Identification of Flavor Compound by Spectrometric Methods

1
Identification of Flavor Compound by
Spectrometric Methods
2
Gas Chromatogram of Flavor Compounds
3
Identification of Compound
O
H3C
CH3
H
H
H2
CH3
H
H2
H
CH3
H
4
SPECTROMETRIC IDENTIFICATION
I. Introduction of Spectrometric
Analyses II. Ultra Violet Spectrometry III.
Infrared Spectrometry IV. Nuclear Magnetic
Resonance Spectrometry V. Mass Spectrometry
5
I. Introduction of Spectrometric Analyses
The study how the sample interacts with
different wavelength in a given region of
electromagnetic radiation is called spectroscopy
or spectrochemical analysis. The collection of
measurements signals (absorbance) as a function
of electromagnetic radiation is called a spectrum.
6
Spectrum of Radiation
7
Electromagnetic Spectrum.
8
Energy Absorption
The mechanism of absorption energy is different
in the Ultraviolet, Infrared, and Nuclear
Magnetic Resonance regions. However, the
fundamental process is the absorption of certain
amount of energy. The energy required for the
transition from a state of lower energy to a
state of higher energy is directly related to the
frequency of electromagnetic radiation that
causes the transition.
9
Spectral Distribution of Radiant Energy
-1
V' Wave number (cm
)
l
Wave length (nm)
V Frequency of Radiation (cycles/sec)
V
1
V'
C
l
-27

(The energy of photon) E V
h
(Planck's Constant 6.62
10
erg - sec)
C
E V
h

h
1 x 107 erg I joule 0.239 calorie Avogadros
number 6.02 x 10 23mol-1
l
l
C V
C
V
l
10
Spectral Properties, Application and Interactions
of Electromagnetic Radiation
Type Quantum Transition
Type spectroscopy
Type Radiation
Frequency ?
Wavelength ?
Wave Number V
Energy
Hz
cm
cm-1
Kcal/mol
Electron volts, eV
Gamma ray
Gamma ray emission
Nuclear
X-ray absorption, emission
Electronic (inner shell)
X-ray
Ultra violet
Electronic (outer shell)
UV absorption
Visible
Infrared
IR absorption
Molecular vibration
Molecular rotation
Microwave absorption
Micro-wave
Magnetically induced spin states
Nuclear magnetic resonance
Radio
11
Molecular Orbital of Triplet Oxygen
Molecular
Atomic
Atomic
s

p

p

p
p
2Pz
2Py
2Px
2Px
2Py
2Pz
s
Energy
s

2S
2S
s
s

1S
1S
s
12
Quantum Numbers and Orbitals
Principal quantum number (n) The average
distance of the electron from the nucleus. 1, 2,
and so on. Azimuthal quantum number (Momentum
quantum number) (l) The shape of the orbital.
1s, 2s, 2p, 3s, 3p, 3d, etc Magnetic quantum
number (Orientational quantum number) (ml) The
orientation of orbital in the space. 2px 2py, 2pz
x, y and z are orientational quantum
number Electron spin quantum number (ms) The
two possible orientation of the electron in a
magnetic field. 1/2 or 1/2
13
One S and Three P Orbitals
14
The 5 d Orbitals
15
II. Ultra Violet Spectrometry
The absorption of ultraviolet radiation by
molecules is dependent upon the electronic
structure of the molecule. So the ultraviolet
spectrum is called electronic spectrum.
16
Spectral Properties, Application and Interactions
of Electromagnetic Radiation
Type Quantum Transition
Type spectroscopy
Type Radiation
Frequency ?
Wavelength ?
Wave Number V
Energy
Hz
cm
cm-1
Kcal/mol
Electron volts, eV
Gamma ray
Gamma ray emission
Nuclear
X-ray absorption, emission
Electronic (inner shell)
X-ray
Ultra violet
Electronic (outer shell)
UV absorption
Visible
Infrared
IR absorption
Molecular vibration
Molecular rotation
Microwave absorption
Micro-wave
Magnetically induced spin states
Nuclear magnetic resonance
Radio
17
Electronic Excitation
The absorption of light energy by organic
compounds in the visible and ultraviolet region
involves the promotion of electrons in ?, ?, and
n-orbitals from the ground state to higher energy
states . This is also called Energy Transition.
These higher energy states are molecular orbitals
called antibonding.
18
Types of Bonds
Antibonding
s

p
Antibonding





p
s
p
s




Energy

p
n
s
n


Nonbonding
n
Bonding
p

Bonding
s

19
Electronic Molecular Energy Levels
The higher energy transitions (? ??) occur a
shorter wavelength and the low energy transitions
(???, n ??) occur at longer wavelength.
20
Electronic Ground and Excitation States
s
s


s
s
hv
Energy

p
p
p

p
p

p


hv
hv
p
p
p


p
hv
n
n
21
Chromophore
Chromophore is a functional group which absorbs a
characteristic ultraviolet or visible region.
210 nm Double Bonds 233 nm Conjugated Diene
268 nm Conjugated Triene 315 nm Conjugated
Tetraene
22
III. Infrared Spectrometry
Radiation energy in the infrared region is
absorbed by the organic compound and converted
into energy of molecular vibration. The energy
absorption pattern thus obtained is commonly
referred to as an infrared spectrum which has the
plot of intensity of radiation absorption versus
wavelength of absorption.
23
Spectral Properties, Application and Interactions
of Electromagnetic Radiation
Type Quantum Transition
Type spectroscopy
Type Radiation
Frequency ?
Wavelength ?
Wave Number V
Energy
Hz
cm
cm-1
Kcal/mol
Electron volts, eV
Gamma ray
Gamma ray emission
Nuclear
X-ray absorption, emission
Electronic (inner shell)
X-ray
Ultra violet
Electronic (outer shell)
UV absorption
Visible
Infrared
IR absorption
Molecular vibration
Molecular rotation
Microwave absorption
Micro-wave
Magnetically induced spin states
Nuclear magnetic resonance
Radio
24
Some Molecular Vibrations
25
Atom, Group, and Molecular Rotations
Z
Y
H atom rotation
COOH group rotation
O
H
H
C
X
Molecular rotation
C
O
CH
group rotation
H
H
3
OH group rotation
Center of gravity of the
molecule is at the origin
26
Infrared Spectrum
27
Infrared Absorption and Functional Groups
3.4 ?m Alkane 6.0 ?m cis-Double Bond 10.3
?m trans-Double Bond 5.8 ?m Carbonyl 3.7
?m Hydroxyl Stretching of Acid Group 2.9
?m Hydroxyl
28
Nuclear Magnetic Resonance Spectrometry
29
Identification of Compound
O
H3C
CH3
H
H
H2
CH3
H
H2
H
CH3
H
30
Spectral Properties, Application and Interactions
of Electromagnetic Radiation
Type Quantum Transition
Type spectroscopy
Type Radiation
Frequency ?
Wavelength ?
Wave Number V
Energy
Hz
cm
cm-1
Kcal/mol
Electron volts, eV
Gamma ray
Gamma ray emission
Nuclear
X-ray absorption, emission
Electronic (inner shell)
X-ray
Ultra violet
Electronic (outer shell)
UV absorption
Visible
Infrared
IR absorption
Molecular vibration
Molecular rotation
Microwave absorption
Micro-wave
Magnetically induced spin states
Nuclear magnetic resonance
Radio
31
Number of signals Position of signals Intensity
of signals Splitting of signals
4
3
14
2
2
4
1
2
2
2
2
4
32
Nuclear Spins in Absence (a) and Presence (b) of
External Magnetic Field
CH3-CHCH-CH2-CHO
33
Methyl Ester of Fatty Acid
O
C
H
C
C
H
O
R
C
H
C
H
C
H
C
H
C
H
3




2
2



34
Nuclear Magnetic Resonance Principle
Precession orbit of nuclear
mass (Precession angular velocity or precession
frequency W0)
W0 2p v
Spinning proton
Nuclear magnetic dipole moment ? ghI
Ho
Spinning charge in proton generates magnetic
dipole moment
Ho
Proton precess in a magnetic field Ho
35
Principles of NMR
2p radian/sec 1 Hz, 1 sec 3 1010 cm
High energy precession
Precessional orbit
Axis of nuclear rotation

Low energy spin state (1/2)
Nuclear Spin (Dipole moment)
Low energy precession
Nuclear Spin (Dipole moment)
Reference axis
Energy Difference
Oscillator Coil (Radio Frequency)
Ho
Precessional orbit
High energy spin state (-1/2)
CH3-CHCH-CH3
Ho
CH4
Precession -Energy Relationship
Oscillator generates rotating component of
magnetic field
36
Types of Bonds
Antibonding
s

p
Antibonding





p
s
p
s




Energy

p
n
s
n


Nonbonding
n
Bonding
p

Bonding
s

37
Magnetic Properties of Nuclei
Nuclei of certain atoms posses an angular
momentum. The total angular momentum depends on
the spin number (spin quantum number I). The
spin number ( I ) is related to the mass number
and the atomic number. Each proton has its own
spin and I is a result of these spins.
38
Fundamental NMR Equation and Magnetic Field
Strength
The energy difference between the high energy
spin state and low energy spin state is V
?H0/ 2?, W0 2?V is precessional frequency As
H0 increases, precessional frequency
increases. ? (Magnetogyric Ratio) a
fundamental nuclear constant 267.512 x 106
radians T-1s-1 V Electromagnetic frequency in
radio frequency H0 An external magnetic
field W0 ?H0 , ?H0 2?V, Therefore W0
2?V W0 Precession frequency (Larmor
frequency)
39
Nuclear Magnetic Dipole Moment Angular
Momentum
Nuclear magnetic dipole moment is from the
rotating nuclear charge. Angular momentum is from
rotating nuclear mass.
? (Magnetogyric Ratio) a fundamental nuclear
constant 267.512 x 106 radians T-1s-1 V
Electromagnetic frequency in radio frequency H0
An external magnetic field
40
Relationship between Radio Frequency and
Magnetic Field Strength for Proton
Radio Frequency (Mega Hertz) Magnetic Field
(Gauss) 60 14,100 100 23,500 300
70,500 500 117,500
41
Energy Difference between Spin States as a
Function of Magnetic Fields Strength
Energy
42
Relationship between Applied Magnetic Field
Radiofrequency
?E hv
4.7 T 200 MHz
1.4 T 60 MHz
2.35 T 100 MHz
7.0 T 300 MHz
43
Schematic Diagram of NMR Spectrometer
Sweep coils
Sample



R-F receiver


R-F transmitter


R-F detector


Recorder
Transmitter coil
Receiver coil


Sweep generator
Magnet
44
Chemical Shift
The difference in the absorption frequency of a
particular proton of the sample from the
absorption frequency of a reference proton. ?
ppm
(Reference frequency - Sample frequency ) ? 106
Operating instrument frequency
45
Chemical Shift
The protons at the electron rich environments
will feel less external magnetic field strength
because the magnetic field strength generated by
electrons surrounding the proton will counteract
the applied magnetic field strength (Ho)
46
Chemical Shift
The Wo (Precessional angular velocity- Larmor
frequency) of the protons in the electron
rich chemical environments will be less and
require less radio frequency to be resonance
with the applied radio frequency compared to the
protons in the electron poor chemical
environments.
47
Reference Compound TetraMethylSilane (TMS)
d 0
48
Absorbance Frequency
60 MHz----from 59,999,280 Hz to 60,000,000
Hz 14,100 Gauss 300 MHz--- from
299,996,400 Hz to 300,000,000 Hz 70,500
Gauss 600 MHz---from 599,992,800 Hz to
600,000,000 Hz 141,000 Gauss
O
C
H
C
O
C
H
R
C
H
C
H
C
H
C
H
C
H







2
3
2
5.3
d
2.7
3.6
d
d
49
General Regions of Chemical Shifts
Aliphatic alicyclic
?-Substituted aliphatic
Acetylenic
?-Monosubstituted aliphatic
?-Disubstitutid aliphatic
Olefinic
Aromatic and heteroaromatic
Aldehydic
TMS
0 ?
1
3
4
5
6
10
2
7
8
9
50
Spin-Spin Coupling (Spin-Spin Splitting)
Spin-Spin Coupling is the indirect coupling of
proton spins through the intervening bonding
electrons. It occurs because there is some
tendency for a bonding electron to pair its spins
with the spin of the nearest protons. The spin
of a bonding electron having been thus
influenced.
51
Spin-Spin Coupling (Spin-Spin Splitting)
Coupling is ordinarily not important beyond 3
bonds unless there is ring strains as in small
rings or bridged systems, or bond delocalizaion
as in aromatic or unsaturated systems
52
Spin-Spin Splitting
Signal a is split into a doublet by coupling with
one proton signal b is split into a triplet by
two protons. Spacing in both sets is same (Jab).
a
b
b
a
Jab
Jab
Jab
0 ?
10
53
Number of signals Position of signals Intensity
of signals Splitting of signals
4
3
14
2
2
4
1
2
2
2
2
54
a e
e c e
e b
O
C
H
C
H
C
H
C
H
(
C
H
C
H
C
H
)
C
H
(
C
H
)
C
H
C
3
2
2
2
2
2
5
2
CH3
O
d
a
0.97
b
1.33
c
2.80
d
3.67
e
5.38
55
Information from NMR Spectrum

• Number of signals
• Position of signals
• Intensity of signals
• Splitting of signals

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(No Transcript)
57
Summary

• As external applied magnetic filed increases
• Spinning proton magnetic dipole moment
• increases
• spinning proton angular momentum increases
• proton precession frequency increases
• the energy difference between high energy spin
  state and low energy spin state increases

58
The magnetic nucleus may assume any one of ( 2 I
1) orientations with respect to the directions
of the applied magnetic field. Therefore, a
proton (1/2) will be able to assume only one of
two possible orientations that correspond to
energy levels of or - ? H in an applied
magnetic field, where H is the strength of the
external magnetic field.
59
If proper v is introduced, the Wo will be
resonance with the properly applied radio
frequency (Hi) and the proton will absorb the
applied frequency and will be raised to the high
energy spin state. Even though the external
magnetic field strength (Ho) applied to the
molecule is the same, the actual magnetic field
strength exerted to the protons of the molecule
are different if the protons are in the different
electronic chemical environment.