Organic chemistry I
Miroslav Zabadal
Chapter 5
Organic structure analysis
Organic structure analysis
•
•
•
infrared spectroscopy (IR)
nuclear magnetic resonance (NMR)
mass spectrometry (MS)
Literature
• Anslyn E. V., Dougherty D. A.: Modern Physical Organic Chemistry, 2004,
University Science Books, Sausalito, California U.S.A.
• Crews P., Rodríguez J., Jaspars M.: Organic Structure Analysis, 1998,
Oxford University Press, New York U.S.A.
• Solomons T. W. G., Fryhle C. B.: Organic Chemistry, 2004, 8th Ed., John
Wiley & Sons Inc., U.S.A.
• Clayden J., Greeves N., Warren S., Wothers P.: Organic chemistry, 2001,
Oxford University Press, New York U.S.A.
• Voltrová S.: Examples for lessons of structure analysis of organic
compounds, 1996, VŠCHT Praha.
• Prof. M. Holík: Lecture “Applied NMR spectroscopy”, 1997, MU in Brno.
• Doc. R. Marek: Lecture “NMR structure analysis”, 1998, MU in Brno.
• http://www.cis.rit.edu/htbooks/nmr/inside.htm
• http://www.chem.ucla.edu/~webspectra/index.html
• http://www.spectroscopynow.com/
• http://www.fch.vutbr.cz/ictep/index.php?fun=studium&file=studijni_material
y/ISA-NMR/ISA_PISA-NMR
•
IR – frequency of bonding vibrations are characteristic for the
corresponding functional groups in molecules
MS – fragmentation of molecules and the measurement of specific
MW fragments
NMR – detection of 1H signals, carbons and others atoms and
supports identification of conectivity, isomers, etc.
•
•
Electromagnetic radiation
•
typical parts of elmag. radiation and interference with molecule:
-
•
cosmic and γ-ray (nuclear emission)
X-ray radiation (ionization of atoms)
UV-VIS (excitation of electrons)
infrared (molecular vibrations)
microwaves (molecular rotations)
radiowaves (nuclear absorption)
photon energy :
E = hν = hc/λ
whereas c = λν
h – Planck’s constant [≈ 6,626 × 10-34 J.s],
c – speed of light
Interaction time
•
comparison of microscopic and macroscopic events on the time line:
age of
man
fluorescence phosphorescence
τ = 10-15 10-12 10-9 10-6
k = 1015 1012 109
electron
excitation
bonding
rotations
bonding
vibrations
106
106
10-3 100
103
103
10-3 10-6
100
fast
chemical
reactions
109
age of
Earth
age of
universe
1012 1015 1018 [s]
10-9 10-12 10-15 10-18 [s-1]
slow
chemical
reactions
translation
of big
molecules
& the shortest time of electron migration between energy levels is 10-16 s
(→ no chemistry in shorter time )
Interaction methods
•
-
application of two fundamental types of interactions :
Absorption – the irradiation is absorbed by atom, molecule or ion,
and more energetic specie is established
Emission – de-excitation of quantum of the light by atom, molecule
or ion, and its deactivation (less energetic specie)
-
Conception of spectrum
Absorption spectrum
Emission spectrum
Excitation spectrum
Absorption sp.
Excitation
and Emission sp.
Emission spectrum – excitace při jedné λmax
(abs.) a měření závislosti intenzity
emitovaného světla na λ
Excitation spectrum – excitace při různých λ1
− λn a měření intenzity fluorescence při
jedné λ
Jablonski diagram
1. Absorption – interaction of a specie with the irradiation (k ≈ 1015 s-1)
2. Fluorescence – radiative transition between excited states with same multiplicity
(k ≈ 106 - 109 s-1)
3. Phosphorescence – radiative transition between excited states with different
multiplicity (k ≈ 10-2 - 103 s-1)
4. Vibration relaxation – radiationless transition from higher vibration states to the
ground state with current heat output (k ≈ 1012 s-1)
5. Intersystem crossing – radiationless transition between vibaration levels of
electronic states with different multiplicity (k ≈ 106 - 1011 s-1)
6. Internal conversion – nonradiative decay between vibaration levels of electronic
states with the same multiplicity (k > 1012 s-1)
Infrared spectroscopy (IR)
•
•
the energy of IR irradiation (MIR: 6 – 46 kJ/mol) is insufficient for
electron exciation
the absorption is limited by transition between vibration and rotation
levels of molecule
molecular rotation is unrealizable in liquids and solids → vibration is
realized
IR data are represented by absorption or transmitance spectra
•
the absorption follows Lambert-Beer law:
•
•
I = I0 × 10-εdc
I0 – intensity of incident light; I – intensity of light pass through;
ε0 – molar absorption coefficient; d – thickness of analyzed sample (solution)
and c – its concentration
•
•
•
Absorbance (optical density):
A = log(I0 / I)
Transmitance:
T = I / I0
the energy of IR radiation corresponds the energy of bonding
vibrations:
Stretching
vibrations ν
symmetric
νs
asymmetric
νas
Bending
vibrations δ
rocking
scissoring
wagging
twisting
Skeletal
vibrations
accordion
Torsional
vibrations
Associates - dipole-dipole, H-bond interactions
circular
IR spectrum
•
IR spectra are too complex in due to many vibrations:
wavenumber [cm-1]
4000 - 1500 cm-1
Characteristic vibrations
(possible differentiation of each
vibration)
1500 - 200 cm-1
Finger Print
(overlap of vibrations)
•
the absorption bands in IR spectrum correspond on each vibrations of
the functional groups:
O
C
O H
C2H5
C2H5
H O
O
C C2H5
C
O
O H
intermolecular
H-bond
O
H
OCH3
O
H
intramolecular
H-bond
IR spectrometer
•
•
single-beam or double-beam spectrometer
dispersion × FT (Fourier-transform) IR spectroscopy
IR sample
•
•
•
gas, liquid or solid sample
sample: (l) or (g) is measured in cuvettes → transparent in detection
spectral region:
Material
ν~ (cm-1)
LiF
1600
BaF2
750
NaCl
600
KBr
370
AgCl
350
polyethylene
200 - 500
- 4000
preparation of sample (= tablet) - pressing of sample and material
that is plastic and transparent at high pressure (e.g. KBr, AgCl),
sample ~ 1mg
IR Sources
•
heating of inert solid material to temperature 1000 - 2200K that
emits infrared radiation:
-
Nernsts source – cylinder from oxides of metals of noble earths
(ZrO2 + oxides Y) + Pt wire; T = 2200 K; ν~ = 500 - 25000 cm-1
Globars source – karborundum (Si-C), electrically heated to 1100 –
1500 K; ν~ = 250 - 8300 cm-1
Heated Ni-Cr wire – electrically heated to temperature 1100 K; ν~ =
500 - 13300 cm-1
IR Detectors
•
three fundamental detectors:
-
-
Thermal – thermocouple (e.g. Bi + Sb); measurement of potantial
changes between metals
Pyroelectric – small plate from monocrystal of pyroelectric material
(e.g. triglycerine-sulfate), its polarizibility depends in electric field on
temperature
Photoelectric – evacuated vessel contains lazer of semiconductor
on glass surface (Hg-Cd-Te) → IR absorption → decrease of
electric resistance of semiconductor
Nuclear magnetic resonance (NMR)
•
•
structure analytic method
NMR method can be applied for atoms with nonzero nuclear spin:
isotope
Representation
in nature [%]
nuc. spin
(I)
1H
99.9844
1/2
2H
0.0156
1
11B
81.17
3/2
13C
1.108
1/2
17O
0.037
5/2
19F
100.0
1/2
29Si
4.700
1/2
31P
100.0
1/2
-
the nuclear spin depends on
composition of nucleus and nucleons:
• odd number of nucleons or even
number of nucleons with odd number
of neutrons and protons
→ nonzero nuclear spin
• even number of nucleons with odd
number of neutrons and protons
→ zero nuclear spin
isotopes with zero nuclear spin (e.g.
spectroscopy
12C, 16O)
are inactive in NMR
•
-
nuclear behaviour in the magnetic field :
vector of magnetic moment μ of nuclear
spin is spinning with Larmor’s frequency ν
→ generation of magnetic field around the
nucleus
• the application of external magnetic field B0 (magnetic induction)
resolves atoms into energy levels:
spin = + ½ (β)
a) vector μ in the same direction as B0 →
spin = - ½ (α)
b) vector μ in the opposite direction as B0 →
• the energy difference between spin states (+½ and -½; I = ½)
depends on the value of external mag. field B0 → increasing B0
increases energetic difference between spin levels
• population difference of nucleus in energetic levels +½ (Nα)
and -½ (Nβ) is small (excess nucleus → +½)
Nβ = 106
Nβ - Nα ≈ 100
při B0 = 12T, T = 300K
•
the application of the radiofrequency radiation (20-900 MHz) leads to
excitation of nucleus from +½ state into -½ state → absorption is
possible when resonance condition is fulfilled:
2πν = γB
•
ν – Larmor’s frequency
γ –gyromagnetic constant (constant
typical for each isotope)
B – magnetic induction
the difference between spin levels for nucleus I = ½ is equal to
magnetic moment μ in the case of constant B0:
isotope
μ [J/T]
ν (B0 = 2,35T)
[MHz]
1H
2,7927
100,0
13C
2,6273
25,3
19F
1,1305
94,1
31P
0,7022
40,5
15N
-0,1000
10,1
•
electrons interference with other electrons and atoms:
B0
B = B0
e-
B = B0 - σ B0
electrons
around
nucleus make a
mag.
field
that
affects
opposite
external mag. field
B0
→
lower
excitation energy
B0
- σ B0
B0
Bm Bm
e-
e-
B = B0 - σ B0 ± Bm
relative shieldning
of atoms → splitting
of signal
J
σ – shielding constant
J – coupling constant [Hz]
B0
NMR spectrum
•
Chemical shift δ – peak position in spectrum relative to internal
standard TMS (tetramethylsilane)
TMS
(CH3)4Si
δ = (νi / νs) × 106
δ = 0 ppm
[ppm]
νi – frequency of observed signal in comparison to TMS
νS – operating frequency of the instrument in Hertz
•
•
•
chemical shift δ is independent on B0 of the external magnetic field
therefore δ(H) benzene = 7.27 ppm is similar for 60Hz and 300MHz
NMR spectrometer
Coupling constant J – depending on external magnetic field B0
Multiplicity of signal (splitting) – 2N + 1 (N = number of nonequivalent nucleus of similar atoms ≈ max. over 3 bonds)
•
•
Number of signals – number of chemical non-equivalent nucleus
Intensity of signal – number of chemical equivalent nucleus for each
signal (integration of signal)
Intensity of peaks for splitted
signal of chem. equivalent
atoms (N = number of atoms)
→ stříškový efekt
I=½
1
1 1
1 2 1
1 3 3 1
1 4 6 4 1
I=1
1
1 1 1
1 2 3 2 1
1 3 6 7 6 3 1
N
0
1
2
3
4
NMR sample
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•
•
sample – liquid or solid state
solvents - deuterated liquids (e.g. CDCl3, DMSO-d6, D2O, benzened6, …)
sample is measured in glass cuvette (Ø 5 or 10mm)
NMR spectrometer
•
•
CW (continuous-wave) spectrometer – sample is continously
irradiated by radiofrequency radiation and magnetic field is variable
FT (Fourier Transform) NMR spectrometer – magnetic field is
permanent and sample is irradiated by radiofrequency pulses
Mass spectrometry (MS)
•
•
destructive structure analytic method
ionization of molecules and following separation and detection of ions
according to ratio m/z (mass(m)-to-charge(z))
ionization: e-, M+, hν (laser), plasm, …
ionization
source
fragmentation
M
M
L
+ N*
L
+ N*
Alison E. Ashcroft: Ionization Methods in Organic Mass Spectrometry, The Royal
Society of Chemistry, UK, 1997 and references cited therein.
•
ions are too reactive → generation and handling in vacuum 10-5 - 10-8
torr (1 atm ≈ 760 torr) → without hitting with other molecules (e.g. air)
MS Sample
•
•
•
gas phase → evaporation (at high temperature)
direct ionisation of sample in MS → disposition of sample (solid or
liquid) in ionisation compartment
integration of MS spectrometer in HPLC, GC or CE → separation of
sample and following analysis of components by MS → sample =
gas, liquid or solid
Ionization
•
several ionisation methods → application depends on sort of sample:
-
EI (electron impact) – e- bombardment of sample, that induces
ionisation of molecule or fragmentation (higher energy)
-
vacuum ~ 10-7 torr
energy of e- ~ 40-70 eV
ionisation of molecule
and only partial
fragmentation
-
ionisation of molecule
and most of molecules
is fragmentated
CI (chemical ionisation) – sample bombardment with M+ = ions of
another molecule that are produced by different methods
-
M+ = CH3+, C2H5+, H3O+, H2+, HeH+, NH4+, ...
sensitive sample ionisation method → only few molecules
are fragmented
M
+
NH4+
→
MH+
+
NH3
-
FI (field ionisation) & FD (field desorption) – sensitive ionisation
method, sample is introduced to electrode surface with positive
potential → electric field ionisation (107-108 V/cm)
-
-
FI - sample in gas phase
FD - solid sample is introduced directly to electrode
surface → desorption and ionisation
LD (laser desorption) – direct ionisation of sample in solid state by
laser (hn) and subsequent secondary ionisation by heating
sample is evaporated by laser (e.g. N2 pulse laser) and
ionisated in gas phase
- fragmentation of molecules depends on energy of laser
beam (N2 laser, l = 337nm)
MALDI (matrix assisted laser desorption ionisation)
- sample is mixed with matrix (material with high ε) →
secondary ionisation of sample by matrix ions
-
matrix - e.g. 4-hydroxya-cyanocinnamic acid for
AA, peptides, NA
-
ESI (electrospray ionisation) – dissolved sample in volatile solvent is
introduced with narrow capillary (Ø 75-150 μm, stainless steel), high
voltage is on spike (3-4 kV) → formation of aerosol and current
ionisation
liquid particles of aerosol are stopped by drying gas (e.g.
hot N2) outside of input into another parts of spectrometer
Analysis and ions deflection
•
ions are accelerated between slits with decreasing potential (from
104 V to 0 V) into a finely focused beam
acceleration of ions
ionisation
chamber
104 V
ions
beam
104 V ~102 V 0 V
•
hmotnostní analyzátory:
-
-
Magnetic separation – different ions are deflected by the
magnetic field by different amounts (radius of curvature ~ mass
and charge of ion, intensity of magnetic field)
TOF (time of flight) – accelerated ions are separated by different
time of flight depending on ratio m/z (motion equations)
Quadrupole – change of amplitude in rods with direct voltage →
only one ion type has fixed trajectory depending on m/z
Quadrupole analyzer
Magnetic analyzer
MS Detectors
•
ions impacts on photoactive surface:
- Photographic plate
ions collide with the walls
where they will pick up
electrons and be neutralized
and
electric
current
is
registered
MS spectrometer
•
fundamental segments of spectometer:
- Photomultiplier tube
photoemission cathode
produces electrons after
collision of ions with wall →
dynodes
GC-MS
•
•
•
integration of gas chromatograph (GC) and mass spectrometer
(MS):
GC – separation of each sample compopnents
MS – detection of components and their identification
MS spectrum
•
the plot of signal intensity (amount in sample) on mass/charge =
m/z:
Main peak = 100 rel. int.
(ion peak with the higher
intensity)
Molecular peak = M+
(ion non-fragmentated molecule)
Organic structure analysis
•
the strategies of determining structure of organic molecules:
MS, NMR
Pure
compound
NMR, IR,
UV
MS, NMR
Working
2D structures
Molecular
formula
Functional
groups
Unsaturation
number
List of working
2D structures
Substructures
3D molecular
structure
draw all
2D isomers
draw all
3D isomers
NMR, IR,
MS
Final
2D structures
•
Unsaturation number (UN) – the sum of the number of multiple bonds
and rings present, calculated from molecular formula:
UN =
[(2a + 2) − (b − d + e)]
2
for a compound
CaHbOcNdXe
(X = F, Cl, Br, I)
Determining structure of unknown compound
•
deriving structure from spectroscopic data:
Exact MS (EI) = 162,0681