecule is obtained by summing the average atomic masses of the constituent
elements.
182
2.4. Determination of the molecular formula

2.4.1. The molecular ion
The molecular ion corresponds to the intact molecule (no fragmentation).
The corresponding m/z ratio directly yields the molecular weight of the compound.
Certain precautions are necessary to ensure that the molecular ion is
correctly identified :
1 With EI sources, the molecular ions can be absent or of very low intensity. Other ionisation
techniques are then required.
2 Measuring the position of the peaks corresponding to the molecular ion with precision requires the
use of high resolution (HR) spectrometers which are able to detect mass differences of a few
1,000 th of a unit.
Low resolution spectrometers can only discriminate between ions which differ by at least on weight
unit.
183
2.4.2. Molecular formula using HR spectrometers

The exact position of the molecular ion peak gives access to the
molecular formula.
Example :
Measured m/z ration of the molecular ion: 120, 070 ( 0.005)
Possibilities :
C5H4N4 : m = 120.044
C7H8N2 : m = 120.069
C9H12 : m = 120.096
C 7H 8N 2
C8H8O : m = 120.058
Nitrogen rule : organic molecules of even molecular weight contain an even number of
nitrogen atoms (for molecules composed only of CHNO atoms).
184
2.4.3. Molecular formula using isotope peaks

The presence of isotope peaks enables to distinguish between compounds which
have molecular weights which only differ by a few 1000th of a unit, even if their
spectra are recorded on a low resolution spectrometer.
Example :
Dinitrobenzene C6H4N2O4 and the alkene C12H24 both have a molecular weight of
168 g/mole.
The (M + 1)/M ratio of the two compounds can be calculated using the isotopic
abundances of 13C, 2H, 15N and 17O :
for C6H4N2O4
for C12H24
13C
6 * 1.11 = 6.66 %
12 * 1.11 = 13.32
2H
4 * 0.016 = 0.06 %
24 * 0.016 = 0.38
15N
2 * 0.38 = 0.76 %
17O
4 * 0.04 = 0.16 %
7.64 %
Measuring the relative intensities of

the M and (M +1) peaks it is
possible to distinguish between the
two compounds.
13.70 %
185

CHIM H407 Part B MolStructure 2016

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CHIM H407 Part B MolStructure 2016

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Molecular Structural

Characterization and Analysis

Molecular Structure Characterization

Spectroscopy: General considerations

Practicals and exercises illustrating both parts

Spectroscopy : General considerations

Ib. Spectroscopy : General

Representation of the electronic, vibrational and

The nuclear and electronic spin levels are also quantized.

The transitions depend on the wavelength of the emitting source.

X-ray spectroscopy (= 0,1-100 ):

UV absorption and emission

NMR spectrum of isobutylamine

2.2. Transition probabilities and Einstein coefficients

Transition probability per unit of time

The rate of induced absorption from a fundamental to an excited state :

= d/d : radiation density at a given frequency

N F (t ) = population of the fundamental state

FE = transition dipole moment

BEF : Einstein induced emission coefficient

The rate of spontaneous emission from an excited to a fundamental state :

AEF : Einstein spontaneous emission coefficient

The net variation of a state is given by :

Considering the radiation density of a blackbody (Plancks law) :

IIb. Electronic spectroscopies

II. Electronic spectroscopies

Absorption spectrum of Na vapour :

For a molecule, the number of transitions is

In the condensed phase, the fine structure is no

II. Electronic spectroscopies

1.2. Beer-Lambert Law

I0 = intensity of incident light

= molar absorptivity constant

c = concentration of the absorbing species

II. Electronic spectroscopies

1.3. Franck-Condon Principle

Classical Formulation : Vertical Transition

II. Electronic spectroscopies

II. Electronic spectroscopies

Transition rates of spontaneous and stimulated emission

Knowing the ratio between the Einstein coefficients and

II. Electronic spectroscopies

2.1. Fluorescence and phosphorescence

Fluorescence: spontaneous emission from

Phosphorescence: spontaneous emission from

(The Franck-Condon principle also applies in emission.)

II. Electronic spectroscopies

2.2. Jablonski diagramme

II. Electronic spectroscopies

2.3. Emission intensity

II. Electronic spectroscopies

2.4. Stokes shift

This overlap is however rare :

the spectra rarely exhibit any fine structure;

The Stokes shift :

II. Electronic spectroscopies

: 103 - 104 l.cm-1.mol-1

200 < < 700nm

Allowed Transitions : > 103 l.cm-1.mol-1

* are difficult to exploit in solution.

II. Electronic spectroscopies

Section of molecule which can undergo electronic transition

Shift to a longer wavelength due to substitution or solvent

Shift to a shorter wavelength due to substitution or solvent