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Introduction
Nuclear Magnetic Resonance, discovered 1946 Purcell & Bloch
EnergieEo Grundzustand
E1 Angeregter Zustand Absorption
Emission
Difference in energyE1 - E0 = h.ν
Frequency (Hz)ν = c / λ
BASICS in NMR-Spektroskopie
h...Planck‘sconstant6,63 . 10 -34 Js
I = 0 for nuclei of even mass- and atomic numbers, the even-even nucleie.g. 12
6C, 168O, 32
16S
Isotope spin I abundance (%) γ (gyromagnetic ratio)__________________________________________________
1H 1/2 99.98 26.82H 1 0.016 4.113C 1/2 1.108 6.719F 1/2 10031P 1/2 100 10.815N 1/2 0.37___________________________________________________
Nuclear Spin (I) and natural Abundance
Magnetic Moments and Energy Levels in the Magnetic Field
EnergieEo
E1 E = -μ . B0 μ = magnetic Moment= γ . I . h/2π
B0 = magnetic Density
Possible energy levels : 2 I + 1for I = 1/2
Energie
Eo + 1/2
E1 - 1/2
„parallel“
„antiparallel“
Energy Separations in the Magnetic Field
E = -μ . B0
B0 = magnetic Density
Energie
Eo + 1/2
E1 - 1/2 106
106 + 10
Energie
Eo -1/2
E1 +1/2
B0
Δ E
increasing difference in energy separations = increasing sensitivity
400 MHz
80 MHz
Free Induction Decay (FID) and
Fourier-Transformation
Decrease in trans-Versal Magnetization
Spin-Lattice and Spin-Spin Relaxation
FID - Timedomain
Frequency domain
Fourier transformation
1H NMR Spectrum of Ethylalcohol
NMR-Parameters are
Integral : relative amount of H-Atoms
Chem. Shift : Elektronic Environment
Spin-Spin Coupling : Sphärical Environment
Deep field shift High field Shift
B0 +
Chemical Shielding
ν Substanz - ν Referenz (Hz)δ = ----------------------------------
ν0 (MHz)
ppm, Parts per million, 10-6
B0
H-Atom
1s Orbital+
Blokal = B0 (1 - σ)σ = Shielding Constant
For Spectra Calibration:Tetramethylsilane (TMS), δ = 0Acetone, Acetonitrile.....
Multiple Bond Anisotropy
Aromatic Ring Current
+
+
O
+
+
H HH R ++- - - --
-
H
+
+-
H
H
H
H
H
H
H
H
H
HH
HH
H
H
H
H
H
[18]-Annulen
8.9 ppm -1.8 ppm
B0
Multiplicity of Resonance Lines due to the Spin-Spin For n magnetic equivalent Nuclei (n+1) Lines
Doublett (d) 1:1
Spin-Spin Coupling
HH
Orientation of Nuclear Spins ppm
δ H
Kopplungskonstante J (Hz)
HH
H
Orientation of Nuclear Spins
ppm
δ H
Triplett (t) 1:2:1
Spin-Spin Coupling
H
HH
H
Orientation of Nuclear Spins
ppm
δ H
Quartett (q) 1:3:3:1
11 1
1 2 11 3 3 1
1 4 6 4 1
Pascals Rule only valid for first Order spectra
J / ν0 . δ < 0.1
bzw. δ H ≠ δ H
Intensity Distribution according to Pascals Triangle
Coupling Constant
Coupling Nuclei Bonds J (Hz) ____________________________________________________
Direkt 13C-H, =13CH 1 JCH 120-170Geminal H-C-H, =CH2 2 2JHH 0 - 20
Vicinal H-C-C-H 3 3JHH 3 - 20„Long-range“ H-C=C-C-H 4 4JHH < 5____________________________________________________
HH
H
Jtrans (11-19) > Jcis (5 -14)
H HH H
Long range coupling < 1 Hz
Dependence of the Coupling constant of the Dihedral Angle „Karplus Equation“
H
Konformationen (Newman-Projektion)
HH
H
H
H
Φ
gauche-Konformation Φ = 60 °
anti-Konformation Φ = 180 °
Homonuclear Decoupling Double Resonance: Nuclear Overhauser Enhancement (nOe)
HH
ppm
δ H δ HSpin-Decoupling by irradiating with anadditional frequency
HH
ppm
δ H δ H
→Simplification of NMR Spectra and increase in intensity of neighbouringprotons
NOe ~ 1/ r6
Distance < 2.5 A
Literatur
Pretsch, Ernö : Spektroskopische Daten zurStrukturaufklärung organischer Verbindungen . - Berlin [u.a.] :Springer , 2001 ISBN: 3-540-41877-6 kart. : DM 79.90
Breitmaier, Eberhard : Vom NMR-Spektrum zurStrukturformel organischer Verbindungen . - Weinheim : WILEY-VCH , 2005ISBN: 3-527-31499-7 Pb. : EUR 39.90, sfr 64.00
Breitmaier, Eberhard : Structure elucidationby NMR in organic chemistry . - Chichester [u.a.] : Wiley , 2004 ISBN:978-0-470-85007-7
13C NMR Spectroscopy Isotope I nat. Abundance (%) γ______________________________
1H 1/2 99.98 26.813C 1/2 1.108 6.7
C13 OHH
H
HH
H
ppm
δ 13C
JC,H
C13 OHH
H
HH
H
ppm
δ 13C
Broad band decoupling→ Increase in intensity(NOe!)
13C NMR Data (ppm) of Hydrocarbons / Alcohols
n -Hexane:
13.7 22.8 31.9 31.9 22.8 13.7
H3C--CH2--CH2--CH2--CH2--CH3
n -Hexanol: γ β α
14.2 22.8 32.0 25.8 32.8 61.9
H3C--CH2--CH2--CH2--CH2--CH2-OH
3 -Hexanol:
14.0 19.4 39.4 72.3 30.3 9.9
OH|
H3C--CH2--CH2--CH--CH2--CH3
13P NMR Spectroscopy
O
HO OH
O P
OO
OO
P
OO
__
NH2
N
N
N
N
O
P
OO
__
γ β α
Isotope I abundance (%) γ__________________________1H 1/2 99.98 26.831P 1/2 100 10.8
0 -10 -20 ppm
adenosintriphosphate
2D NMR Spectroscopythe total correlated Spectroscopy (TOCSY)homonuclear Hartman-Hahn Echo HOHAHA
preparation
evolution
spinlockdetection
2D NMR Spectroscopythe total correlated Spectroscopy(TOCSY)homonuclear Hartman-Hahn Echo HOHAHA
strychnine
HR-MAS High Reslolution Magic Angle Spinning
The line width of an NMR resonance depends strongly on the microscopic environment of the nucleus under study. Interactions such as the chemical shift and dipole-dipole coupling between neighboring spins are anisotropic and impose a dependence on the NMR frequency based on the orientation of a spin or molecule with respect to the main magnetic field direction. Furthermore, the magnetic susceptibility of the sample and susceptibility differences within the sample lead to broadening of the resonances.
HR-MAS Introduction
In liquid state the rapid isotropic motion of the molecules averages the anisotropic interactions, resulting in an isotropic chemical shift frequency and a disappearance of the line broadening due to dipolar couplings. Furthermore the sample geometry, a cyclinder parallel to the main magnetic field, is chosen such that the susceptibility broadening is minimized.
HR-MAS Introduction
In solids on the other hand, the lack of molecular mobility results in broad lines.Because the magnitude of the coupling between two nuclear spins depends on the internucleardistance, the dipolar coupling is a through-space interaction. In contrast, J coupling requires the presence of chemical bonds. It is transferred through the electrons engaged in these bonds and thus is confined to nuclei within a molecule. Through-space dipolar coupling, however, also occurs between nuclei in different molecules.
HR-MAS Introduction
The two coupling mechanisms are therefore complementary in information content.
Three properties of the heteronuclear dipolar coupling Hamiltonian stand out:1) The magnitude of the coupling is proportional to the product of the gyromagnetic ratios. 2) The dipolar coupling is inversely proportional to the cube of the inter-nuclear distance, so the interaction falls off rapidly as the nuclei are moved farther apart.
HR-MAS Introduction
3) The dipolar coupling is dependent on the orientation. This means that for two nuclei of spins I and S which are separated by a fixed distance, the magnitude of the dipolar interaction will be greater for certain orientations of the I ± S internuclear vector than for others.
HR-MAS Introduction
In a static solid sample comprised of randomly oriented crystallites, however, the internuclear vector remains invariant over time, and the resonance frequency produced by each crystallite depends on its orientation with respect to the external field. In a polycrystalline powder sample in which the crystallites are oriented in all possible directions, the presence of a heteronuclear dipolar coupling produces a spectrum such as that shown next;
HR-MAS Introduction
Proton spectra of a human Lipoma tissue. The top spectrum is acquired in a conventional high resolution probe (spinning at 20 Hz), while the lower spectrum is acquired in a HR-MAS probe (spinning at 5 kHz)
HR-MAS Introduction
Dipolar pattern for two coupled spins in a powder sample; the two signals correspond to a positive (parallel spins) and a negative (antiparallel spins) value.
- d -d/2 0 d/2 d
The points with maximum intensity corresponds to Φ 90–a perpendicular orientation to B0 is adopted by a majority of the crystals.At the magic angle of Φ 54.7o the dipolar coupling is zero.
HR-MAS IntroductionThe line broadening can be reduced by spinning the sample rapidly around an axis which is oriented at an angle Φ 54.7o with the direction of the magnetic field. By spinning at this so-called Magic Angle, at a rate larger than the anisotropic interactions, these interactions are averaged to their isotropic value, resulting in substantial line narrowing.
The proton spectra of HMW (high molecular weight) subunits of wheat in D2O
a) Under static conditionsb) Obtained with a HR-MAS probe at 10 kHz
spinning rate
HR-MAS Introduction
In addition to pure solids or pure liquids there is a wide range of materials which exhibit either reduced or anisotropic mobility. Examples include polymer gels, lipids, tissue samples, swollen resins, plant and food samples. While these samples generally have sufficient mobility to greatly average anisotropic interactions, the spectral resolution for the static samples is still much lower than that which achieved for liquid samples.
HR-MAS Introduction
The excess broadening under static conditions is due to a combination of residual dipolar interactions and variations in the bulk magnetic susceptibility. For a variety of samples, including the aforementioned examples, magic angle spinning is efficient at averaging this left-over components of the solid state line width and leads to NMR spectra that display resolution approaching that of liquid samples. Such methods have been termed HIGH Resolution MAS NMR
HR-MAS ApplicationUpper probe chamber with MAS stator in magic angle position.(pneumatic switch)
MAS pneumatic control unit for sample spinning (up to 17 kHz)
HR MAS Application
Detection of natural abundance 1H–13C correlations of cholesterol in its membrane environment
Dependence on the spinning frequency the 1H MAS NMR spectrum of a sample of multilamellar vesicles of DMPC-d54/cholesterol
HR MAS Application
More applications; Fruitsanalysis of the ripening processespenetration of chemicals: insectizides, herbizidesanalysis of changes in parts damaged by transport etc.investigation of the rot processes
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